Final Report Summary - STAR-T REK (Set up and comparison of multiple stem cell approaches for kidney repair)
Executive Summary:
Stem cells have the remarkable potential to develop into many different cell types. In many tissues they serve as an internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or to become another type of cell with a more specialized function.
All stem cells—regardless of their source—have three general properties: they are capable of dividing and renewing themselves for long periods, they are unspecialized, and they can give rise to specialized cell types.
Regenerative medicine (which is also referred to as Cell therapy) is devoted to treatments in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed cells or tissues. Cell therapy depends on an understanding of how stem cells establish, maintain, and regenerate tissues and organs. There is much that we don’t yet know – about stem cells, about development, about what happens in disease and injury at a cellular level.
Chronic Kidney Disease (CKD) is a leading cause of mortality and morbidity in western countries and is estimated to affect 11% of the adult population. It can progress towards End Stage Renal Disease (ESRD), which has no cure and requires renal replacement therapy i.e. dialysis or renal transplantation. The number of patients with ESRD is growing consistently with rising cumulative costs that are even greater than the treatment costs of cancer.
Earlier stages of CKD are expected to be treatable with cell therapy.
Stem cells (SC), able of self renewal and to intervene in building/maintaining the structural and functional integrity of tissues, are especially attractive for such a purpose.
The focus of this project is to assess the regenerative potential of stem cells derived from different sources, to investigate the obstacles to their clinical utilization and their potential side effects.
Star-t rek is aiming at establishing whether stem cell therapy might offer an alternative approach to organ transplantation for patients suffering from kidney failure and at giving an insight on stem cells, their development, on what happens in the disease at a cellular level.
Successful clinical application of stem cell research requires a joined-up and well –planned approach. STAR-T REK has taken this role, in bringing together leading groups of basic and applied researchers and clinicians.
The novelty of the project STAR-T REK is:
• Use of different markers to isolate cell preparations
• Adoption of heterogeneous protocols of injection
• Study on distinct experimental models of renal failure
• Assessment of all potential safety concerns and rigorous toxicology study, with particular regard to the risk of anomalous differentiation and long-term possibility of tumorigenicity
• Possibility of tissue bioengineering a de novo replacement organ using fetal renal stem cells, with an ultimate aim of being able to implant functional renal replacements in patients with end stage renal disease
The results have been collected, compared, evaluated and converged in optimized protocols for phase I/II clinical trials in humans.
Project Context and Objectives:
In details, the project objective are the following:
1) Isolation of adult bone marrow derived stem and standardization of protocols for autologous cell therapy of renal failure
This first objective of the project has been achieved through coordinated discussion and work on shared protocols among different group leaders. First of all, among the different stem cell populations that can be derived from the bone marrow, we decided to focus our research on mesenchymal stem cells (MSC), which is the only bone-marrow derived stem cell type whose renoprotective/regenerating activity has been widely demonstrated in several preclinical models of acute and chronic kidney injury. Shared protocols were standardized for mouse MSC, rat MSC and human MSC. The protocol for human MSC preparation was also established taking into account GMP requirement and procedures.
2) Isolation and characterization of already known types of adult or fetal renal resident stem cells and standardization of protocols for their isolation for cell therapy of renal failure
The second objective of this project is focused on the possible use of resident renal stem cells as therapeutic tool for acute or chronic kidney disorders. To this aim a deep knowledge of the properties of renal stem cells is required. We have characterized CD133+CD24+ human renal progenitors and described their use in preclinical models of acute kidney injury. We demonstrated that CD133+CD24+ renal progenitors can be distinguished in distinct subpopulations from normal human kidneys based on the surface expression of vascular cell adhesion molecule 1 (VCAM1), also known as CD106. CD133+CD24+CD106+ cells were localized at the urinary pole of Bowman’s capsule, while a distinct population of scattered CD133+CD24+CD106- cells was localized in the proximal tubule as well as in the distal convoluted tubule. Once injected in SCID mice affected by acute tubular injury, both these populations displayed the capacity to engraft within the kidney, generate novel tubular cells and significantly improve renal function. These properties were not shared by other cell types of the adult kidney.
The glomerular tuft comprises three resident cell types, mesangial cells, endothelial cells, and podocytes. Some studies have shown that primary injury to each of these cell types is associated with glomerular disease. However, injury to endothelial and mesangial cells can be repaired by proliferation of adjacent cells. By contrast, podocytes cannot divide, and podocyte depletion is a common determining factor that results in glomerulosclerosis. Indeed, as long as the podocyte loss is limited, restitution or repair is possible.
By contrast, 20 to 40% podocyte loss results in a scarring response, until at greater than 60% podocyte loss, glomeruli become globally sclerotic and nonfiltering. During the course of this project, we provided the first evidence that podocytes can be regenerated by the CD24+CD133+ renal progenitors of the Bowman's capsule. In addition, we demonstrated that CD133+CD24+ renal progenitors are a heterogeneous and hierarchical population of undifferentiated and more differentiated cells that are arranged in a precise sequence within the Bowman's capsule of adult human kidneys. A subset of more undifferentiated cells expressing renal progenitor markers in the absence of podocyte markers localized at the urinary pole of Bowman's capsule acts as bipotent progenitor for both tubular cells and podocytes and exhibits self-renewal potential. A transition population expressing both renal progenitors and podocyte markers is localized between the urinary and the vascular pole of Bowman's capsule, exhibits a committed differentiative potential only toward the podocyte lineage and lacks self-renewal properties. Finally, differentiated cells, that do not express renal progenitor markers, but display high levels of the podocyte specific markers, localizes at the vascular pole of Bowman's capsule. Similar findings were observed in a parallel study performed in rodents by the group of Juergen Floege, who also demonstrated that transitional cells with morphologic and immunohistochemical features of both parietal epithelial cells and podocytes could be detected in proximity of the glomerular vascular stalk. More importantly, using an elegant model of genetic tagging of parietal epithelial cells in a triple-transgenic doxycycline-inducible mouse line, the same authors unequivocally demonstrated that podocytes are recruited from parietal epithelial cells, which proliferate and differentiate from the urinary to the vascular stalk generating novel podocytes. In summary, a large body of evidence now indicates that the Bowman’s capsule of adult kidneys contains a population of glomerular epithelial stem/progenitor cells which can replace lost podocytes through multiple mechanisms and allow glomerular regeneration. In a further series of studies, we have also demonstrated that injection of human CD24+CD133+ renal progenitors reduce proteinuria and restore podocyte depletion in preclinical model of focal segmental glomerulosclerosis. The related article (Ronconi et al. J Am Soc Nephrol., 2009), represents the first demonstration that renal stem cells can repair glomerular injury and was considered as a major breakthrough in the field of nephrology. It was the subject of an Editorial and was also selected by the Faculty of Thousands as a “must read” article with a score of 6.4. Taken together, these results also suggest the evidence for the existence of a stem cell system within adult human kidneys and that this renal progenitors cell population may represent a good candidate for cell therapy of chronic renal injury.
3) Identification of novel, unknown types of adult or fetal renal stem cells in mouse/rat and human adult kidney
The third objective of this project was to develop protocols to purify and identify putative further populations of resident stem cells in the developing kidney and to develop assay systems to test the competence of putative multipotent cells. Mouse models that allow identification and purification of embryonic and adult kidney related stem cells were developed. Also an experimental set up where stem cells or pluripotent cells can be effectively incorporated to the developing kidney were developed. By this set up embryonic stem cells can be in part targeted to the lineages of the embryonic kidney. Finally toxin related systems have been achieved to identify functional role of the stem cells and their roles in the renewal of the kidney structures in the adult. These strategies should allow identification and characterization of putative unknown types of renal stem cells in mouse.
4) Development and upgrading of techniques of de novo kidney organogenesis
Several novel assays to culture the embryonic kidney have been developed in the project. This approach has already been useful for three purposes; (i) for the study of the mechanisms that regulate normal renal development, (ii) for testing the ability of different stem cell types to make renal tissue (we have, for example, published the ability of human amniotic fluid-derived stem cells to do so) and (iii) for tissue engineering of transplantable kidney rudiments. Finally also a set up that allows reconstitution of the Bowman’s capsules with its associated major cell types was achieved. These advances are the starting points for a further development, including vascularization. We have preliminary results suggesting that this tissue engineered kidney has the ability to connect properly to a pumped blood system.
During the life of the Star-t-rek project, the basic techniques have been improved so that they generate an engineered equivalent of a fetal kidney that is organized around a single, branched collecting duct system that is capable of attracting blood vessels to itself and of constructing a glomerulus (paper in preparation by the group of Edinburgh). The results have been used as a foundation for further grant applications to the UK BBSRC to develop the technique towards practical transplant.
5) Comparison of in vivo renal regenerative potential of stem cells obtained from different sources in animal models of acute and chronic renal failure to set up standardized protocols for clinical trials in humans.
We analysed the regenerative capacity of mesenchymal stem cells (MSC) for injured renal tissue in different models of acute and chronic renal disorders related to tubulointerstitial or glomerular injury. This allowed us to understand that MSC may represent good tools for treatment of patients with acute kidney injury. In addition, we demonstrated that the activity of resident renal stem cells can be modulated to enhance the endogenous regenerative capacity of the kidney in preclinical models of chronic kidney injury related to glomerular damage.
6) Evaluation of the mechanisms at the basis of the renal regenerative potential of stem cells obtained from different sources in animal models of acute and chronic renal failure to set up standardized protocols for clinical trials in humans.
One of the most relevant problems related to cell therapy of acute kidney injury using mesenchymal stem cells is the reported possibility of their maldifferentiation into intraglomerular adipocytes in preclinical models of chronic Thy1.1 nephritis. Results achieved during the first period of the project showed surprisingly that MSC when injected in healthy kidneys were not detectable and left no trace of maldifferentiation. In contrast, the same cell preparation led to focal adipogenic intraglomerular maldifferentiation in rats with chronic Thy1.1 nephritis on day 60, thus suggesting that MSC maldifferentiation is probably related to growth factors produced during the glomerular disorder. Similarly, using confocal microscopy, laser capture microdissection, and real-time quantitative RT–PCR we also demonstrated that a deregulated activation of APEMP can induce the generation of hypercellular lesions in different podocytopathies and crescentic glomerulonephritis. These results provide an explanation for the pathogenesis of hyperplastic lesions in podocytopathies and crescentic glomerulonephritis and suggest that a deeper knowledge of the biology and growth properties of APEMP is required to set up cell therapy of kidney injury using these cells. These results are very important to set up cell therapy strategies of renal injury using renal progenitors. However, the mechanisms that regulate the growth and differentiation of renal progenitors are still mostly unknown. In STAR-T REK, we demonstrated also that a regulated activation of the Notch pathway in renal progenitors during their phases of differentiation toward the podocyte lineage is critical to balance injury and regeneration in glomerular disorders. The results of this study establish the novel concept that renal re generation can be achieved through paharmacological modulation of renal stem cell growth and differentiation. In addition, we demonstrated in different models of acute and chronic kidney injury, that the beneficial effect of cell therapy using MSC is mostly related to paracrine and anti-inflammatory effects and not to regeneration of resident epithelial cells of the kidney. However, the beneficial effects of MSC-treatment in different models of acute kidney injury can be relevant, which moved us to explore their possible clinical use in phase I/II clinical trials.
7) Evaluation of safety and immunogenicity of stem cells obtained from different sources in animal models of acute and chronic renal failure to set up standardized protocols for clinical trials in humans.
Results from multiple preclinical models of acute and chronic renal disease suggest that administration of MSC for cell therapy of acute kidney injury is safe in preclinical models of acute kidney injury, but administration of the same cells in conditions of chronic inflammation represents a possible risk factor for maldifferentiation. Currently, no risk of tumorigenicity were observed in all the preclinical models of renal injury analysed. More importantly, preliminary results in pilot phase I clinical trials suggest that injection of MSC in patients who underwent kidney transplantation is associated with a transient, mild and reversible acute renal failure related to factors released by MSC. After longer follow-up, patients are however in good health.
8) Set up standardized and optimized protocols for phase I/II clinical trials in humans
Results on the renal functional and histomorphological analysis and the evaluation of side effects have been collected and shared by all the groups of the consortium, allowing a standardized analysis of the data. The potential clinical efficiency of treatment with different stem cell types in the same experimental models has been directly compared, allowing to establish which could be the best type of treatment in each renal disease in terms of clinical efficacy and optimize protocols for phase I/II clinical trials in humans. Finally, pilot phase I trials of cell therapy using MSC were performed in patients who underwent kidney transplantation.
In summary, the outcomes of the project are:
- the identification of which cell type is better suitable for beneficial effect in preclinical models of acute and chronic renal failure;
- the assessment whether the beneficial (or not) effects are mediated directly by the transplanted cells or indirectly through involvement of other cell types;
- Insights on the mechanisms of stem cells-mediated regenerative effects, which are essential to set up cell therapies that should be effective and safe;
- novel concept that renal regeneration can be achieved through paharmacological modulation of renal stem cell growth and differentiation;
- Possibility of tissue bioengineering a de novo replacement organ using fetal renal stem cells or other strategies of kidney tissue engineering, with an ultimate aim of being able to implant functional renal replacements in patients with end stage renal disease.
The results of this project led to the a characterization of renal stem cells in adult human kidney and their capacity to regenerate injured renal tissue. In addition, tissue bioengineering of a de novo replacement organ using fetal renal stem cells and other strategies of kidney tissue engineering were explored. We analysed the regenerative capacity of mesenchymal stem cells (MSC) for injured renal tissue. These different strategies were compared and the mechanisms at the basis of their beneficial effects were assessed. This allowed us to understand that MSC may represent good tools for treatment of patients with acute kidney injury, although their beneficial effect is mostly related to paracrine and anti-inflammatory effects and not to regeneration of resident epithelial cells of the kidney. On this basis, a detailed protocol for clinical trials was prepared and pilot phase I clinical trials were performed in patients undergoing kidney transplantation. In addition, we demonstrated in preclinical models of acute and chronic kidney injury, that the activity of resident renal stem cells can be modulated to enhance the endogenous regenerative capacity of the kidney. Thus, the results of this project provided the scientific basis for treatment of patients with acute kidney injury by stem cell therapy, and for treatment of patients with chronic kidney disorders through pharmacological modulation of renal stem cell function.
Project Results:
WP1 Isolation and characterization of adult progenitors/stem cells for renal regeneration: main S&T results/foregrounds
1. Isolation of adult bone marrow derived stem cells (CD133+/CD34+ or mesenchymal stem cells) and standardization of protocols for autologous cell therapy of renal failure.
The results obtained are reproducible and the MSC populations, isolated from the bone marrow of different species by using these techniques, represent a good tool to test in preclinical models of renal failure.
Murine bone marrow-derived MSC: primary culture
Isolation and purification of murine MSC Bone marrow (BM) was obtained from 2-mo-old male C57BL6/J mice. Mice were killed, and femurs and tibias were aseptically removed. BM was flushed from the shaft of the bone with DMEM medium containing 2% FCS (Invitrogen) plus penicillin/streptomycin (100 U/ml to 0.1 mg/ml) and then filtered through a 100-µm sterile filter to produce a single-cell suspension. Murine MSC were recovered from BM by their tendency to adhere tightly to plastic culture dishes. Filtered BM cells were plated in DMEM plus 10% FCS and penicillin-streptomycin (100 U/ml to 0.1 mg/ml) and after 30 h non-adherent cells were removed. Medium was changed regularly every 3 d. After 2 to 3 wk, subconfluent (80%-90%) cells were detached by trypsin-EDTA (0.5 to 0.2 g/L) and murine MSC were purified by immunodepletion of CD45 positive cells. Briefly, after blocking with PBS that contained 0.5% BSA, cells were incubated for 20 min with rat anti-mouse CD45 antibody, 0.2 µg/106 cells (Caltag). Then, cells were incubated with magnetic microbeads coated with goat anti-rat IgG (Miltenyi Biotec), and CD45 negative MSC were isolated by magnetic cell-sorting separation system. For in vivo and in vitro experiments cells were used within first-second passages.
Characterization of murine MSC Cells were characterized for their capability to differentiate toward osteocytes, adipocytes, and chondroblasts. Murine MSC were grown until confluence, and the growth medium was replaced with the inductive medium consisting of Iscove’s modified Dulbecco’s medium, 20% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.05 mM β-mercaptoethanol supplemented with specific differentiation reagents as follows.
Osteogenesis: Cultures were fed twice a week for 3 wk with 10 mM β-glycero-phosphate, 50 μg/ml ascorbic acid 2-phosphate, and 10-9 M dexamethasone. Then cells were fixed with 10% formalin for 20 min at room temperature and mineralization—presence of calcium-rich hydroxyapatite—of the extracellular matrix was assessed by staining for 20 min with 2% wt/vol Alizarin Red S, adjusted to pH 4.1 with ammonium hydroxide.
Adipogenesis: Cells were incubated for 3 wk with 5 μg/ml insulin and 10-9 M dexamethasone. Adipogenic differentiation was visualized in phase-contrast microscopy by the presence of highly refractive intracellular lipid vacuoles. Oil Red O staining was used to assay the accumulation of lipid droplets in these vacuoles.
Chondrogenesis: Murine MSC were harvested and 6X105 cells were centrifuged to form a pellet on the bottom of a 15-ml polypropylene tube. The micromass was cultured in 500 μl of chondrogenic medium that consisted of 50 μg/ml ascorbic acid 2-phosphate and 1 ng/ml TGF-β1. After 3 wk of culture, cell clumps were harvested, embedded in paraffin, cut into 3-μm sections, and stained for glycosaminoglycans using 0.1% safranin O.
Results showed that murine MSC were able to differentiate toward osteocyte, adipocytes, and chondrocytes when exposed to specific inductive media.
Murine bone marrow-derived MSC: expanded culture
Isolation and growth of murine MSC We cultured murine MSC derived from bone marrow and expanded by using the protocol reported by Peister (Peister A. et al., Blood 103: 1662-1668, 2004). Following this protocol, by means of plating cells at very low densities, we obtained single-cell derived colonies with a rapid growth. The rate of expansion of the cultures was extremely sensitive to the initial plating densities. Cells obtained from each bone were first seeded into 75 cm2 flasks. After plating, the medium was changed for the following 4 weeks and, during this period, the non-adherent cells together with many detaching cells were removed. Clones were evident, although small, as early as 1 week of culture and, after 4 weeks, cells were almost confluent with a morphology resembling the heterogeneous cell population obtained by the traditional method already described, with a predominance of spindle-shape cell types. Once trypsinized, cells were split 1:2 and grown for 3 weeks. At the second passage, cells were seeded at 50 cells/cm2. At the following passages, cells reached confluence after 2-3 weeks of culture and were trypsinized and expanded up to the 9th passage. The analysis of the morphology of expanded murine MSC after nine passages in culture showed that cells represented a homogeneous population.
Characterization of murine MSC The cytofluorimetric analysis of expanded murine MSC revealed that they were negative for the haematopoietic cell markers CD11b and CD45. The hematopoietic marker stem cell antigen 1 (Sca-1) was expressed by the 81% of the cells, whereas c-kit expression was absent. At each passage, cells were able to form colonies and to differentiate into adipocytes.
Moreover, cells were able to produce mineral deposition when induced to differentiate with an osteogenic medium, suggesting a differentiation towards osteocytes had occurred. The expanded culture of murine MSC were able to differentiate into adipocytes and osteocytes at comparable extent than primary culture of murine MSC.
Set up of mouse MSC cultures
1. Bone marrow collection:
a) BM are obtained from 2-mo-old male C57BL6/J mice. Mice are killed, and femurs and tibias are aseptically removed. BM is flushed from the shaft of the bone with DMEM medium (Sigma) containing 2% FCS (Invitrogen) plus penicillin/streptomycin (100 U/ml and 0.1 mg/ml; Invitrogen) and then filtered through a 100-µm sterile filter (Falcon) to produce a single-cell suspension. MSC are recovered from BM by their tendency to adhere tightly to plastic culture dishes.
b) The viability will be evaluated by the percentage of negative cells after staining by blue trypan.
2. Culture set-up:
a) Filtered BM cells are plated at the concentration of 20.000/ml (25ml/Flask)
b) Non-adherent cells are removed after 30 hours and fresh medium is added.
• Medium: DMEM medium (Sigma) containing 2% FCS (Invitrogen) plus penicillin/streptomycin (100 U/ml and 0.1 mg/ml; Invitrogen)
• Equipment to be used: Container of culture: T175. To put in the incubator in wet atmosphere with 37°C with CO2 5%.
3. Medium exchange:
a) Medium is changed regularly every 3 days until the ending of the culture. The cell growth is appreciated by observation of the lower plateau under a microscope. The steps where the cellular layer is not covered any more with medium must be shortest as possible in order to avoid damaging the cells by desiccation.
• Medium: DMEM medium (Sigma) containing 2% FCS (Invitrogen) plus penicillin/streptomycin (100 U/ml and 0.1 mg/ml; Invitrogen)
4. Cell detachment and mouse MSC purification:
a) After 2 to 3 weeks, subconfluent (80-90%) cells are detached by trypsin-EDTA (0.5 and 0.2 g/L; Invitrogen).
b) MSC are purified by immunodepletion of CD45 positive cells. Briefly, after blocking with PBS that contained 0.5% BSA (Sigma), cells are incubated for 20 min with rat anti-mouse CD45 antibody, 0.2 µg/106 cells (Caltag). Then, cells are incubated with magnetic microbeads coated with goat anti-rat IgG (Miltenyi Biotec), and CD45 negative MSC are isolated by magnetic cell-sorting separation system.
c) For in vivo and in vitro experiments cells were used within first-second passages. in DMEM plus 10% FCS and penicillin-streptomycin.
Rat bone marrow-derived MSC
Isolation and growth of rat MSC BM was obtained from male Lewis rats weighting 180-200g. Briefly, rats were killed, and femurs and tibias were aseptically removed. BM was flushed from the shaft of the bone with α-MEM medium containing 2% fetal calf serum (FCS) and 1% penicillin/streptomycin (P/S) and then filtered through a 100 μm sterile filter to produce a single-cell suspension. Filtered BM cells were plated (200000 cell / cm2) in α-MEM plus 20% FCS and 1%P/S. Non-adherent cells were removed after 72 hours and rat MSC were recovered by their capacity to adhere highly to plastic culture dishes. Medium was changed regularly every 3d; after 2 to 3 wk, adherent cells were detached by trypsin-EDTA (0.5 to 0.2g/L) and used for the experiments.
Characterization of rat MSC Cells were characterized for their capability to differentiate toward adipocytes, and osteocytes. MSC were grown until confluence, and the growth medium was replaced with the inductive medium consisting of α MEM medium (Invitrogen) containing 20% FCS and 1% penicillin/streptomycin supplemented with specific differentiation reagents as follows.
Adipogenesis: Cells were incubated for 3 wk with 5 μg/ml insulin, 10-6 M dexamethasone, 0.5 μM isobutylmethylxanthine, 50 μM indomethacin. Then cells were fixed with 10% formalin for 10 min at room temperature and adipogenic differentiation was visualized in phase-contrast microscopy by the presence of highly refractive intracellular lipid vacuoles. Oil Red O staining was used to assay the accumulation of lipid droplets in these vacuoles.
Osteogenesis: Cultures were fed twice a week for 3 wk with 10 mM β-glycero-phosphate, 0.2mM ascorbic acid 2-phosphate, and 10-8 M dexamethasone. Then cells were fixed with 4% paraformaldehyde for 10 min at 37°C and mineralization—presence of calcium-rich hydroxyapatite—of the extracellular matrix was assessed by staining for 20 min with 2% wt/vol Alizarin Red S, adjusted to pH 4.1 with ammonium hydroxide.
We have observed that rat MSC were able to differentiate towards adipogenic and osteogenic lineage after 3 weeks of culture with specific inductive media.
Set up of rat MSC cultures
1. Bone marrow collection:
c) BM is obtained from Lewis rats (Harlan, France) weighting 180-200g. BM from femur cavity was flushed (10 times) with MEM medium (ABCYs, France) containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (P/S) (Invitrogen, USA).
d) Cell suspension is centrifuged (400 g, 5 min).
e) The viability will be evaluated by the percentage of negative cells after staining by blue trypan.
2. Culture set-up:
c) Cell pellet is re-suspended in complete medium (MEM + 10 % FCS + 1% P/S) and cells are plated in culture flasks at a concentration of 2x105 cells/cm2).
d) Non-adherent cells are removed after 72 hours and fresh medium is added. MSCs are recovered by their capacity to adhere highly to plastic culture dishes.
• Medium: MEM medium (Lonza) containing 10% FCS (Bodinco) plus penicillin/streptomycin (100 U/ml and 0.1 mg/ml; Sigma )
• Equipment to be used: Container of culture: T175 . To put in the incubator in wet atmosphere with 37°C with CO2 5%.
3. Medium exchange:
b) Medium is changed regularly every 3 days until the ending of the culture. The cell growth is appreciated by observation of the lower plateau under a microscope. The steps where the cellular layer is not covered any more with medium must be shortest as possible in order to avoid damaging the cells by desiccation.
• Medium: MEM medium (Lonza) containing 10% FCS (Bodinco) plus penicillin/streptomycin (100 U/ml and 0.1 mg/ml; Sigma)
4. Cell detachment and rat MSC purification:
d) After 2 to 3 weeks, subconfluent (70-80%) cells are detached by trypsin-EDTA (0.5 and 0.2 g/L; Sigma)
e) For in vivo and in vitro experiments cells are used at passage 3.
Human bone marrow-derived MSC
Isolation and growth of human MSC BM aspirates were collected within the Transplantation Program of the Haematology Division. Two–five milliliter of heparinised diagnostic samples were obtained during the clinical follow up of patients previously treated by autologous or allogeneic BMT who were in complete confirmed hematologic remission after informed consent and separated by Ficoll-Hypaque gradients centrifugation to yield mononuclear cells. In the case of bag washouts, total nucleated cells from filter and bag residues were obtained after two washings with 250 ml saline and centrifugation at 2000 r.p.m. for 5 min. Two protocols for the expansion of human MSC were compared: cells were plated at 200 000 cells/cm2 in DMEM low glucose medium containing gentamicin 0.1mM and heparin 1000 IU in the presence of 5% platelet lysate or 10% fetal bovine serum (FBS). We used the batch of FBS selected from the EBMT MSC Expansion Consortium (Perbio Science, Etten Leur, Netherlands). Non-adherent cells were removed after 3–4 days and fresh medium was added. At subconfluency, cells were recovered after Trypsin-EDTA treatment and subsequently replated at 3000 cells/cm2 in the same medium.
We obtained a significantly better expansion of human MSC when cultured in medium containing platelet lysate in respect to FBS. At variance, no difference were observed in terms of gross morphology, differentiation potential, surface markers of human MSC expanded with platelet lysate or FBS as describe below (Capelli C. et al. Bone Marrow Transplant.: 40, 785-791, 2007).
Characterization of human MSC The immunophenotype of human MSC were studied by using the following conjugated monoclonal antibodies: CD45-FITC, CD34-PE, CD14-FITC, HLA-DR FITC, CD29-PE, CD90-FITC, CD73 -PE, HLA-ABC-PE, CD105-PE or isotype-matched IgG-FITC and IgG-PE control antibodies. Data showed that cells expanded in platelet lysate or FBS exhibited a similar immunophenotype. In fact, both populations were positive for CD90, CD73, CD105 and negative for CD45, CD34, CD14 and HLA-DR markers.
Adipogenesis: Human MSC were used at third passage in culture. 5X104 cells derived from FBS and platelet lysate cultures were washed with PBS and plated in cell culture dishes, with adipogenic medium consisting of MesenCult Basal medium and 10% Adipogenic Stimulatory supplements (StemCell Technologies). Medium was changed weekly and dishes were observed under the microscope for the development of lipid droplets. Their lipid nature was confirmed by staining with Sudan Black IV after 21–28 days culture in these conditions (Capelli C. et al. Bone Marrow Transplant.: 40, 785-791, 2007).
Osteogenesis: Human MSC at third passage in culture were washed with PBS and plated with osteogenic medium consisting of Mesen-Cult Basal medium, 10% Osteogenic Stimulatory supplements consisting of 1X10-8M dexamethasone, 2X10-4M ascorbic acid and 7X10-3M β-glycerophosphate. The presence of alkaline phosphatise activity in differentiated samples was confirmed by staining with immunohistochemical alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique after 21–28 days of culture in these conditions (Capelli C. et al. Bone Marrow Transplant.: 40, 785-791, 2007).
Results showed that both populations of human MSC grown in platelet lysate or FBS were able to differentiate in a comparable extent toward adipocytes and osteocytes as demonstrated by specific staining with Sudan Black IV and APAAP (Capelli C. et al. Bone Marrow Transplant.: 40, 785-791, 2007).
For the development of a reagent enabling the large scale enrichment of MSCs from bone marrow CD271 and W8B2 antibodies were chemically coupled to paramagnetic particles. These conjugates were used in several optimization experiments to improve purity and yield of CFU-F forming target cells. In the future the focus will be on CD271 Microbeads.
To transfer the procedure to the closed clinical large scale CliniMACS system the sample preparation, labeling and elution of MSCs will be optimized. Feasibility was already shown.
Standardization of the characterization of MSCs is a prerequisite for comparative clinical studies involving isolated and/or cultivated MSCs. To provide an easy to handle reliable quality control a phenotyping protocol based on flow cytometry using the most important markers was developed. The new protocol includes the markers CD73, CD90, CD105 as positive markers and CD14, CD20, CD34, CD45 as negative markers. Only 2 cell samples with about 1x105 cells are needed to fully characterize the cells.
2. Characterization of CD24+CD133+ adult renal resident stem cells (APEMP) and standardization of protocols for isolation of these cells for cell therapy of renal failure.
A large body of evidence currently indicates that APEMP represent a population of renal progenitors which can potentially regenerate tubular as well as glomerular injury. To obtain CD24+CD133+ APEMP a protocol was standardized.
1. Kidneys are minced and digested with Collagenase type IV 750 U/ml (Sigma) for 45 minutes at 37°C.
2. Total kidney is mechanically disaggregated using sieving through 60 and 80 mesh screens or using the Medimachine System (BD Biosciences).
3. The pass-through is recovered, washed with PBS and red cells are removed through lysis buffer (NH4Cl 0.087% ) for 4 minutes at 37°C or through depletion with anti–glycophorin A MicroBeads (Miltenyi).
4. The suspension is depleted of leukocytes, using anti-CD45 MicroBeads (Miltenyi), following the manufacturer’s instructions using the VarioMACS column (Miltenyi).
5. The CD45-depleted fraction undergoes a second magnetic separation for CD133 (CD133 Cell Isolation Kit, containing the anti-CD133/1 mAb, clone AC133, also used for hematopoietic stem cell sorting). The separation is performed by magnetic cell sorting using LS columns (Miltenyi). The positive cell fractions usually consists of >97% of CD24+CD133+ cells.
Cells are cultured on dishes in EGM-MV 20% FBS. Cultures are checked for simultaneous expression of CD133 and CD24 by flow cytometry and routine cell passaging is performed. Medium is changed twice a week.
In addition, this population was largely characterized, the mechanisms at the basis of their homing toward injured kidneys were identified, and their beneficial effect in acute as well as chronic preclinical models of glomerular injury was addressed. In particular, we demonstrated that APEMP represent an heterogeneous and hierarchical population of progenitors with different commitment toward the podocyte or tubular lineage. We indeed demonstrated that APEMP can regenerate podocytes and improve proteinuria in in vivo models of glomerular injury, and we also demonstrated that APEMP can be distinguished in bipotent as well as tubular committed progenitors on the basis of their expression of the surface marker CD106. Consistently, both these populations could regenerate injured tubular tissue and improve renal function in mice modela of acute renal failure related to tubular injury. Finally, we investigated the mechanisms that mediate APEMP migration, homing, and repopulation, which are crucial for the success of a clinical strategy of SC-based kidney regeneration. To identify APEMP homing factors, the chemokine receptor expression pattern of these cells was investigated. APEMP exhibit high expression of the two receptors for SDF-1, CXCR4, and CXCR7. To assess whether the interaction of SDF-1 with CXCR4 and/or CXCR7 was involved in the regenerative capacity of APEMP, glycerol-treated SCID mice were injected with the red fluorescent dye PKH26 APEMP labeled into the tail vein after pretreatment with a neutralizing anti-CXCR4 antibody, or a neutralizing anti-CXCR7 antibody. BUN levels, as well as areas of necrosis found 4 d after injury, were significantly higher in mice injected with saline or with PKH26-stained APEMP pretreated with either an anti-CXCR4 or -CXCR7 antibody in comparison with those found in mice injected with PKH26-stained APEMP, which had been pretreated with the respective isotype-matched antibody with irrelevant specificity. Similar results were obtained on day 14 after injury. Accordingly, evaluation of the fibrosis score with Masson's trichrome staining in mice injected with APEMP pretreated with either an anti-CXCR4 or -CXCR7 antibody, demonstrated a higher fibrosis score in comparison with the respective control mice injected with PKH26-stained APEMP, which had been pretreated with isotype-matched control antibody with irrelevant specificity. Quantitation of the number of APEMP over the total number of renal cells demonstrated a significantly higher percentage of APEMP in the kidney of mice injected with cells pretreated with the isotype-matched control antibody with irrelevant specificity in comparison with mice injected with cells pretreated with an anti-CXCR4 antibody or with an anti-CXCR7 antibody, 4 d after injury as well as 14 d after injury. The role of interaction between SDF-1 and its receptors CXCR4 and CXCR7 was also investigated in in vitro culture of APEMP. SDF-1 –induced migration of renal progenitor cells was only abolished by an anti-CXCR4 antibody, whereas transendothelial migration required the activity of both CXCR4 and CXCR7. CXCR7 resulted essential for renal progenitor cell adhesion to endothelial cells and renal progenitor cell survival. Taken together, these results suggest that APEMP may represent a tool for treatment of acute kidney injury.
3. Identification of Sca-1+Lin- “like” adult renal resident stem cells in adult human kidneys and standardization of protocols for isolation of these cells for cell therapy of renal failure.
In mouse, we focused on two major sources of stem cells for kidney repair. One was described in 2006 in adult kidneys known as the SCA+ Lin- cells that seem to represent in mice a mesenchymal renal residing stem cells. We have now continued characterizing the attributes of SCA+ Lin- cells in adult and embryonic mouse kidneys. In particular we attempted to examine the niche of these putative stem cells, which as in the bone marrow might comprise innervating and endothelial neighbouring cells. Our new data clearly shows that indeed the SCA+ Lin- cells reside in the kidney in a typical 3 dimensional niche as previously shown for these cells in the bone marrow. One important marker is Nestin, the expression of which in mature adult tissues is restricted usually to areas of regeneration. Nestin has been shown to interact with other cytoskeleton proteins, suggesting a role in regulating cellular cytoskeletal structure. In developing kidney, nestin is expressed in vascular cleft of the S-shaped body and vascular tuft of capillary loop–stage glomerulus. The nestin-positive structures are also labeled by endothelial cell marker CD31 in immature glomeruli. Nestin is not detected in epithelial cells of immature glomeruli. Likewise we identified similar niches in human fetal kidney by staining for nestin and CD34, co-expressed in glomerular and tubular structures, which are negative for panepithelial markers. This results will help to better understand the mechanisms of kidney regeneration in rodents models of renal injury.
Identification of bone marrow or kidney origin of Sca-1+Lin- cells
We have previously characterised a multipotent organ stem/progenitor cells in the adult mouse kidney (Dekel et al, 2006). This cell population is stem cell antigen-1 (Sca-1) positive and lacks CD45 and lineage markers and resides in the renal interstitial spaces. Since the same phenotype can also be found in the BM it was of interest to define whether these unique cells arrive in the kidney from the BM or develop endogenously in the kidney.
We now addressed this question by using BM radiation chimera derived by transplantation of GFP+ BM into lethally irradiated (10Gy TBI) recipients. Thus, when kidneys were harvested from these mice in which the blood cells were predominantly of donor type, we found that the majority (93%)of the SCA+CD45- CD31-CD326- population was of host origin ( GFP-) as opposed to almost 70 % of the hematopoietic SCA+CD45+ CDD31-CD326- cell population found to be GFP+ (i.e. originating from BM cells) . Taken together, these results suggest that the renal Sca1+Lin- stem cells likely represent an endogenous population developing in the kidney in-situ and not arriving from the BM, which has important implications to understand the regenerative mechanisms of the kidney.
4. Identification of novel, unknown types of renal stem cells in mouse/rat and adult human kidneys:
Unknown types of stem cells in kidney have been identified on the basis of their clonogenic properties. The presence of clonal cells in kidney tissue homogenates has been demonstrated by using colony-forming assay. In this in vitro culture system, only single renal progenitor forms colonies. Colonies have been analyzed for their multidifferentiation capacity and self-renewal potential. In a second strategy, cells expressing marker of metanephric mesenchyme which are down-regulated during the differentiation or expressing markers of stem cell derived from different tissues have been isolated using FACS. Their clonogenicity, multidifferentiation and self-renewal potential have been analyzed by limiting dilution assay and culture in differentiating medium.
Recent studies reported the existence of scattered CD133+CD24+ cells distributed within the proximal tubules in adult human kidneys, and suggested they may represent a subset of progenitors destined to tubular regeneration. However, a phenotypic and functional characterization of tubular CD133+CD24+ cells was not reported, because specific markers allowing distinction from the CD133+CD24+ cells of Bowman’s capsule are unknown. To perform phenotypic and functional studies separately on CD133+CD24+ cells of Bowman’s capsule as well as on CD133+CD24+ tubular cells, we sought to identify a surface marker that allowed to distinguish these two populations. To this aim, CD133+CD24+ cells were prepared from outgrowth of capsulated glomeruli or from the tubular portion of renal tissue and screened for mRNA expression of 279 genes, using TaqMan low-density array (TLDA) technology. More than 95% of the investigated genes showed similar expression in CD133+CD24+ cells obtained from glomerular outgrowths compared with CD133+CD24+ cells obtained from tubular tissue. However, the expression of Vascular cell adhesion molecule-1 (VCAM-1), also known as CD106, was about 300-fold higher in CD133+CD24+ cells obtained from glomerular outgrowths than in CD133+CD24+ cells obtained from tubular tissue.
In the human kidneys, CD133+CD24+CD106+ cells were localized at the urinary pole of Bowman’s capsule, while a distinct population of CD133+CD24+CD106- scattered cells was localized in the proximal tubule, as well as in the distal convoluted tubule.
CD133+CD24+CD106+ cells exhibited a high proliferative rate and could differentiate toward the podocyte as well as the tubular lineage. By contrast, CD133+CD24+CD106- cells showed a lower proliferative capacity and displayed a committed phenotype toward the tubular lineage. Interestingly, both CD133+CD24+CD106+ and CD133+CD24+CD106- cells showed higher resistance to injurious agents in comparison to all other differentiated cells of the kidney. Finally, once injected in SCID mice affected by acute tubular injury, both these populations displayed the capacity to engraft within the kidney, generated novel tubular cells and significantly improved renal function, a property that was not shared by other cell types of the adult kidney.
Isolation of “untouched” cell populations from human whole blood.
The magnetic bead-mediated isolation of rare cells is challenging because of the very rare nature of target cells, e.g. CD271+ mesenchymal stromal cells show a frequency of 0.02% in human bone marrow aspirate. The isolation protocol of a certain stem cell population from human or mouse tissue using specific monoclonal antibodies conjugated to superparamagnetic nanoparticles was optimized with regard to the following points:
1. Optimization of magnetic beads focused on the reduction of unspecific binding:
Unspecific binding is always a challenge when utilizing magnetic particles for the isolation of certain stem cell populations within heterogeneous cell suspensions. This background labeling is thought to occur as a result of non-specific antibody binding to endogenous Fc receptors, ionic effects, and hydrophobic interactions.
2. Optimization of a protocol for isolation of cell populations using whole blood:
Whole blood is an attractive source for lymphocytes and progenitor stem cells due to it's availability. Current technology requires isolation of leukocytes from whole blood, e.g. by a Ficoll density gradient separation, before specific cell populations can be further purified. We previously developed a novel cell separation technology to isolate target cells directly from whole blood without proceeding bulk separation steps such as density gradient centrifugation. This technology removes unwanted non-target cells. Monoclonal antibodies have been covalently conjugated to novel magnetic beads (200nm - 500 nm). Antibody-bead conjugates have been evaluated for depletion performance when combined with an erythrocyte aggregation reagent. Both components are added to diluted human whole blood, incubated for 10 minutes and unwanted components of blood (erythrocytes, thrombocytes, lineage positive cells) are allowed to sediment in a magnetic field generated by a specifically designed permanent magnet for 10 minutes.
Achievements:
a.) Several magnet designs have been evaluated and developed. A design of magnet yoke, magnet size and shape and plastics housing could be selected to use 30 ml of human whole blood for cell separation in a 50 ml centrifuge tube.
b.) Several lineage markers were evaluated for depletion performance of non-stem cells, including CD3, CD19, CD56, CD14, CD15, CD16, CD2, CD7, CD36, CD4, CD8. Most of the conjugates allowed removing 99% of the cells positive for a lineage marker from whole blood.
c.) A method has been developed for further reducing erythrocyte content in the final stem cell preparation. Especially for rare target cell populations such as progenitor cells the 99.6% removal of erythrocytes in the single step cell separation method results in a huge excess of erythrocytes over target cells. An additional removal of 99% of erythrocytes can be achieved by use of an erythrocyte specific antibody conjugated to magnetic beads.
d.) Combinations of lineage markers have been evaluated for optimized depletion of unwanted lineage positive cells.
e.) A lyophilisation process has been established to stabilize cocktails of cell separation reagents.
5. Preparation of protocols for the characterization of human adult stem cells
1. Characterization of the cell surface expression of human mesenchymal stromal cells (MSCs)
The state of the art marker panel for the characterization of in vitro expanded human MSCs was published by Dominici et al (2006). They defined the markers CD73, CD90 and CD105 to be expressed on MSCs and lack of expression of hematopoietic markers like CD14, CD19, CD34 and CD45. With regard to this minimal criteria we optimized a fast and reliable FACS based assay. It consists of a MSC staining cocktail including CD73-APC, CD90-FITC, CD105-PE, CD14-PerCP, CD20-PerCP, CD34-PerCP, CD45-PerCP and a MSC control cocktail including corresponding isotype controls.
MSCs show a bright auto-fluorescence and the fluorochrome cocktails include four different fluorochrome dyes. Hence, the analysis requires a proper compensation of the used flow cytometer. Therefore, we optimized a procedure for a fast and accurate compensation procedure using single staining of each fluorochrome.
2. Functional analysis of the immunosuppressive potential of human mesenchymal stromal cells (MSCs)
MSCs show immunosuppressive capacity when co-cultured with proliferating Tcells. This in vitro observation may give a certain insight into the interaction of MSCs and immune cells in vivo within several clinical settings and can be used as a functional quality control assay for MSCs used in the clinics. There is a demand for a standardized and optimized assay and therefore we optimized a protocol for the examination of the immunosuppressive effect of MSCs on proliferating T cells. The protocol is based on MACSiBead particles pre-loaded with biotinylated CD2, CD3, and CD28 antibodies. We optimized the loading conditions of MACSiBead particles with antibodies and titrated the MACSiBeads for an optimal T cell stimulation. This process was embedded into a protocol including the optimized concentration of MACSiBeads, T-Cells and MSCs within a 96 well platform for an accurate high throughput analysis.
WP2 Isolation of fetal renal progenitors/stem cells and assessment of their ex-novo kidney organogenesis potential
1 Characterization of CD24+CD133+ human fetal renal resident stem cells and standardization of protocols for isolation and expansion of these cells for cell therapy of renal failure.
To obtain CD24+CD133+fetal renal resident stem cells, the embryonic kidneys were minced and digested with collagenase IV (500U/ml) for 20 minutes at 37°C. The renal suspensions were collected and checked for simultaneous expression of CD133 and CD24 by flow cytometry. CD133 was evidenced by using a PE-conjugated anti CD133/2 mAb combined with an anti CD24 mAb (IgG1) followed by an Alexa Fluor® 633-labelled goat anti-mouse IgG1. Since CD133+ cells represented a subset of whole CD24+ cells, the CD24+CD133+cells were isolated by using the CD133 Cell Isolation Kit (Miltenyi Biotec GmbH). The positive cell fractions consisted of >99% of CD24+CD133+ cells; the recovered cells were plated in EGM-MV (Cambrex Bio Science, East Rutherford, NJ) 20% FBS (Hyclone, Logan, UT) and then used for characterization. FACS analysis showed that such a population homogeneously exhibited not only the presence of CD24 and CD133, but also expressed CD106, CD44, CD54 and CD29, while haematopoietic markers (CD34, CD45) were not detectable. The effect of CD133+CD24+ cells obtained from adult human kidneys was compared with that of cells obtained from fetal human kidneys. When injected into mice with ARF related to tubulointerstitial injury (mice models of glycerol-induced acute kidney injury in SCID mice), both cell types regenerated cells of different portions of the nephron, reduced tissue necrosis and fibrosis and significantly improved renal function with similar efficiency, regenerating tubular structures.
2 Identification of novel, unknown types of renal stem cells in mouse, rat and human fetal kidneys.
a. through phenotypic and functional screening. The lab of partner 8 has focused during 2009 to develop assay systems to analyze reliable the capacities of specific cell types, especially their potential to reconstitute specific structures in the developing kidney. For this purpose the mouse embryonic kidney has been prepared at midgestation and the ureteric bud has been mechanically separated from the adjacent metanephric mesenchyme. Thereafter the kidney mesenchymal cells have been dissociated to single cells. Followed by this the cells have been incubated in a variety of mixtures consisting of specific factors that are known to be important in the control of kidney development. The aim here has been to maintain the competence of the kidney mesenchymal cells to form the nephrons. During 2009 the so called kidney mesenchyme dissociation and reaggregation model has been established. We can now efficiently dissociate the kidney mesenchyme to single cells and maintain the competence in these cells at least until 48 hrs prior to experimental induction of nephrogenesis in these cells when reaggregated. Such system has enabled us to screen the potential of a variety of cells in their capacity to integrate efficiently to the structures that are derived from the kidney mesenchyme, namely the nephrons with is numerous cell types and the stromal cells. We have also screened the reconstitution potential of such reaggregates by analyzing to what degree the reaggregated and induced kidney mesenchyme can indeed form a segmented nephron. As it seems based on the data, the induced nephron developing in such reaggregates can form the major segments of the nephron. We have used this system to address the potential of variety of pluripotent cells such as genetically labeled embryonic stem (ES) cells and even human embryonic stem cells. These studies are starting to point that the genetically indicator labeled cells can be directed towards the cell lineages of the pretubular cells. We are using this system to assay the requirements of the ES cells or other the pluripotent cells to be induced towards the structures of the nephron. We are also developing methodology to infect the dissociated mesenchymal cells with a variety of viruses to introduce specific components to the kidney mesenchymal cells or the pluripotent cells. We are using the reaggregation system and genetically GFP labeled cells to address the potential of the individual cells of the kidney to reconstitute the kidney structures. Finally we also develop the same methodologies for the epithelial ureteric bud to address its potential for differentiation and viral infection. This work is in progress.
In addition, partner 7 further developed a system for making fine-grained chimaeric kidneys, into which stem cells can be introduced to test their potential when placed in an appropriate environment. Specifically, while screening potential stem cells from mouse and human, partner 7 has identified an AFSC line that incorporates very well into this system and is currently assessing the degree of renal differentiation using human-specific RT-PCR for renal genes. In addition, partner 7 is also using this system to test the potential of cells isolated from embryos of different developmental stages to contribute to renal tissue, to map the range of cell types into which we could reprogram stem cells and reasonably expect them to produce renal tissue.
b. Identification of novel nephron stem cells with the aid of the Wnt-4 GFP Cre knock in mouse.
In order to fate map the cells that have expressed the Wnt-4 gene we have targeted the CreGFP cassette under the control of the endogenous Wnt-4 promoter. The constructs have been made and successfully targeted to the Wnt-4 locus. The ES cells have been injected to the blastocyst and the mouse line has been established. We have used the Wnt-4GFPCre mouse line to verify that the Wnt-4 promoter drives correctly the GFP and Cre. Indeed the GFP becomes expressed by the cells that normally express Wnt-4 gene. Given this success we have used a floxed indicator mouse line, namely the floxed Rosa26 where LacZ becomes permanently activated upon the Cre reaction. These studies have allowed us to map the fate of the cells that have expressed the Wnt-4 gene. The in vivo fate mapping studies have indicated that the cells that have expressed the Wnt-4 gene generate the nephrons. However on top of that we also found some LacZ positive cells in the stroma suggesting that the Wnt-4+ cells may have also other fates. To address this possibility in detail we established an organ culture based system where the fate of the cells was analyzed by a time-lapse set up. This set up allows almost real-time monitoring of the fates generated by those cells that have expressed the Wnt-4. The time-lapse studies revealed the dynamic nature of the cells that express the Wnt-4 gene. The stem cells become motile. Also the dynamics how the nephrons are generated from the nephron stem cells can be visualized. Interestingly we also could show that the first few nephrons are transient and that these cells likely transform to generate the medullary stromal cells. These medullary stromal cells are however transient in their nature but are likely essential for further development of the smooth muscle actin positive stromal cells induced by the signals that are expressed by the transient stromal precursor cells. Fate mapping with other Cre lines should be done to obtain additional evidence for this possibility.
The work has been published during 2009 (Shan J, Jokela T, Skovorodkin I, Vainio S. Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development. Differentiation. 2010 Jan;79(1):57-64). To address the role of the Wnt-4 protein itself in the control of the nephron stem cells we have also generated a floxed allele of the Wnt-4 gene. This allows detailed studies of the role of Wnt-4 in specification of the different cell lineages of the nephron (Shan J, Jokela T, Peltoketo H, Vainio S. Generation of an allele to inactivate Wnt4 gene function conditionally in the mouse. Genesis. 2009 Nov;47(11):782-8).
c. Purification and replacement of novel fetal nephron stem cells through Avidin-LDL fusion protein, Lodavin approach. In order to purify and replace the nephron stem cells we have generated and applied two different mouse lines in the laboratory. We have established the constructs to target Lodavin to the Rosa26 locus. This has been achieved during 2009. We have also generated the Lodavin mouse like and confirmed that Lodavin expression can be activated by the Cre mediated approach. We have successfully obtained activation and expression of Lodavin by using several Cre lines including the Wnt-4 Cre to target the nephron stem cells. Lodavin expression is indentified with a specific antibody against avidin. We are in a process of testing if Lodavin can be used as a vehicle to also purify the nephron stem cells. We have recently obtained a FACS machine to our university. We will use this in trials to try to purify the Wnt-4+ cells from the embryonic kidney by using Lodavin or the GFP that is expressed from the genetically manipulated Wnt-4 locus. In conjunction with the aims to purify the nephron stem cells we have also applied a mouse line where human diphtheria toxin receptor can be activated and directed to those cells that express the Cre recombinase. This mouse line called iDTR has been targeted to the Rosa26 locus. Indeed we have used the organ culture setting to assay if the iDTR expression and thus susceptibility to diphtheria toxin (DT) can be directed to the nephron stem cells to ablate them specifically. Here iDTR expression has been activated to the nephron stem cells with the aid of Wnt-4 Cre. The embryonic kidneys have been prepared from the compound positive embryos thus expressing iDTR and Wnt-4Cre and DT has been applied in organ culture. In this setting we can successfully delete the nephron stem cells. We are currently using the set up to develop in vivo models for renal cell replacement therapies based on the use of the nephron stem cells.
Assessment of the developmental potential of renal stem cells using a novel and rapid in vitro system.
We have tested the ability of a number of different stem cell types to produce renal tissue in chimaeric engineered kidneys, made from suspensions of mouse foetal kidney stem cells (direct from the foetus) and labeled test cells. These test cells have included embryonic stem (ES) cells, ES cells treated in various ways, for example with BMPs, to try to encourage them towards the renal lineage, human amniotic fluid stem cells (AFSCs), mouse AFSCs, and putative stem cells isolated from mature kidneys. We have found (and published) that human AFSCs seem to show a strong ability to do this, and further have found that TOR signaling is important to that. We have found the ability of the other stem cells to be disappointing. In an effort to find out why, we have gone back in developmental time, testing the ability of cells from successively earlier stages of mouse development, in the lineage that will eventually give rise to kidney, to contribute to renal tissue in this assay. This work has identified a critical ‘switch’ – everything from ES cells to embryonic day 8 intermediate mesoderm has a weak ability to contribute to kidney formation, while everything later performs well. We will now (in other projects) concentrate on understanding what mediates this change, to see if we can being ES cells to the capable state ‘manually’ by treatment with defined molecules.
3 De novo kidney organogenesis in vitro.
A novel organ culture set up was developed that allows 3D imaging of the stem cell behaviour to be monitored close to real time by time lapse approach. Here the kidney is cultured under a certain pressure and this pressure improves the exchange of gases and allows also good visualization of the developing kidney rudiment. The set up is excellent to follow the fate and developmental capacity of the locally injected stem cells that are either derived from the embryonic kidney or inserted from other sources. Such flattened culture set up is beneficial likely due to reduction of hypoxia. These aspects will be published as a result of the project. Moreover it has been developed and published a novel ‘low volume’ technique for culturing intact mouse fetal kidneys that allows proper formation of cortico-medullary zonation and loops of Henle. Second, The development of techniques for engineering fetal kidneys from simple suspensions of cells has been continued: now it is possible to build kidneys properly arranged around a single branched collecting duct system, and kidneys that can attract blood vessels from living tissue and form glomeruli.
Routine in vitro kidney cultures are hampered by the absence of a normal vascular network. As a result glomeruli do not develop beyond the late S-shaped body stage and filtration never occurs. To circumvent this problem, we catheterized the dorsal aorta of embryos at various stages and perfused the developing kidneys with oxygenated growth medium (micropump-controlled flow rate and pressure). Control kidneys showed a high rate of necrosis and apoptosis due to the absence of oxygen in deeper tissue layers. By contrast, perfused kidneys displayed only very few apoptotic cells and appeared to develop glomeruli with an intact podocyte/endothelial interface. In conclusion, this approach may represent a significant improvement over existing techniques of kidney organ culture and may in future pave the way for the growth of human kidneys in vitro.
4 De novo kidney organogenesis in vivo.
During this project, we set up a technique to dissociate the separated constituent parts of the early embryonic kidney rudiment and recombine the separate tissue and obtain de novo kidney organogenesis. Similarly we can use the set up to introduce differentiation directed originally pluripotent cells to the system and obtain directed differentiation towards the cell lineages of the kidney. By such mechanism and the developed diphtheria toxin system we are heading towards the ultimate goal of being able to obtain a complete kidney from originally pluripotent cells. The objective to obtain de novo kidney organogenesis was approached by using a specific toxin based approach. The diphtheria toxin (DT) system was selected as the compound since normally the mouse cells are not susceptible for the DT but can be converted responsive if a human DT receptor has been introduced to the kidney stem cells. Novel mouse lines and crosses were generated and the system tested. Indeed evidence was provided that the nephron stem cells can be specifically ablated by the DT mediated approach. The developed protocols and systems serve as the essential basics to obtain de novo kidney organogenesis in vivo.
Significant results:
The Wnt4+ cells indeed serve as the stem cells to generate the whole nephron. The first few epithelialized embryonic kidney mesenchymal cells undergo a second cell transition and form a transient interstitial stromal cell population. This population is important as an inductive signalling source to contribute to secondary stroma development. The evidence for this is that if Wnt-4 gene is inactivated the secondary stroma cell differentiation fails completely.
WP3 Comparison of in vivo renal regenerative potential of stem cells obtained from different sources in animal models of acute and chronic renal failure
1 Comparison of the in vivo renal regenerative potential of stem cells obtained from different sources in animal models of acute kidney injury with tubulointerstitial damage.
The ability of CD133+CD24+CD106+ and CD133+CD24+CD106- cells to regenerate injured renal cells was then assessed in a model of rhabdomyolysis-induced acute kidney injury in SCID mice, generated by intramuscular injection of glycerol. To this end, CD133+CD24+CD106+, CD133+CD24+CD106- cells or saline were injected into the tail vein of SCID mice with rhabdomyolysis-induced acute kidney injury 4 and 20 h after glycerol injection. As an additional control, glycerol-treated SCID mice were injected with a mixture of CD133−CD24− renal cells, which represent 90-95% of the total cells of the kidney. Measurement of blood urea nitrogen (BUN) levels demonstrated that mice injected with CD133+CD24+CD106+ cells or CD133+CD24+CD106- cells showed a significantly reduced severity of ARF at day 5, 8, 11 and 15 that was not observed in mice treated with saline or with CD133−CD24− renal cells. Interestingly, only injection of CD133+CD24+CD106+ cells resulted in a significant reduction of the severity of ARF at day 3, as revealed by the lower BUN levels in comparison to mice treated with CD133+CD24+CD106- cells or saline. Treatment with CD133+CD24+CD106+ cells or CD133+CD24+CD106- cells was also associated with a better preservation of renal structure and a reduction of renal fibrosis in comparison to mice treated with CD24-CD133- cells or saline at day 15 after induction of ARF, as assessed with Masson's trichrome and scoring for the presence of renal scarring. To further investigate their ability to regenerate injured tubular cells, CD133+CD24+CD106+, CD133+CD24+CD106- or CD133−CD24− renal cells were labeled with the red fluorescent dye PKH26 before their injection into glycerol-treated SCID mice. Labeled CD133+CD24+CD106+ cells engrafted within the tissue and generated novel tubular cells, acquiring the proximal tubule specific marker LTA or the distal nephron marker Dolichos Biflorus Agglutinin (DBA). Identical results were obtained when CD133+CD24+CD106- cells were used. Quantitation of the number of PKH26-labeled cells over the total number of proximal tubular cells was performed on sections that were stained with LTA and demonstrated that in mice injected with CD133+CD24+CD106+ cells, 7.81±3.1% of PKH26-labeled/LTA-stained and 4.92±1.8% of PKH26-labeled/DBA-stained tubular cells were observed, while in mice injected with CD133+CD24+CD106- cells, 6.7±2.2% of PKH26-labeled/LTA-stained and 5.23±2.13% of PKH26-labeled/DBA-stained tubular cells were observed at day 15 after injury. By contrast, red labeling was never observed in mice injected with CD133−CD24− renal cells or with saline solution. Furthermore, human HLA-I antigen was detected in mice that were administered an injection of CD133+CD24+CD106+ cells or CD133+CD24+CD106- cells, whereas in SCID mice that received CD133-CD24- cells, HLA-I human antigen expression was never found, as demonstrated by double-label immunohistochemistry for human HLA-I and pan-cytokeratin. Taken together, these results suggest that only CD133+CD24+ cells display the property to differentiate into tubular cells of distinct portions of the nephron.
Taken together, the results of this study provide evidence that the tubular compartment of the nephron contains scattered renal progenitors that display functional regenerating capacity for injured renal tubular cells, as well as increased resistance to apoptotic stimuli.
The existence of tubular progenitors suggests that novel treatments aimed at promoting the regenerative capacities of the kidney could conceivably be possible, and be used to prevent and treat tubular injury.
A model of Acute Kidney Injury (AKI) induced by the nephrotoxic drug cisplatin in immunodeficient NOD-SCID mice was set up to test regenerative effects of human mesenchymal stem cells (MSCs) isolated from different sources including bone marrow (BM), adipose tissue and cord blood (CB). Infusion of CB-MSCs in immunodeficient mice with cisplatin-induced AKI ameliorated both renal function and tubular cell injury, and remarkably prolonged survival in respect to mice injected with BM-MSC. By contrast, infusion of adipose tissue-derived MSC failed to ameliorate renal function and structure in mice with AKI. Transplanted CB-MSCs localized in peritubular areas, limited capillary alterations and neutrophil infiltration. Apoptosis was reduced and tubular cell proliferation increased by virtue of stem cell capacity to produce growth factors. The reno-protective effect of CB-MSCs was further confirmed by their ability to inhibit oxidative damage and to induce the pro-survival factor Akt in tubular cells. The evidence that CB-MSCs in vitro increased the production of growth factors and inhibited IL-1b and TNFa synthesis when co-cultured with damaged-proximal tubular cells indicates a regenerative and anti-inflammatory action of stem cell treatment. Altogether these results highlight the potential of human CB-MSCs as future cell therapy for testing in human AKI.
The effect of stem cell therapy was evaluated on renal functional parameters and on renal cell proliferation, apoptosis and animal survival. Quantification of the engraftment of the different human MSC populations in damaged renal tissue and in other organs as lung, liver, brain, heart was performed. Results have evidenced cord blood-derived MSCs as the most powerful population for cell therapy in the process of kidney regeneration in experimenthal AKI.
These results suggest that both MSC and renal stem cells can be used to treat AKI, although they exert their beneficial effect through different mechanisms.
MSC’s in animal models of acute kidney injury
Kidney ischemia-reperfusion injury was induced in a model of bilateral warm renal ischemia in mice followed by three days of observation.
Mouse model of renal I/R injury
Normal male C57BL6 mice of 10 weeks old were used in this study. Isoflurane anaesthesia was used. A baseline blood sample was obtained from the tail vein. Via a midline laparotomy the renal pedicles were identified and bilaterally clamped for 30 minutes using microvascular clamps (S&T, Neuhausen, Switzerland). In the sham group identical surgical procedures were used, except that clips were not applied. After the renal pedicle clamps were removed, the kidneys were observed for color change indicating reflow. One milliliter of warm saline was left in the abdominal cavity before the incision was sutured in two layers and the mice were allowed to recover from anesthesia. As analgesic 0.05mg/kg buprenorfine was administered s.c. After 72 hours the mice were anesthetized again. The final blood sample was obtained using cardiac puncture. Immediately after blood collection, the kidneys were removed and sagitally cut. One halve was snap frozen in liquid nitrogen and the other halve kept on formalin. Blood samples were collected in heparin coated capillary tubes. Plasma urea concentration was measured using the reflotron system (Roche diagnostics, Almere, The Netherlands) as indicator of kidney function at baseline and 72 hours after reperfusion. Throughout the whole experimental period the mice were maintained on standard diet and given water ad libitum. The study was approved by the veterinary authorities of the LUMC.
Murine MSC’s combined with ARA290 in IR injury
The beneficial effects of MSCs in renal IR have repeatedly been shown, but recently it appeared that these effects may even be increased by culturing MSCs under hypoxic conditions. This effect is most probabaly mediated by an increase in hypoxia inducible factor (HIF)-1 that regulates secretion of erythropoietine (EPO) from MSC's. When EPO or EPO receptor is inhibited this positive effect on MSC’s is diminished and in myocardial I/R it is already shown that the combination of EPO and MSCs worked synergistically. Here we used ARA290, a synthetic EPO analogue that does not have the hematopoietic characteristics of EPO.
Mice were divided into the following four groups: 1) I/R treated with ARA290 only (n=6); 2) I/R treated MSC’s combined with ARA290 (n=6); 3) I/R treated with PBS (n=4) and 4) sham treated with PBS (n=2). Bilateral kidney ischemia was induced by clamping the renal vessels for 30 minutes, followed by reperfusion. Six hours after reperfusion the treatment (ie MSC and/or ARA290) was administered by i.p injection. Kidney function was measured by plasma urea concentration. Kidneys were harvested at 72 hours after reperfusion. There were no significant differences in kidney function between mice treated with ARA290 alone and combined ARA290 and MSC’s.
2 Comparison of the in vivo renal regenerative potential of stem cells obtained from different sources in animal models of acute kidney injury with glomerular damage.
The rat model of adriamycin (ADR) induced nephropathy was employed as a model of kidney injury with glomerular damage, serving as a tool to evaluate the efficacy of cell therapy with bone marrow-derived mesenchymal stem cells (MSCs). The regenerative potential of treatment with bone marrow-derived mesenchymal stem cells (MSCs) on glomerular resident cells of distinct compartments was evaluated. Nephrosis was induced in male Lewis rats by ADR (5mg/kg i.v.). Rat MSCs (2x106 cells) were injected into the tail vein at 36, 60 hours, 3, 5, 7, 14 and 21 days after ADR. Rats were sacrificed at 3, 9, 16 and 30 days. MSC treatment did not affect the development of proteinuria nor serum creatinine levels of nephrotic rats. Morphometric analysis revealed an early decrease of WT1+podocytes in response to ADR in respect to control rats associated with a decreased glomerular expression of nephrin and CD2AP. Increased rate of apoptotic podocytes and a decreased expression of glomerular VEGF expression were additional features of the model. As a consequence of ADR-induced podocyte injury, glomerular adhesions of the capillary tuft to the Bowman’s capsule (synechiae) were observed from 16 days, followed by crescents-like lesions and glomerulosclerosis. Cellular components of sinechiae were either NCAM+parietal progenitor cells or nestin+podocytes. Engraftment of MSCs in the damaged kidney, documented by the presence of PKH-26-labeled cells in glomeruli of ADR-rats, limited podocyte loss and apoptosis, and partially preserved nephrin and CD2AP. Stem cell infusion attenuated the formation of glomerular podocyte-PEC bridges and normalized the distribution of NCAM+progenitor cells along the Bowman’s capsule. Consistent with the restoration of Bowman’s capsule architecture, a decrease of glomerulosclerosis was observed. Finding that MSCs increased glomerular VEGF expression and preserved microvascular rarefaction may explain the pro-survival effect displayed by stem cell therapy. MSC infusion also lessened the inflammation with lower glomerular and interstitial accumulation of ED1-positive monocytes/macrophages.
These findings suggest that bone marrow-derived MSC by virtue of their tropism for damaged kidney and their ability to provide a local pro-survival environment may represent a useful strategy to preserve podocyte viability and reduce glomerular inflammation and sclerosis.
3 Comparison of the in vivo renal regenerative potential of stem cells obtained from different sources in animal models of chronic renal failure with tubulointerstitial injury.
Glomerular and tubular interstitial damage was assessed during time in the experimental model of 5/6 nephrectomy in the rat to establish the optimal timing of cell therapy. While studying models of chronic renal failure available in the lab, we identified a new renal progenitor cell population in the Bowman’s capsule of Munich Wistar Frömter (MWF) adult rats, which develop progressive glomerular injury with time. In this model, we previously reported that angiotensin-converting enzyme inhibitor (ACEi) renoprotection was associated with podocyte repopulation and preservation of glomerular architecture. Here, we studied the time course of the lesions, their cellular components and the effect of ACEi. Early glomerular lesions were synechiae followed by extracapillary crescents and glomerulosclerosis later on. Majority of cells forming crescents were claudin1+ parietal epithelial cells and to a lesser extent WT1+ podocytes, both in active proliferation. In crescents, cells expressing the metanephric mesenchyme marker NCAM, were also found. Three distinct populations of parietal epithelial cells were identified in the rat Bowman’s capsule at different stages of differentiation from immature progenitor cells to differentiated parietal podocytes. A large population expressing stemness markes including NCAM and CD24, transitional cells expressing NCAM+and the podocyte marker WT1+ and NCAM-WT1+cells, the latter population representing parietal podocytes were found. After exposure to inductive medium, cultured parietal epithelial cells obtained by capsulated glomeruli generated podocytes, documenting their progenitor nature. Mitotic activity of cultured renal progenitors was induced by Angiotensin II through the downregulation of cell-cycle inhibitor C/EBPδ expression. In vivo, chaotic migration and proliferation of the Bowman’s capsule progenitor cells paved the way to crescent formation and subsequent sclerosis. An unprecedented result of this study was that ACEi, beside their effect of lowering blood pressure and proteinuria, reduced the formation of crescents and the evolution toward glomerulosclerosis in MWF rats. A new mechanism of renoprotection by ACEi through the limitation of NCAM+ progenitor proliferation and migration via the modulation of C/EBPδ has been disclosed leading to restoration of glomerular architecture. These data provide a clue for designing specific molecules targeted to novel players of renal repair that can possibly foster the intrinsic capacity of the kidney to regenerate. These observations were further supported by results obtained through injection of APEMP in mice models of chronic glomerular injury improved proteinuria and generated novel podocytes within injured glomeruli. This means that strategies aimed at controlling the growth and differentiation of APEMP can lead to glomerular and tubulointerstitial regenration in mice models of chronic renal failure.
At the university of Aachen, three different protocols of MSC treatment were tested in 5/6 nephrectomized rats: Daily i.v. injection of 2 ml of MSC conditioned medium for 40 consecutive days, weekly i.v. injection of 1 mio MSC for 5 weeks or for 11 weeks vs. respective controls. None of the treatments resulted in significant amelioration of disease course, nevertheless a beneficial trend was noted in the most intensive treatment group (11 weekly injections). The group of Prof. Floege and Prof. U. Kunter assessed the kidneys for adverse, i.e. profibrotic effects of MSC treatment but found no differences in collagen accumulation between the groups
In the renal IR model in mice at LUMC, a longer follow-up time of 3 weeks was chosen to assess chronic kidney injury after IR. Consecutive urea measurements during the follow up period showed regain of kidney function with time. Within three weeks of follow up, a considerable amount of kidney fibrosis developed, which was quantified by Sirius red (SR) staining. Histological tissue injury was scored on the PAS staining with semi-quantitative score on amount of proximal tubule necrosis (score 0-3), band necrosis of cortex and medulla (score 0-1) and protein casts in the tubules (score 0-1).
Murine MSC’s administered late after IR injury
It is unclear whether MSCs act more efficiently when applied after induction of tissue damage, as compared to administration before induction of damage. Therefore we used the same model of murine MSCs that were administered to Bl6 mice undergoing renal IR, with the modification that MSCs were administered 7 days after renal IR. No ARA290 was administered. No difference between untreated mice and mice treated with MSC’s in terms of renal function could be demonstrated.
The group at INSERM has previously shown that bone marrow MSCs (BMMSCs) may inhibit fibroblast activation through the secretion of paracrine factors (Mias C and al, Stem Cells. 2009 Nov;27(11):2734-43). Based on these results, we aimed to investigate the effects of bone marrow MSCs on progression of post-ischemia reperfusion renal remodeling and dysfunction and the mechanisms potentially involved. With this scope, we used an original rodent model that we have recently designed (Chaaya R and al, Nephrol. Dial. Transplant. 2011 Feb;26(2):489-98) mimicking the development of chronic renal disease occurring in clinic after ischemia-reperfusion and immunosuppressive therapy.
Using this model, we investigated (i) whether cell-based therapy using bone marrow MSCs directly injected in the kidney prevents or reduces the development of fibrosis; (ii) the impact of paracrine factors secreted by bone marrow MSCs on epithelio-mesenchymal transition of kidney proximal tubular cells in vitro.
In order to mimic better the clinical frame of post ischemia-reperfusion chronic renal disease occurring in humans, we used uninephrectomized rats submitted to ischemia-reperfusion and chronic cyclosporine treatment. The use of cyclosporine has a double interest: firstly, as observed for immunosuppressive therapy in human, cyclosporin aggravate the outcome of renal disease and increases interstitial fibrosis and tubular damage; second, its immunosuppressive properties minimize the previously described immunomodulation activity of grafted MSCs. Using this original model we have been able to determine the mechanisms of prevention of renal fibrosis by BMMSCs in chronic renal failure.
4 Establishment of which stem cell type displays the best regenerative properties in mice models of chronic renal failure with glomerular injury
Among bone marrow derived stem cells, we tested rat mesenchymal stem cells. We tested different MSC preparations in the model of Thy1.1 nephritis, a model of mesangioproliferative glomerulonephritis. MSC were isolated from healthy rats (H-MSC), healthy trackable hPLAP transgenic rats (TG-MSC) and from CKD donors (CKD-MSC). In addition, given our negative outcomes in the remnant kidney model, we tested healthy MSC after 7 days incubation in a novel “uremic medium” (UM-MSC). Only healthy donor MSC (H-MSC, TG-MSC) significantly improved renal repair (reduction in mesangiolysis score and proteinuria, increase in glomerular mitoses) whereas CKD-MSC and UM-MSC did not. These findings suggest that heterologous MSC from healthy donors might be more beneficial than autologous MSC in patients with progressive CKD. In addition, the regenerative potential of APEMP at different stages of differentiation was evaluated in mouse model with adriamycin-induced nephropathy. Adriamycin nephropathy was induced in female SCID mice at the age of 6 wk by a single intravenous injection of adriamycin on day 0 in the tail vein. On day 1, and again on days 4, 9, 18, and 25 after adriamycin injection, two groups of mice received intravenous administration of saline or of PKH26-labeled CD133+CD24+PDX- cells. Additional groups of mice were treated with saline, CD133-CD24-, CD133+CD24+PDX+, CD133+CD24+PDX-, or with clonally expanded PKH26-labeled CD133+CD24+PDX- cells. CD133+CD24+PDX- cells, but not CD133+CD24+PDX+ or CD133-CD24- cells, into mice with adriamycin-induced nephropathy reduced proteinuria and improved chronic glomerular damage, suggesting that CD133+CD24+PDX- cells could potentially treat glomerular disorders
5 Models of chronic renal failure related to genetically inheritable diseases:
Frasier animals carrying point mutations in the Wilms’ tumor suppressor gene WT1 and which mimic the human Frasier syndrome have been better characterized. To generate this mouse model and to clarify the function of the + and −KTS proteins in development, a specific mutations has been introduced into the endogenous Wt1 locus, designed to interfere with the production of one of the two variants. Heterozygous mice with a reduction of +KTS levels develop glomerulosclerosis and represent a model for Frasier syndrome. To better characterize the Wt1-Frasier (Wt1 (+KTS)-/+) mice phenotype on a molecular level we carried out exome array analysis and identified a set of genes deregulated in Frasier syndrome. We reasoned this was important information that needed to be obtained for any kind of approaches in cell replacement therapies. Incidentally, the identified target genes represent excellent candidates for genes involved in glomerular disease. To further establish the ideal genetic background to test out replacement therapies, we crossed the Frasier mutant mice with a variety of different genetic strains. While heterozygous mice on a CBA or Bl6 background survived up to 15 months, crosses on the FVBN background developed fulminant proteinuria and died as early as 6 weeks after birth. We therefore have established a colony of Wt1 (+KTS)-/+ mice, which will now be suitable for transplantation experiments of stem cells, and that can potentially be useful to evaluate the effects of cell therapy using MSC or APEMP in genetically inheritable renal diseases.
6 Functional and histomorphometric analysis of stem cells regenerative potential Stem cell therapy
Both CD133+CD24+ cells obtained from glomerular and tubular fraction were tested for their effect in inducing renal function improvement and tubular regeneration in glycerol-induced acute renal failure, which mimics rhabdomyolisis-induced acute tubular necrosis. Rhabdomyolysis-induced ARF was studied in 6 week old female SCID mice (Harlan, S. Pietro al Natisone, Italy), by intramuscular injection with hypertonic glycerol (8 ml/kg body weight of a 50% glycerol solution; Sigma-Aldrich) into the inferior hind limbs. Animal experiments were performed in accordance with institutional, regional, and state guidelines and in adherence with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. On day 0, mice received an i.v. injection into the tail vein of CD133+CD24+CD106+ cells (0.75 × 106 cells 4 h after glycerol injection and 0.75 × 106 cells 20 h after glycerol injection) and CD133+CD24+CD106- cells (0.75 × 106 cells 4 h after glycerol injection and 0.75 × 106 cells 20 h after glycerol injection). The cells, obtained from five different human donors (2 men and 3 women) were labeled with the PKH26 Red Fluorescence Cell Linker kit (Sigma-Aldrich) immediately before injection.
Blood samples were obtained from the submandibular venous sinus at day 3, 5, 8, 11 or 15, and BUN levels were measured by Reflotron System (Roche Diagnostics, Rotkreuz, Swiss). Normal range in our experiments was between 30 and 37 mg/dl, as calculated in 10 additional untreated mice (day 0). BUN levels that exceeded 40 mg/dl were considered abnormal.
For histomorphometric analysis kidney sections of mice were stained with a Masson-Goldner trichromic kit (Bio-Optica). Nonoverlapping fields of the entire section (20 fields for each mouse) were independently analyzed using a 20X objective by two observers. The severity of renal scarring was assessed as follows: normal tubulointerstitium scored 0; mild tubular atrophy with interstitial edema or fibrosis affecting up to 25% of the field scored 1; moderate tubular atrophy with interstitial edema or fibrosis affecting 25–50% of the field scored 2; and severe tubular atrophy with interstitial edema or fibrosis affecting > 50% of the field scored 3.
Using the model of chronic renal failure (nephrectomy/ischemia-reperfusion/cyclosporine), we showed that a single intrarenal administration of bone marrow MSCs 7 days after ischemia-reperfusion significantly improved renal function and modified renal remodelling. The improvement of renal function was associated to reduced extracellular matrix accumulation as shown by the decrease in collagen staining and mRNA expression measured by histomorphological analysis and semiquantitative RT-PCR, respectively. In addition, MSCs administration also reduced tubular dilation, which is a classical feature of progressive renal failure.
WP4 Evaluation of mechanisms of in vivo regenerative potential as well as safety, potential side effects and immunogenicity of stem cells obtained from different sources in animal models of acute and chronic renal failure
1. Evaluation of the mechanisms at the basis of stem cell-mediated regenerative potential.
A large effort was devoted to understand the mechanisms at the basis of stem- cell mediated regenerative potential. First of all we analysed the mechanisms at the basis of MSC-induced beneficial effects in mice models of glomerular and tubular injury. In view of the podocyte protective effect of bone marrow-derived mesenchymal stem cells (BM-MSC) in adriamycin (ADR)-treated rats, vascular endothelial growth factor (VEGF), highly produced by rat MSC and known to exert pro-survival and angiogenic activity was assessed. VEGF staining was significantly reduced in glomeruli of ADR-treated rats as compared to control rats. Indeed, mice treated with adriamycin develop a nephrotic syndrome related to podocyte injury. Infusions with MSC enhanced glomerular VEGF expression in rats receiving MSC were significantly higher than those of rats given saline at days 9 and 16. Furthermore, a marked reduction in volume density of endothelial cells in ADR-treated rats given saline compared with control rats was found. The degree of microvessel rarefaction was significantly limited in glomeruli of ADR-rats infused with MSC versus glomeruli of nephrotic rats on saline. In addition, an increased number of ED1-positive monocytes/macrophages was found in the glomeruli of ADR-treated rats given saline as compared to control rats. Infusion of MSC resulted in an important anti-inflammatory effect as shown by a significant reduction of glomerular cell infiltrates with respect to ADR-rats on saline. In addition, treatment with repeated injections of MSC limited early podocyte depletion, as indicated by the significantly higher number of podocytes per glomerulus in the group of ADR-treated rats receiving MSC as compared to saline. MSC displayed a marked anti-apoptotic effect as indicated by the lower number of apoptotic podocytes in renal tissue of MSC-treated rats with respect to ADR-rats on saline at the corresponding times. Consistent with the ability of infused MSC to reduce podocyte dysfunction, MSC-treatment also significantly limited the presence of glomerular podocyte-APEMP bridges. Specifically, in ADR rats receiving MSC the percentage of glomeruli with area occupied by >50% adhesion of the tuft to the Bowman’s capsule was significantly lower than that observed in rats given saline. In control animals, no glomeruli with adhesions more than 50% were found. Following MSC therapy, the distribution of APEMP was restored along the Bowman’s capsule to a pattern similar to controls. MSC treatment decreased the extension of sclerotic lesions at day 30 as indicated by a significant reduction of the glomerulosclerosis index compared to ADR-rats receiving saline.
A further mechanism of protection was identified in mice models of cisplatin-induced Acute kidney injury (AKI). Oxidative damage caused by reactive oxygen species has been implicated in the pathogenesis of cisplatin-induced renal failure. Peroxynitrite, the reaction product of nitric oxide with superoxide anion, has been taken as key oxidant species involved in the direct nitration of tyrosine residues causing protein oxidation. In cisplatin-mice with AKI, the expression of nitrotyrosine was significantly increased at 4 days in tubules of both the cortex and the medulla in respect to control mice. A marked reduction of nitrotyrosine staining at tubular levels was found when cisplatin-mice were treated with human MSC, indicating that human stem cells protect renal tissues from oxidative damage. Since in vitro and in vivo models of cisplatin–induced toxicity have shown that apoptosis represents one of the major causes of tubular cell loss, we studied whether human MSC treatment could exert an anti-apoptotic action on renal cells in mice with AKI. A significant increase in the number of apoptotic cells was observed in renal tissue of mice treated with cisplatin with respect to control mice. Human MSC infusion resulted in a significant reduction of apoptotic cells as compared to cisplatin-mice given saline, thereby suggesting a pro-survival effect of the stem cell therapy in mice with AKI. Tubular cell proliferation as an index of renal regeneration in the context of acute renal failure was also studied. Tubular cell proliferation slightly increased in cisplatin-mice given saline as compared to controls, indicating a spontaneous weak induction of the reparative process. Importantly, the treatment with human MSC strongly induced cell division, thus strengthening the meaning of a powerful induction of tissue recovery.
Taken together, these results suggest that MSC exert their beneficial effects through different mechanisms that involve, paracrine and anti-inflammatory effects, but do not exhibit the capacity to differentiate into renal tubular or glomerular cells. By contrast, APEMP could differentiate into tubular epithelial cells, as well as into glomerular epithelial cells. Once injected in SCID mice affected by ADR-induced nephropathy or glycerol-induced acute kidney injury, APEMP displayed the capacity to engraft within the kidney, generated novel podocytes as well as tubular cells and significantly improved renal function, a property that was not shared by other cell types of the adult kidney. Blocking APEMP engraftment through anti-CXCR4 and anti-CXCR7 antibodies, completely abolished APEMP-mediated beneficial effects on kidney function, suggesting that cell therapy using APEMP may help induce regeneration of injured resident renal cells through differentiation into podocytes or tubular cells. In addition, we analysed the mechanisms that regulate the growth and differentiation of APEMP, and we demonstrated that a regulated activation of the Notch pathway in APEMP during their phases of differentiation toward the podocyte lineage is critical to balance injury and regeneration in glomerular disorders and may be modulated through inhibitors of the Notch pathway. Taken together, these results suggest that MSC may represent a good tool for cell therapy of renal injury because of their paracrine and anti-inflammatory effects, while APEMP growth and differentiation may be pahrmacologically modulated to enhance the endogenous regenerative capacities of the kidney. These results have important implications for treatment of patients affected by acute or chronic renal disorders.
2 Evaluation of the potential risk of stem cell maldifferentiation.
We planned initially to follow-up our MSC-treated remnant kidney rats for histological signs of maldifferentiated MSC, a phenomenon we first described in 2007. In 5/6 nephrectomized rats that had received intraarterial MSC injection, kidneys at timepoint of kill exhibited frequent intraglomerular accumulation of finely dispersed lipids, mostly in the mesangium, that did not resemble the large lipid vacuoles that we observed in 2007 after intrarenal MSC injection. Rather, these lipid accumulations occurred in non-MSC treated control kidneys as well and thus cannot be linked to MSC treatment. The lipids were so ubiquitious that additional underlying rare “true maldifferentiation” could not be distinguished in light microscopy. In retrospective, it could be speculated whether our observation of intraglomerular maldifferentiation (exclusively observed in MSC treated rats with chronic Thy1 nephritis) in 2007 was caused by intraglomerular MSC that had undergone senescence due to the CKD environment, thus possibly explaining why no further maldifferentiation was reported by other groups who mainly inject MSC in acute renal failure, which does not cause CKD.
It has been known that a high inflammatory environment, acute or chronic, promotes adipogenic differentiation from MSC, confirmed by the fact that inflammatory states are associated with bone loss. As uremia implicates chronic inflammation, increased adipogenesis in our CKD-MSC seems well in line.
Impairment of stem/progenitor cells during pathological as well as physiological (aging) conditions has been acknowledged before). Senescence means that dividing cells upon extended cell culture passaging ultimately start to enlarge, become more granular, show reduced proliferation until they ultimately stop to divide although remaining metabolically active. They can be maintained in this state for years. Senescence occurs in vivo as well and increases with aging, it is considered a tumor-suppressing mechanism. We conclude that theoretically, all conditions that increase DNA damage (e.g. reactive oxygen species) can lead to stem/progenitor cell senescence and thus to increased spontaneous adipogenesis in MSC.
In the kidneys of cisplatin-treated mice infused with CB-MSCs, no sign of maldifferentiation of human CB-MSCs into other cell types of non-renal origin or adipocytes was observed in animals which survived 40 days after cell injection.
In summary, our current data suggest progressive CKD to be a risk factor for unwanted MSC differentiation events, be it osteogenic (Kramann et al. ATVB 2011) or adipogenic maldifferentiation.
Human renal Progenitors (APEMP) APEMP displayed the potential to generate tubular cells when injected into SCID mice models of acute tubular necrosis induced by glycerol injection. In these models differentiation toward glomerular cells or extrarenal cell types was not observed. Nevertheless, when injected into SCID mice affected by adriamycin nephropathy, which displayed tubular as well as podocyte injury, APEMP generated novel tubular cells as well as novel podocytes, as demonstrated through different methods: (i)l abelling before injection of different types of stem cells with the red fluorescent dye PKH26 (ii) evaluation of the presence of the Y chromosome by using fluorescence in situ hybridization (FISH) following injection of stem cells derived from male subjects in female mice (iii) staining by immunohistochemistry of the HLA-I human antigen performed on kidney sections.
However, abnormal regenerative responses of APEMP may occur during chronic inflammatory processes. Indeed, using confocal microscopy, laser capture microdissection, and real-time quantitative reverse transcriptase–PCR we also demonstrated that hypercellular lesions of different podocytopathies and crescentic glomerulonephritis consist of these three distinct populations: CD133+CD24+PDX- or nestin- (renal progenitors), CD133+CD24+PDX+ or nestin+ (transitional cells), and CD133-CD24-PDX+ or nestin+ (differentiated podocytes). These results suggest that glomerular hyperplastic lesions derive from the proliferation of renal progenitors at different stages of their differentiation toward mature podocytes, providing an explanation for the pathogenesis of hyperplastic lesions in podocytopathies and crescentic glomerulonephritis.
3 Prevention of maldifferentiation by particular experimental conditions, in vitro pretreatment, or in vivo pharmacological interventions
The importance of reactive oxygen species (ROS) in death of grafted cells has been supported by studies showing that prevention of oxidative stress through pharmacological approaches or genetic modifications allowed increasing MSCs survival after graft. At present, the intracellular sources of ROS and their involvement in MSCs death have not been clearly identified. We have shown that the generation of H2O2 in MSC after a treatment with serotonin, 5-HT (through its monoamine oxidase-dependant degradation) leads to an apoptosis of these cells. These results indicate that the exposure of MSCs to en environment susceptible of producing ROS may be deleterious for the efficiency of the cell therapy (Trouche E and al. Stem Cells Dev. 2010 Oct;19(10):1571-8). In order to improve cell survival after intraparenchymal graft, we have developed a pharmacological strategy based on melatonin pre-treatment. Exposure of MSCs to this pineal hormone induced an overexpression of anti-oxidant defences such as catalase and superoxide dismutase (SOD-1). This effect is associated with an improvement in the functional recovery after a renal injury and an increase in grafted-cells survival (Mias C and al. Stem Cells. 2008 Jul;26(7):1749-57).
4 Evaluation of Tumorigenicity.
We evaluate the tumorigenicity of human APEMP preparations by injection in NOD/SCID mice subcutaneously and observed no tumorigenic potential.
We also evaluated the tumorigenicity of human MSC preparations by injection in NOD/SCID mice subcutaneously and follow potential tumour growth for 4 months.
During the course of the project data was obtained showing that simple subcutaneous injection of MSCs in mice leads to scattering of the injected MSCs throughout the mice. Using luciferase transfected MSCs in numbers equalling those of cell numbers used in clinical trial and 10x this amount, we observed that one week after injection MSCs could not be traced back. These findings prompted us to initiate studies on the use of MSCs encapsulated in matrigel to analyse whether encapsulated MSCs placed subcutaneously would persist long enough to reliably exclude tumorigenicity. So far we have tested in matrigel encapsulated luciferase transfected mouse MSC’s in a syngeneic mouse model. MSC’s were traceable up to 8 weeks after injection without signs of tumorigenicity.
To establish the magnitude of the problems that might arise using the clinical products we performed karyotyping on MSC preparations prepared for clinical use. In over 50 MSC preparations derived from healthy individuals karyotypic abnormalities have not been observed. To avoid HLA-reactivity the use of autologous MSCs is preferable in the setting of kidney transplantation. When analysed bone-marrow derived MSC preparations from end stage renal disease (ESRD) patients show similar expansion potential and immunosuppressive potential compared to MSC preparations from healthy individuals. However, karyotypic anomalies were observed in 2 out of 12 expanded MSC preparations of ESRD patients. MSC preparations showing abnormalities in karyotype were omitted and not used in clinical trials. This observation requires further investigation.
Future research will focus on the tumorigenic potential of MSC’s derived from ESRD patients. The aim is to prepare encapsulate MSCs from these patients in matrigel and test these in NOC/SCID mouse models.
5 Evaluation of immunogenicity
When rats were treated with acute Thy1.1 nephritis with intrarenal injection of MSC preparations, it was assessed all kidneys for consecutive influx of monocytes/macrophages. It was not observed significant influx of inflammatory cells in the treated vs. control kidneys, although MSC secrete many cytokines that induce chemotaxis. One has to keep in mind that injured intrinsic renal cells express many inflammatory chemokines themselves providing a strong “background” signal. Interestingly, even kidneys that were treated with TG-MSC did not show increased inflammatory cell influx although the TG-MSC carry the human transgene on the outer cell membrane.
Pre-transplant infusion of syngeneic MSC significantly prolongs graft survival in a murine kidney transplant model.
We set up a severe and clinically relevant kidney transplant model. Recipient C57 mice (H-2b) were sensitized toward donor antigens by the infusion of donor Balb/c splenocytes (H-2d, 1x106 i.v. seven days before kidney transplantation). Transplantation of Balb/c kidneys into sensitized mice (group 1) resulted in a rapid increase in BUN levels (and acute graft rejection within 10 days in all transplanted mice. We next assessed the effect of pre-transplant MSC infusion in prolonging kidney graft survival. To this purpose donor-sensitized C57 recipient mice were given syngeneic MSC infusion (0.5x106 i.v.). Mice of group 2 and 3 received a single pre-transplant MSC infusion at day -1 (n=5) and at day -7 (n=5) respectively, and mice of group 4 received double pre-transplant MSC infusion at days -7 and -1 (n=5). All mice received a Balb/c kidney transplant at day 0.
A single (either day -7 or day -1) and double (at day -7 and at day -1) pre-transplant infusion of MSC significantly prolonged kidney graft survival as compared with untreated transplanted. In mice from groups 2, 3 and 4 surviving more than 20 days post-transplant, kidney graft function was well preserved during the 60 day-follow up.
Thus, pre-transplant syngeneic MSC represents a promising cell-based immunotherapy in solid organ transplantation.
We previously described in adult kidneys that the SCA+ Lin- cells represent in mice a mesenchymal renal residing stem cell. The second approach indicated already in 2004 in a study detailed in Nature Medicine is based on our ability to define optimal 'window'' of gestation ideally suited for harvest of embryonic pig or human embryonic tissue for transplantation. A major question related to these two stem cell sources is how immunogenic they are and how much immune suppression is required to enable long term engraftment of these cells in allogeneic recipients. Thus we found at the first part of the grant period that SCA+ Lin- mesenchymal stem cells although able to some degree to suppress immune response; they still exhibited significant immunogenicity upon transplantation into allogeneic recipients, inducing memory T cells that would reject promptly a second infusion of fibroblasts from the same donor. Thus, the prevailing arguments that MSC can evade the immune system and therefore it might be possible to use 'off-the-shelve' MSC for renal repair without immune suppression altogether, is highly unlikely in our opinion.
Unfortunately, we have reached similar conclusions also regarding the use of embryonic precursor tissues that are grown and develop within the recipients post transplant. Although they are capable to be predominantly vascularised by host blood vessels they can still be rejected through the indirect pathway (Cross priming). Thus for each source we might need some level of immune suppression if transplanted in allogeneic or in xenogeneic recipients. Otherwise, the best option will ber to use MSC from autologous sources, which is feasible in patients with kidney disorders, where the bone marrow is usually healthy.
WP5 Data Management and readouts to set up protocols for phase I/II clinical trials in humans
1 To set up protocols in view of phase I/II clinical trials in humans following standardization of results obtained from preclinical models, choosing the best type of stem cell for cell therapy of distinct clinical renal disorders.
A shared protocol for Good Manufacturing Practice (GMP)-expansion of human MSC preparation and use of human MSC was discussed among all the partners of the Consortium and developed in direct interaction with the European Group for Blood and Marrow Transplantation (EBMT)-MSC expansion consortium. and is reported below:
Set up of human MSC cultures
All the steps are carried out in circuit closed in a laminar flow hood located in a clean room of classroom ISO7. Cross contamination is prevented by separation of production rooms, one-way routing and double-layer clothing.
1. Bone marrow collection:
a) Bone marrow cells are obtained by suction of the medullar cavity of a flat bone (illium).
b) A numeration of the cellular starting suspension will be carried out by an automat of haematology before any manipulation.
c) 2–5 ml heparinized samples are separated by Ficoll-Hypaque gradients centrifugation (Lympholyte-H, Cedarlane, Ontario, Canada) to yield mononuclear cells. In the case of bag washouts, total nucleated cells from filter and bag residues are obtained after two washings with 250 ml saline and centrifugation at 2000 r.p.m. for 5 min.
d) The viability will be evaluated by the percentage of negative cells after staining by blue trypan.
e) A bacteriologic analysis will be carried out by sowing 0,5 ml of the suspension of bone marrow in each of 2 bottles of hemoculture (aerobic and anaerobic). Lastly, an evaluation of the CFU-F will be carried out (5x105 cells/25 cm² flask in complete medium).
2. Culture set-up:
a) The cells are put in culture at the concentration of 2x105 cells/cm2
b) Non-adherent cells are removed after 3–4 days and fresh medium is added.
• Medium: DMEM low glucose medium (Gibco Invitrogen,Carlbad, CA, USA) containing gentamicin 0.1mM (Mayne Pharma, Napoli, Italy) and heparin 1000 IU (Pfizer Italia, Latina, Italy) in the presence of 10% FBS (selected batch conform the EBMT MSC Expansion Consortium).
• Equipment to be used: Container of culture: T175 flaskor one floor culture chambers (surface area 636 cm2: CellSTACKTM, Corning) identified with the code of the donor, the date of culture initiation. To put in the incubator in wet atmosphere with 37°C with CO2 5%.
3. Medium exchange:
a) The medium is changed 2 times per week until the ending of the culture. The cell growth is appreciated by observation of the lower plateau under a microscope. The steps where the cellular layer is not covered any more with medium must be shortest as possible in order to avoid damaging the cells by desiccation.
b) Medium: DMEM low glucose medium (Gibco Invitrogen,Carlbad, CA, USA) containing gentamicin 0.1mM (Mayne Pharma, Napoli, Italy) and heparin 1000 IU (Pfizer Italia, Latina, Italy) in the presence of 10% FBS (selected batch conform the EBMT MSC Expansion Consortium).
4. Cell detachment and washing:
a) At subconfluency, cells are recovered after Trypsin-EDTA (StemCell Technologies, Vancouver, British Columbia, Canada) treatment and subsequently replated at approximately 3000 cells/cm2 in the same medium.
5 Products quality controls and release criteria
The final cell products undergo all the established quality control tests that are planned to adhere to specific criteria, including:
• Viability >80% measured by 7-amino-actinomycin D (7-AAD) vital dye staining and FACS analysis on a FacsScan (Becton Dickinson, San Jose`, CA, USA), according to standard procedure assays.
• Sterility of final product and culture supernatant performed by direct inoculation of at least 1% of the product or 5ml of pooled culture supernatant in BactAlert FA and FN media and automated measurement (Biomerieux, Marcy l’Etoile, France). Adequacy was reached when all cultures scored negative after a cultureof approximately 14 days (we use 7 days)
• Endotoxin content performed by the Limulus amebocyte lysate (LAL) test according to European Pharmacopeia (PBI S.p.A. Milano, Italy). Adequacy o2.5 EU/ml. This value is strictly dependent on the final volume of the product to be administrated, and therefore is subject to change according to the application.
• Mycoplasma contamination, detected with the MycoAlert test (Cambrex Corporation, Verviers, Belgium). Adequacy: no contamination detected.
• FACS analysis: hMSCs are detached with Trypsin-EDTA, washed and stained with the following conjugated monoclonal antibodies: CD45-FITC, CD34-PE, CD14-FITC, HLA-DRFITC, CD29-PE, CD90-FITC, CD73-PE, HLA-ABC-PE, CD105-PE or isotype-matched IgG-FITC and IgG-PE control antibodies. CD90 expression should be >80%, CD105 >80%, CD45 <10%.
• Genetic stability: We analyze the kariotype of the patient before the expansion, and the kariotype of the cells at the end of the culture. No genetic anomalies are detected after cell expansion.
• BSA: since FBS is used during expansion, we test BSA content in the final product using ELISA. Our release criterium is BSA < 75 EU/ml but, as for endotoxins, this value is dependent on the final volume of the product and it is subject to change according to the application (systemic or local delivery of the cells).
After incubation, cells are washed, resuspended in PBS and then analyzed by standard flow cytometry on a FacsScan following standard procedures.
Localization of infused MSC into the graft are associated with transient acute renal insufficiency in kidney transplant patients
We recently investigated safety and feasibility of MSC infusion in two living-related donor kidney recipients who were given ex-vivo expanded, autologous, bone marrow derived-MSC at day 7 post-transplant, after induction therapy with Basiliximab/low dose thymoglobulin. Few days after cell infusion, both MSC-treated patients developed acute renal insufficiency and we were able to perform a kidney biopsy of patient 2. Histologic and immunohistochemic analysis of graft infiltrating cells did exclude an acute cellular or humoral rejection. Indeed intragraft CD4+, CD8+ T cells, CD14+ monocytes, CD20+ B cells and CD68+ macrophages were very low as compared with those in control kidney graft biopsies from patients given the same immunosuppression who experienced acute cellular-rejection. We found a high number of granulocytes in the peritubular inflammatory infiltrate and increased complement C3 deposition in patient 2 than in grafts with early acute cellular rejection. MSC, identified as CD105-CD44 double positive cells, were found in the graft interstitium of patient 2, but not in naïve untransplanted kidneys nor in a renal graft with acute cellular-rejection.
Thus, autologous MSC given post-transplant in kidney transplant patients was associated with transient renal insufficiency associated with intragraft recruitment of neutrophils and complement C3 deposition. Despite the well known anti-inflammatory properties of MSCs, the various soluble factors produced by MSCs also include proinflammatory mediators, which eventually may have contributed to the intragraft recruitment of granulocytes and slow progressive deterioration of renal function. The safety concern of posttransplant MSCs anticipated the need to modify the study protocol moving cell infusion pretransplant, an approach being effective in experimental models of solid organ transplantation. We would also like to underline that after one year of follow-up the two patients are in good health and exhibit a good kidney function.
Preparation of a risk analysis table for the cellular expansion according to GMP guidelines
Risk management is an important aspect of the GMP pharmaceutical production, and its application is even more critical for products such as cell-based therapies that cannot be terminally sterilized after manufacturing.
Moreover, patient-specific cell therapies often start from a sample coming from a donor (the patient itself or an allogeneic source) that can transport viral or bacterial contamination. In a production facility where several batches of cells are manufactured at the same time, several other procedures must be put in place in order to prevent cross-contamination or sample mix-ups.
In order to facilitate the management and execution of clinical-grade expansions of the different cell populations identified in the project, we have performed a risk analysis of all the steps involved in the production process of a typical cell-based medicinal product, generated from a donor.starting sample.
The main productions steps were divided as follows:
1) Receipt and control of incoming sample
2) Sample handling during admission to the GMP facility
3) Sample handling during cellular expansion
4) Preparation and labelling of the finished product
For each of these passages we have identified the possible risks and proposed solutions that could decrease the chance of deviations and non-conformities, as well as mitigating the damage caused by unexpected events such as contaminations and leakages.
The table below summarizes all the aspects analyzed and the related risks and solutions:
Step / material Risk for the product Solution(s) Risk for operator/facility Solution(s)
1) Incoming patient sample
• Vial is broken
• Loss of traceability
• Check sample at receipt and register material integrity
• Check conformity on documents
• Vial is broken
• Contamination of facility
• Wear gloves
• Verify presence of Certificate of Analisys testing the absence of viruses
2) Sample handling during admission to the GMP facility
• Contamination during manipulation steps
• Cross-contaminations
• Work in GMP facility, perform environmental monitoring, train operators
• Use a dedicated incubator for each batch; clean surfaces after each manipulation
• Accidental contact with sample
• Leakage during storage at 4°C (quarantine)
• Wear gloves and general laboratory protection equipment; expose the product only under biohazard cabinet
• Keep sample in a sealed plastic bag inside the refrigerator
3) Sample handling during cellular expansion
• Contamination during manipulation steps
• Mycoplasma contamination
• Bacterial contamination
• Incubation at suboptimal temperature
• Work in GMP facility, perform environmental monitoring, train operators
• Check Mycoplasma contamination once a week
• Check medium for turbidity, changes in color, presence of molds; in case of contamination, isolate and identify microorganism and activate dedicated cleaning procedure
• Keep incubators under regular calibration schedule; keep incubators under remote alarm system
• Leakage of flask / bioreactor
• Bacterial contamination spread by sample
• Dispose of broken flask, clean biohazard cabinet or incubator with suitable detergent; sterilize incubator
• Identify microorganism and activate dedicated cleaning procedures
4) Preparation of the finished product
• Loss of traceability
• Wrong transport temperature to the clinic
• Labeling procedure is performed immediately after final filling. Procedure is monitored by QC personnel. A dedicated labelling form is filled during the operation and a copy of the label is kept together with the Batch Production Record.
• Use validated transport box; include datalogger in the package
WP6 Dissemination and Training
1 Dissemination of results
Web site: a project dedicated web site has been created and updated, in accordance to the project participants’ needs, in particular with the period reports provided to the EC and with the material prepared for the project meetings.
Publications: the project results have been described in several publication on peer reviewed journal with very high impact.
Meetings and conferences: PI, experienced researchers and young and experienced scientists involved in STAR-T REK project participated to national and international scientific courses, workshop and congresses. These events were important opportunities for the dissemination of project results and also useful tools of training for the young scientists.
2 Training activities will be substantially the following:
Exchange of key staff members from one lab to another (short-term fellowships) in order to enhance cohesion and implementation of project activities, to share skills, expertise and reagents and to standardise techniques used in the different laboratories.
Dr. Ilya Skovorodkin from Prof. Seppo Vainio laboratory has visited Dr. Paola Romagnani laboratory for a week during 2010.
During the Final meeting held in Milan, at Istituto di Ricerche Farmacologiche Mario Negri, a round table for young scientists involved in the project was organized together with the visit to the labs. The young scientists, that were the one involved in the experimental part of the project and were the main actors of the experiments.
WP7 Project management
The management activities have been conducted aiming at the successful progress of the project within the agreed time, cost and quality limits as defined by the project contract signed with the EU and the Consortium Agreement signed among the beneficiaries. The coordinating team worked also to establish effective communication among the consortium beneficiaries, to guarantee appropriate administrative and scientific management of the project and correct dealing of ethical issues and to maintain a good flow of information and data.
1 Set up of project organizational structure to establish a common baseline for task and responsibility allocation. The project responsibility were allocated at the beginning of the project.
Project coordinator- general coordination of the project
Non scientific Management – coordination of the non-scientific activities of the project (meeting, training, dissemination, daily management of the project, amendments, guidance on FP7 rules and duties)
List of WP leaders –for the coordination and supervision of the scientific activities.
2 Administrative Issues Management to ensure on-time provision of periodic management reports, and cost statements. Handle the EC project reviews, payment issues and Amendments to the GA. ALTA supported the project coordinator in the organization of the non-scientific activities: meetings; amendments to the consortium agreement; interim reports; in the payments (collecting the bank account, checking the distribution of the money); setting up and management of the web site; preparation and coordination of the EC project reporting.
Tracking: to guarantee control, validation and verification of project results, to ensure that plans are fulfilled and implement necessary corrective actions. The scientific coordinator constantly checked the work of each beneficiary and intervened in to solve problems causing delays.
Task 4. Intellectual property management
Achievements: A Consortium Agreement was prepared to define consortium members’ rights and duties. The final draft has been signed by all the project participants.
For protecting and safeguard the IPR of the participants, the abstract of the manuscripts, posters and of any dissemination document, together with sufficient information concerning the planned dissemination activity and the envisaged data of dissemination, were circulated within the consortium before the disseminating action. Any objection to the planned activity would have been made in accordance with the EC-GA in writing to the Coordinator and to any Party concerned within 30 days after receipt of the notice. If no objection was made within the time limit stated above, the publication was permitted.
Potential Impact:
The STAR-T REK project has given a fundamental contribution towards the expected impacts listed in the work programme HEALTH-2007-1.4. Innovative therapeutic approaches and intervention. The objectives of the topic were to research, consolidate and ensure further developments in advanced therapies and technologies with broad potential application. The focus of STAR-T REK project was on regenerative medicine. The specific topic of the call addressed was HEALTH-2007-1.4-8: Stem cells for kidney regeneration.
The project was aimed at increasing the developing European capability in regenerative medicine of injured organs in general and of damaged kidneys in particular.
This project has generate many data, new insights into important mechanism of cell based regenerative processes and created synergies among scientists working in the field of kidney repair for developing novel therapeutic strategies based on stem cell therapies for several types of acute and chronic kidney disorders, which affect about 11% of the adult population.
International leader groups joined their resources and efforts for setting up standardized protocols of SC isolation and administration in patients affected by renal failure. The information produced during the project is a very important baseline for further studies and will influence deeply the scientific community towards further improvements and new directions of regenerative medicine and cell therapy.
The novelty of this proposal is that it assesses the regenerative potential of stem cells derived from different sources and investigate the possible obstacles to their utilization, as well as their potential side effects in preclinical models of acute and chronic renal failure. Indeed, the clinical usefulness of the treatment, as well as the need to understand currently discrepant results, need comparative experimental studies that have never been performed before either in SC therapy of kidney injury, or in that of other organs.
The expected outcome of the studies has been the identification of which cell type (between bone marrow derived stem cells, adult renal stem cells fetal renal stem cells) is better suitable for beneficial effect in preclinical models of acute and chronic renal failure, and whether these effects are mediated directly by the transplanted cells or indirectly through involvement of other cell types.
In summary, the main project outcomes are the following:
- identification of which cell type is better suitable for beneficial effect in preclinical models of acute and chronic renal failure;
- assessment whether the beneficial (or not) effects are mediated directly by the transplanted cells or indirectly through involvement of other cell types;
- insights on the mechanisms of stem cells-mediated regenerative effects, which are essential to set up cell therapies that should be effective and safe;
- novel concept that renal re generation can be achieved through paharmacological modulation of renal stem cell growth and differentiation;
- possibility of tissue bioengineering a de novo replacement organ using fetal renal stem cells or other strategies of kidney tissue engineering, with an ultimate aim of being able to implant functional renal replacements in patients with end stage renal disease.
The contribution of the project to each of these single aims was critical. The first most direct impact of our work is the development of a method of engineering foetal kidneys from simple suspensions of foetal kidney stem cells. For the rest of the Star-t-rek group, this has provided a test system for assessing the abilities of different types of stem cells to contribute to kidneys. More broadly, the system suggests a means of practical tissue engineering for a range of tissues, and it has already been taken up by other researchers. The eventual societal impact, if all goes well, will be a practical clinical technique. The work has been presented at a number of Public Understanding events and has been covered by international newspapers, confirming that its potential impact is seen by public commentators and not just by the Star-t-rek researchers involved. This project finally lead to clarify that MSC can represent a helpful tool for treatment of acute kidney injury, although their main mechanisms of their beneficial effect is mostly related to their capacity to secrete benficial factors and to reduce inflammatory processes. We also did not observe tumor formation in our preclinical models of acute kidney injury, and we however observed that these cells can be immunogenic and thus their administration would probably require some immunosuppressive treatment. Based on the standardized protocol for human MSC preparation and injection that was set-up during the project, we also started a phase I/II clinical trial in patients who underwent kidney transplantation. We demonstrated for the first time that MSC can be safely administered to kidney-transplanted patients. We observed a mild, transient acute renal failure related to MSC administration about 20 days after their administration, which anticipated the need to modify the study protocol moving cell infusion pretransplant, an approach being effective in experimental models of solid organ transplantation. However, after long term (one year) follow-up, patients are in good health and exhibit a normal renal function, suggesting that MSC may indeed represent a source for induction of tolerogenic responses in kidney transplanted-patients. Thus, the STAR-TREK project has lead critical contribution to our knowledge of safety and clinical utility of cell therapy using MSC. Finally, the STAR-TREK Project dramaticaaly increased our knowledge of the regenerative capacities of the kidney. Indeed, a renal progenitor system was identified and largely characterized by the partners during the course of this project, that lead to the definition of a novel concept in kidney biology, the one of the existence of a “renopoietic system”. The discovery that adult kidney can be potentially regenerated by an endogenous progenitor system changes our view of kidney biology. Indeed, during the course of the Project we also demonstrated that the regenerative capacity of the kidney can be enhanced through pharmacological agents, which opens a novel perspective for innovative therapeutic tools for patients affected by kidney disorders. We thus believe that STAR-T REK will indirectly contribute to the establishment of Europe as a major player in the regenerative medicine of injured organs and tissue and in the set up of novel therapeutic strategies for treatement of acute and chronic kidney diseases due to the main and important data that generated.
Stem cells have the remarkable potential to develop into many different cell types. In many tissues they serve as an internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or to become another type of cell with a more specialized function.
All stem cells—regardless of their source—have three general properties: they are capable of dividing and renewing themselves for long periods, they are unspecialized, and they can give rise to specialized cell types.
Regenerative medicine (which is also referred to as Cell therapy) is devoted to treatments in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed cells or tissues. Cell therapy depends on an understanding of how stem cells establish, maintain, and regenerate tissues and organs. There is much that we don’t yet know – about stem cells, about development, about what happens in disease and injury at a cellular level.
Chronic Kidney Disease (CKD) is a leading cause of mortality and morbidity in western countries and is estimated to affect 11% of the adult population. It can progress towards End Stage Renal Disease (ESRD), which has no cure and requires renal replacement therapy i.e. dialysis or renal transplantation. The number of patients with ESRD is growing consistently with rising cumulative costs that are even greater than the treatment costs of cancer.
Earlier stages of CKD are expected to be treatable with cell therapy.
Stem cells (SC), able of self renewal and to intervene in building/maintaining the structural and functional integrity of tissues, are especially attractive for such a purpose.
The focus of this project is to assess the regenerative potential of stem cells derived from different sources, to investigate the obstacles to their clinical utilization and their potential side effects.
Star-t rek is aiming at establishing whether stem cell therapy might offer an alternative approach to organ transplantation for patients suffering from kidney failure and at giving an insight on stem cells, their development, on what happens in the disease at a cellular level.
Successful clinical application of stem cell research requires a joined-up and well –planned approach. STAR-T REK has taken this role, in bringing together leading groups of basic and applied researchers and clinicians.
The novelty of the project STAR-T REK is:
• Use of different markers to isolate cell preparations
• Adoption of heterogeneous protocols of injection
• Study on distinct experimental models of renal failure
• Assessment of all potential safety concerns and rigorous toxicology study, with particular regard to the risk of anomalous differentiation and long-term possibility of tumorigenicity
• Possibility of tissue bioengineering a de novo replacement organ using fetal renal stem cells, with an ultimate aim of being able to implant functional renal replacements in patients with end stage renal disease
The results have been collected, compared, evaluated and converged in optimized protocols for phase I/II clinical trials in humans.
Project Context and Objectives:
In details, the project objective are the following:
1) Isolation of adult bone marrow derived stem and standardization of protocols for autologous cell therapy of renal failure
This first objective of the project has been achieved through coordinated discussion and work on shared protocols among different group leaders. First of all, among the different stem cell populations that can be derived from the bone marrow, we decided to focus our research on mesenchymal stem cells (MSC), which is the only bone-marrow derived stem cell type whose renoprotective/regenerating activity has been widely demonstrated in several preclinical models of acute and chronic kidney injury. Shared protocols were standardized for mouse MSC, rat MSC and human MSC. The protocol for human MSC preparation was also established taking into account GMP requirement and procedures.
2) Isolation and characterization of already known types of adult or fetal renal resident stem cells and standardization of protocols for their isolation for cell therapy of renal failure
The second objective of this project is focused on the possible use of resident renal stem cells as therapeutic tool for acute or chronic kidney disorders. To this aim a deep knowledge of the properties of renal stem cells is required. We have characterized CD133+CD24+ human renal progenitors and described their use in preclinical models of acute kidney injury. We demonstrated that CD133+CD24+ renal progenitors can be distinguished in distinct subpopulations from normal human kidneys based on the surface expression of vascular cell adhesion molecule 1 (VCAM1), also known as CD106. CD133+CD24+CD106+ cells were localized at the urinary pole of Bowman’s capsule, while a distinct population of scattered CD133+CD24+CD106- cells was localized in the proximal tubule as well as in the distal convoluted tubule. Once injected in SCID mice affected by acute tubular injury, both these populations displayed the capacity to engraft within the kidney, generate novel tubular cells and significantly improve renal function. These properties were not shared by other cell types of the adult kidney.
The glomerular tuft comprises three resident cell types, mesangial cells, endothelial cells, and podocytes. Some studies have shown that primary injury to each of these cell types is associated with glomerular disease. However, injury to endothelial and mesangial cells can be repaired by proliferation of adjacent cells. By contrast, podocytes cannot divide, and podocyte depletion is a common determining factor that results in glomerulosclerosis. Indeed, as long as the podocyte loss is limited, restitution or repair is possible.
By contrast, 20 to 40% podocyte loss results in a scarring response, until at greater than 60% podocyte loss, glomeruli become globally sclerotic and nonfiltering. During the course of this project, we provided the first evidence that podocytes can be regenerated by the CD24+CD133+ renal progenitors of the Bowman's capsule. In addition, we demonstrated that CD133+CD24+ renal progenitors are a heterogeneous and hierarchical population of undifferentiated and more differentiated cells that are arranged in a precise sequence within the Bowman's capsule of adult human kidneys. A subset of more undifferentiated cells expressing renal progenitor markers in the absence of podocyte markers localized at the urinary pole of Bowman's capsule acts as bipotent progenitor for both tubular cells and podocytes and exhibits self-renewal potential. A transition population expressing both renal progenitors and podocyte markers is localized between the urinary and the vascular pole of Bowman's capsule, exhibits a committed differentiative potential only toward the podocyte lineage and lacks self-renewal properties. Finally, differentiated cells, that do not express renal progenitor markers, but display high levels of the podocyte specific markers, localizes at the vascular pole of Bowman's capsule. Similar findings were observed in a parallel study performed in rodents by the group of Juergen Floege, who also demonstrated that transitional cells with morphologic and immunohistochemical features of both parietal epithelial cells and podocytes could be detected in proximity of the glomerular vascular stalk. More importantly, using an elegant model of genetic tagging of parietal epithelial cells in a triple-transgenic doxycycline-inducible mouse line, the same authors unequivocally demonstrated that podocytes are recruited from parietal epithelial cells, which proliferate and differentiate from the urinary to the vascular stalk generating novel podocytes. In summary, a large body of evidence now indicates that the Bowman’s capsule of adult kidneys contains a population of glomerular epithelial stem/progenitor cells which can replace lost podocytes through multiple mechanisms and allow glomerular regeneration. In a further series of studies, we have also demonstrated that injection of human CD24+CD133+ renal progenitors reduce proteinuria and restore podocyte depletion in preclinical model of focal segmental glomerulosclerosis. The related article (Ronconi et al. J Am Soc Nephrol., 2009), represents the first demonstration that renal stem cells can repair glomerular injury and was considered as a major breakthrough in the field of nephrology. It was the subject of an Editorial and was also selected by the Faculty of Thousands as a “must read” article with a score of 6.4. Taken together, these results also suggest the evidence for the existence of a stem cell system within adult human kidneys and that this renal progenitors cell population may represent a good candidate for cell therapy of chronic renal injury.
3) Identification of novel, unknown types of adult or fetal renal stem cells in mouse/rat and human adult kidney
The third objective of this project was to develop protocols to purify and identify putative further populations of resident stem cells in the developing kidney and to develop assay systems to test the competence of putative multipotent cells. Mouse models that allow identification and purification of embryonic and adult kidney related stem cells were developed. Also an experimental set up where stem cells or pluripotent cells can be effectively incorporated to the developing kidney were developed. By this set up embryonic stem cells can be in part targeted to the lineages of the embryonic kidney. Finally toxin related systems have been achieved to identify functional role of the stem cells and their roles in the renewal of the kidney structures in the adult. These strategies should allow identification and characterization of putative unknown types of renal stem cells in mouse.
4) Development and upgrading of techniques of de novo kidney organogenesis
Several novel assays to culture the embryonic kidney have been developed in the project. This approach has already been useful for three purposes; (i) for the study of the mechanisms that regulate normal renal development, (ii) for testing the ability of different stem cell types to make renal tissue (we have, for example, published the ability of human amniotic fluid-derived stem cells to do so) and (iii) for tissue engineering of transplantable kidney rudiments. Finally also a set up that allows reconstitution of the Bowman’s capsules with its associated major cell types was achieved. These advances are the starting points for a further development, including vascularization. We have preliminary results suggesting that this tissue engineered kidney has the ability to connect properly to a pumped blood system.
During the life of the Star-t-rek project, the basic techniques have been improved so that they generate an engineered equivalent of a fetal kidney that is organized around a single, branched collecting duct system that is capable of attracting blood vessels to itself and of constructing a glomerulus (paper in preparation by the group of Edinburgh). The results have been used as a foundation for further grant applications to the UK BBSRC to develop the technique towards practical transplant.
5) Comparison of in vivo renal regenerative potential of stem cells obtained from different sources in animal models of acute and chronic renal failure to set up standardized protocols for clinical trials in humans.
We analysed the regenerative capacity of mesenchymal stem cells (MSC) for injured renal tissue in different models of acute and chronic renal disorders related to tubulointerstitial or glomerular injury. This allowed us to understand that MSC may represent good tools for treatment of patients with acute kidney injury. In addition, we demonstrated that the activity of resident renal stem cells can be modulated to enhance the endogenous regenerative capacity of the kidney in preclinical models of chronic kidney injury related to glomerular damage.
6) Evaluation of the mechanisms at the basis of the renal regenerative potential of stem cells obtained from different sources in animal models of acute and chronic renal failure to set up standardized protocols for clinical trials in humans.
One of the most relevant problems related to cell therapy of acute kidney injury using mesenchymal stem cells is the reported possibility of their maldifferentiation into intraglomerular adipocytes in preclinical models of chronic Thy1.1 nephritis. Results achieved during the first period of the project showed surprisingly that MSC when injected in healthy kidneys were not detectable and left no trace of maldifferentiation. In contrast, the same cell preparation led to focal adipogenic intraglomerular maldifferentiation in rats with chronic Thy1.1 nephritis on day 60, thus suggesting that MSC maldifferentiation is probably related to growth factors produced during the glomerular disorder. Similarly, using confocal microscopy, laser capture microdissection, and real-time quantitative RT–PCR we also demonstrated that a deregulated activation of APEMP can induce the generation of hypercellular lesions in different podocytopathies and crescentic glomerulonephritis. These results provide an explanation for the pathogenesis of hyperplastic lesions in podocytopathies and crescentic glomerulonephritis and suggest that a deeper knowledge of the biology and growth properties of APEMP is required to set up cell therapy of kidney injury using these cells. These results are very important to set up cell therapy strategies of renal injury using renal progenitors. However, the mechanisms that regulate the growth and differentiation of renal progenitors are still mostly unknown. In STAR-T REK, we demonstrated also that a regulated activation of the Notch pathway in renal progenitors during their phases of differentiation toward the podocyte lineage is critical to balance injury and regeneration in glomerular disorders. The results of this study establish the novel concept that renal re generation can be achieved through paharmacological modulation of renal stem cell growth and differentiation. In addition, we demonstrated in different models of acute and chronic kidney injury, that the beneficial effect of cell therapy using MSC is mostly related to paracrine and anti-inflammatory effects and not to regeneration of resident epithelial cells of the kidney. However, the beneficial effects of MSC-treatment in different models of acute kidney injury can be relevant, which moved us to explore their possible clinical use in phase I/II clinical trials.
7) Evaluation of safety and immunogenicity of stem cells obtained from different sources in animal models of acute and chronic renal failure to set up standardized protocols for clinical trials in humans.
Results from multiple preclinical models of acute and chronic renal disease suggest that administration of MSC for cell therapy of acute kidney injury is safe in preclinical models of acute kidney injury, but administration of the same cells in conditions of chronic inflammation represents a possible risk factor for maldifferentiation. Currently, no risk of tumorigenicity were observed in all the preclinical models of renal injury analysed. More importantly, preliminary results in pilot phase I clinical trials suggest that injection of MSC in patients who underwent kidney transplantation is associated with a transient, mild and reversible acute renal failure related to factors released by MSC. After longer follow-up, patients are however in good health.
8) Set up standardized and optimized protocols for phase I/II clinical trials in humans
Results on the renal functional and histomorphological analysis and the evaluation of side effects have been collected and shared by all the groups of the consortium, allowing a standardized analysis of the data. The potential clinical efficiency of treatment with different stem cell types in the same experimental models has been directly compared, allowing to establish which could be the best type of treatment in each renal disease in terms of clinical efficacy and optimize protocols for phase I/II clinical trials in humans. Finally, pilot phase I trials of cell therapy using MSC were performed in patients who underwent kidney transplantation.
In summary, the outcomes of the project are:
- the identification of which cell type is better suitable for beneficial effect in preclinical models of acute and chronic renal failure;
- the assessment whether the beneficial (or not) effects are mediated directly by the transplanted cells or indirectly through involvement of other cell types;
- Insights on the mechanisms of stem cells-mediated regenerative effects, which are essential to set up cell therapies that should be effective and safe;
- novel concept that renal regeneration can be achieved through paharmacological modulation of renal stem cell growth and differentiation;
- Possibility of tissue bioengineering a de novo replacement organ using fetal renal stem cells or other strategies of kidney tissue engineering, with an ultimate aim of being able to implant functional renal replacements in patients with end stage renal disease.
The results of this project led to the a characterization of renal stem cells in adult human kidney and their capacity to regenerate injured renal tissue. In addition, tissue bioengineering of a de novo replacement organ using fetal renal stem cells and other strategies of kidney tissue engineering were explored. We analysed the regenerative capacity of mesenchymal stem cells (MSC) for injured renal tissue. These different strategies were compared and the mechanisms at the basis of their beneficial effects were assessed. This allowed us to understand that MSC may represent good tools for treatment of patients with acute kidney injury, although their beneficial effect is mostly related to paracrine and anti-inflammatory effects and not to regeneration of resident epithelial cells of the kidney. On this basis, a detailed protocol for clinical trials was prepared and pilot phase I clinical trials were performed in patients undergoing kidney transplantation. In addition, we demonstrated in preclinical models of acute and chronic kidney injury, that the activity of resident renal stem cells can be modulated to enhance the endogenous regenerative capacity of the kidney. Thus, the results of this project provided the scientific basis for treatment of patients with acute kidney injury by stem cell therapy, and for treatment of patients with chronic kidney disorders through pharmacological modulation of renal stem cell function.
Project Results:
WP1 Isolation and characterization of adult progenitors/stem cells for renal regeneration: main S&T results/foregrounds
1. Isolation of adult bone marrow derived stem cells (CD133+/CD34+ or mesenchymal stem cells) and standardization of protocols for autologous cell therapy of renal failure.
The results obtained are reproducible and the MSC populations, isolated from the bone marrow of different species by using these techniques, represent a good tool to test in preclinical models of renal failure.
Murine bone marrow-derived MSC: primary culture
Isolation and purification of murine MSC Bone marrow (BM) was obtained from 2-mo-old male C57BL6/J mice. Mice were killed, and femurs and tibias were aseptically removed. BM was flushed from the shaft of the bone with DMEM medium containing 2% FCS (Invitrogen) plus penicillin/streptomycin (100 U/ml to 0.1 mg/ml) and then filtered through a 100-µm sterile filter to produce a single-cell suspension. Murine MSC were recovered from BM by their tendency to adhere tightly to plastic culture dishes. Filtered BM cells were plated in DMEM plus 10% FCS and penicillin-streptomycin (100 U/ml to 0.1 mg/ml) and after 30 h non-adherent cells were removed. Medium was changed regularly every 3 d. After 2 to 3 wk, subconfluent (80%-90%) cells were detached by trypsin-EDTA (0.5 to 0.2 g/L) and murine MSC were purified by immunodepletion of CD45 positive cells. Briefly, after blocking with PBS that contained 0.5% BSA, cells were incubated for 20 min with rat anti-mouse CD45 antibody, 0.2 µg/106 cells (Caltag). Then, cells were incubated with magnetic microbeads coated with goat anti-rat IgG (Miltenyi Biotec), and CD45 negative MSC were isolated by magnetic cell-sorting separation system. For in vivo and in vitro experiments cells were used within first-second passages.
Characterization of murine MSC Cells were characterized for their capability to differentiate toward osteocytes, adipocytes, and chondroblasts. Murine MSC were grown until confluence, and the growth medium was replaced with the inductive medium consisting of Iscove’s modified Dulbecco’s medium, 20% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.05 mM β-mercaptoethanol supplemented with specific differentiation reagents as follows.
Osteogenesis: Cultures were fed twice a week for 3 wk with 10 mM β-glycero-phosphate, 50 μg/ml ascorbic acid 2-phosphate, and 10-9 M dexamethasone. Then cells were fixed with 10% formalin for 20 min at room temperature and mineralization—presence of calcium-rich hydroxyapatite—of the extracellular matrix was assessed by staining for 20 min with 2% wt/vol Alizarin Red S, adjusted to pH 4.1 with ammonium hydroxide.
Adipogenesis: Cells were incubated for 3 wk with 5 μg/ml insulin and 10-9 M dexamethasone. Adipogenic differentiation was visualized in phase-contrast microscopy by the presence of highly refractive intracellular lipid vacuoles. Oil Red O staining was used to assay the accumulation of lipid droplets in these vacuoles.
Chondrogenesis: Murine MSC were harvested and 6X105 cells were centrifuged to form a pellet on the bottom of a 15-ml polypropylene tube. The micromass was cultured in 500 μl of chondrogenic medium that consisted of 50 μg/ml ascorbic acid 2-phosphate and 1 ng/ml TGF-β1. After 3 wk of culture, cell clumps were harvested, embedded in paraffin, cut into 3-μm sections, and stained for glycosaminoglycans using 0.1% safranin O.
Results showed that murine MSC were able to differentiate toward osteocyte, adipocytes, and chondrocytes when exposed to specific inductive media.
Murine bone marrow-derived MSC: expanded culture
Isolation and growth of murine MSC We cultured murine MSC derived from bone marrow and expanded by using the protocol reported by Peister (Peister A. et al., Blood 103: 1662-1668, 2004). Following this protocol, by means of plating cells at very low densities, we obtained single-cell derived colonies with a rapid growth. The rate of expansion of the cultures was extremely sensitive to the initial plating densities. Cells obtained from each bone were first seeded into 75 cm2 flasks. After plating, the medium was changed for the following 4 weeks and, during this period, the non-adherent cells together with many detaching cells were removed. Clones were evident, although small, as early as 1 week of culture and, after 4 weeks, cells were almost confluent with a morphology resembling the heterogeneous cell population obtained by the traditional method already described, with a predominance of spindle-shape cell types. Once trypsinized, cells were split 1:2 and grown for 3 weeks. At the second passage, cells were seeded at 50 cells/cm2. At the following passages, cells reached confluence after 2-3 weeks of culture and were trypsinized and expanded up to the 9th passage. The analysis of the morphology of expanded murine MSC after nine passages in culture showed that cells represented a homogeneous population.
Characterization of murine MSC The cytofluorimetric analysis of expanded murine MSC revealed that they were negative for the haematopoietic cell markers CD11b and CD45. The hematopoietic marker stem cell antigen 1 (Sca-1) was expressed by the 81% of the cells, whereas c-kit expression was absent. At each passage, cells were able to form colonies and to differentiate into adipocytes.
Moreover, cells were able to produce mineral deposition when induced to differentiate with an osteogenic medium, suggesting a differentiation towards osteocytes had occurred. The expanded culture of murine MSC were able to differentiate into adipocytes and osteocytes at comparable extent than primary culture of murine MSC.
Set up of mouse MSC cultures
1. Bone marrow collection:
a) BM are obtained from 2-mo-old male C57BL6/J mice. Mice are killed, and femurs and tibias are aseptically removed. BM is flushed from the shaft of the bone with DMEM medium (Sigma) containing 2% FCS (Invitrogen) plus penicillin/streptomycin (100 U/ml and 0.1 mg/ml; Invitrogen) and then filtered through a 100-µm sterile filter (Falcon) to produce a single-cell suspension. MSC are recovered from BM by their tendency to adhere tightly to plastic culture dishes.
b) The viability will be evaluated by the percentage of negative cells after staining by blue trypan.
2. Culture set-up:
a) Filtered BM cells are plated at the concentration of 20.000/ml (25ml/Flask)
b) Non-adherent cells are removed after 30 hours and fresh medium is added.
• Medium: DMEM medium (Sigma) containing 2% FCS (Invitrogen) plus penicillin/streptomycin (100 U/ml and 0.1 mg/ml; Invitrogen)
• Equipment to be used: Container of culture: T175. To put in the incubator in wet atmosphere with 37°C with CO2 5%.
3. Medium exchange:
a) Medium is changed regularly every 3 days until the ending of the culture. The cell growth is appreciated by observation of the lower plateau under a microscope. The steps where the cellular layer is not covered any more with medium must be shortest as possible in order to avoid damaging the cells by desiccation.
• Medium: DMEM medium (Sigma) containing 2% FCS (Invitrogen) plus penicillin/streptomycin (100 U/ml and 0.1 mg/ml; Invitrogen)
4. Cell detachment and mouse MSC purification:
a) After 2 to 3 weeks, subconfluent (80-90%) cells are detached by trypsin-EDTA (0.5 and 0.2 g/L; Invitrogen).
b) MSC are purified by immunodepletion of CD45 positive cells. Briefly, after blocking with PBS that contained 0.5% BSA (Sigma), cells are incubated for 20 min with rat anti-mouse CD45 antibody, 0.2 µg/106 cells (Caltag). Then, cells are incubated with magnetic microbeads coated with goat anti-rat IgG (Miltenyi Biotec), and CD45 negative MSC are isolated by magnetic cell-sorting separation system.
c) For in vivo and in vitro experiments cells were used within first-second passages. in DMEM plus 10% FCS and penicillin-streptomycin.
Rat bone marrow-derived MSC
Isolation and growth of rat MSC BM was obtained from male Lewis rats weighting 180-200g. Briefly, rats were killed, and femurs and tibias were aseptically removed. BM was flushed from the shaft of the bone with α-MEM medium containing 2% fetal calf serum (FCS) and 1% penicillin/streptomycin (P/S) and then filtered through a 100 μm sterile filter to produce a single-cell suspension. Filtered BM cells were plated (200000 cell / cm2) in α-MEM plus 20% FCS and 1%P/S. Non-adherent cells were removed after 72 hours and rat MSC were recovered by their capacity to adhere highly to plastic culture dishes. Medium was changed regularly every 3d; after 2 to 3 wk, adherent cells were detached by trypsin-EDTA (0.5 to 0.2g/L) and used for the experiments.
Characterization of rat MSC Cells were characterized for their capability to differentiate toward adipocytes, and osteocytes. MSC were grown until confluence, and the growth medium was replaced with the inductive medium consisting of α MEM medium (Invitrogen) containing 20% FCS and 1% penicillin/streptomycin supplemented with specific differentiation reagents as follows.
Adipogenesis: Cells were incubated for 3 wk with 5 μg/ml insulin, 10-6 M dexamethasone, 0.5 μM isobutylmethylxanthine, 50 μM indomethacin. Then cells were fixed with 10% formalin for 10 min at room temperature and adipogenic differentiation was visualized in phase-contrast microscopy by the presence of highly refractive intracellular lipid vacuoles. Oil Red O staining was used to assay the accumulation of lipid droplets in these vacuoles.
Osteogenesis: Cultures were fed twice a week for 3 wk with 10 mM β-glycero-phosphate, 0.2mM ascorbic acid 2-phosphate, and 10-8 M dexamethasone. Then cells were fixed with 4% paraformaldehyde for 10 min at 37°C and mineralization—presence of calcium-rich hydroxyapatite—of the extracellular matrix was assessed by staining for 20 min with 2% wt/vol Alizarin Red S, adjusted to pH 4.1 with ammonium hydroxide.
We have observed that rat MSC were able to differentiate towards adipogenic and osteogenic lineage after 3 weeks of culture with specific inductive media.
Set up of rat MSC cultures
1. Bone marrow collection:
c) BM is obtained from Lewis rats (Harlan, France) weighting 180-200g. BM from femur cavity was flushed (10 times) with MEM medium (ABCYs, France) containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (P/S) (Invitrogen, USA).
d) Cell suspension is centrifuged (400 g, 5 min).
e) The viability will be evaluated by the percentage of negative cells after staining by blue trypan.
2. Culture set-up:
c) Cell pellet is re-suspended in complete medium (MEM + 10 % FCS + 1% P/S) and cells are plated in culture flasks at a concentration of 2x105 cells/cm2).
d) Non-adherent cells are removed after 72 hours and fresh medium is added. MSCs are recovered by their capacity to adhere highly to plastic culture dishes.
• Medium: MEM medium (Lonza) containing 10% FCS (Bodinco) plus penicillin/streptomycin (100 U/ml and 0.1 mg/ml; Sigma )
• Equipment to be used: Container of culture: T175 . To put in the incubator in wet atmosphere with 37°C with CO2 5%.
3. Medium exchange:
b) Medium is changed regularly every 3 days until the ending of the culture. The cell growth is appreciated by observation of the lower plateau under a microscope. The steps where the cellular layer is not covered any more with medium must be shortest as possible in order to avoid damaging the cells by desiccation.
• Medium: MEM medium (Lonza) containing 10% FCS (Bodinco) plus penicillin/streptomycin (100 U/ml and 0.1 mg/ml; Sigma)
4. Cell detachment and rat MSC purification:
d) After 2 to 3 weeks, subconfluent (70-80%) cells are detached by trypsin-EDTA (0.5 and 0.2 g/L; Sigma)
e) For in vivo and in vitro experiments cells are used at passage 3.
Human bone marrow-derived MSC
Isolation and growth of human MSC BM aspirates were collected within the Transplantation Program of the Haematology Division. Two–five milliliter of heparinised diagnostic samples were obtained during the clinical follow up of patients previously treated by autologous or allogeneic BMT who were in complete confirmed hematologic remission after informed consent and separated by Ficoll-Hypaque gradients centrifugation to yield mononuclear cells. In the case of bag washouts, total nucleated cells from filter and bag residues were obtained after two washings with 250 ml saline and centrifugation at 2000 r.p.m. for 5 min. Two protocols for the expansion of human MSC were compared: cells were plated at 200 000 cells/cm2 in DMEM low glucose medium containing gentamicin 0.1mM and heparin 1000 IU in the presence of 5% platelet lysate or 10% fetal bovine serum (FBS). We used the batch of FBS selected from the EBMT MSC Expansion Consortium (Perbio Science, Etten Leur, Netherlands). Non-adherent cells were removed after 3–4 days and fresh medium was added. At subconfluency, cells were recovered after Trypsin-EDTA treatment and subsequently replated at 3000 cells/cm2 in the same medium.
We obtained a significantly better expansion of human MSC when cultured in medium containing platelet lysate in respect to FBS. At variance, no difference were observed in terms of gross morphology, differentiation potential, surface markers of human MSC expanded with platelet lysate or FBS as describe below (Capelli C. et al. Bone Marrow Transplant.: 40, 785-791, 2007).
Characterization of human MSC The immunophenotype of human MSC were studied by using the following conjugated monoclonal antibodies: CD45-FITC, CD34-PE, CD14-FITC, HLA-DR FITC, CD29-PE, CD90-FITC, CD73 -PE, HLA-ABC-PE, CD105-PE or isotype-matched IgG-FITC and IgG-PE control antibodies. Data showed that cells expanded in platelet lysate or FBS exhibited a similar immunophenotype. In fact, both populations were positive for CD90, CD73, CD105 and negative for CD45, CD34, CD14 and HLA-DR markers.
Adipogenesis: Human MSC were used at third passage in culture. 5X104 cells derived from FBS and platelet lysate cultures were washed with PBS and plated in cell culture dishes, with adipogenic medium consisting of MesenCult Basal medium and 10% Adipogenic Stimulatory supplements (StemCell Technologies). Medium was changed weekly and dishes were observed under the microscope for the development of lipid droplets. Their lipid nature was confirmed by staining with Sudan Black IV after 21–28 days culture in these conditions (Capelli C. et al. Bone Marrow Transplant.: 40, 785-791, 2007).
Osteogenesis: Human MSC at third passage in culture were washed with PBS and plated with osteogenic medium consisting of Mesen-Cult Basal medium, 10% Osteogenic Stimulatory supplements consisting of 1X10-8M dexamethasone, 2X10-4M ascorbic acid and 7X10-3M β-glycerophosphate. The presence of alkaline phosphatise activity in differentiated samples was confirmed by staining with immunohistochemical alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique after 21–28 days of culture in these conditions (Capelli C. et al. Bone Marrow Transplant.: 40, 785-791, 2007).
Results showed that both populations of human MSC grown in platelet lysate or FBS were able to differentiate in a comparable extent toward adipocytes and osteocytes as demonstrated by specific staining with Sudan Black IV and APAAP (Capelli C. et al. Bone Marrow Transplant.: 40, 785-791, 2007).
For the development of a reagent enabling the large scale enrichment of MSCs from bone marrow CD271 and W8B2 antibodies were chemically coupled to paramagnetic particles. These conjugates were used in several optimization experiments to improve purity and yield of CFU-F forming target cells. In the future the focus will be on CD271 Microbeads.
To transfer the procedure to the closed clinical large scale CliniMACS system the sample preparation, labeling and elution of MSCs will be optimized. Feasibility was already shown.
Standardization of the characterization of MSCs is a prerequisite for comparative clinical studies involving isolated and/or cultivated MSCs. To provide an easy to handle reliable quality control a phenotyping protocol based on flow cytometry using the most important markers was developed. The new protocol includes the markers CD73, CD90, CD105 as positive markers and CD14, CD20, CD34, CD45 as negative markers. Only 2 cell samples with about 1x105 cells are needed to fully characterize the cells.
2. Characterization of CD24+CD133+ adult renal resident stem cells (APEMP) and standardization of protocols for isolation of these cells for cell therapy of renal failure.
A large body of evidence currently indicates that APEMP represent a population of renal progenitors which can potentially regenerate tubular as well as glomerular injury. To obtain CD24+CD133+ APEMP a protocol was standardized.
1. Kidneys are minced and digested with Collagenase type IV 750 U/ml (Sigma) for 45 minutes at 37°C.
2. Total kidney is mechanically disaggregated using sieving through 60 and 80 mesh screens or using the Medimachine System (BD Biosciences).
3. The pass-through is recovered, washed with PBS and red cells are removed through lysis buffer (NH4Cl 0.087% ) for 4 minutes at 37°C or through depletion with anti–glycophorin A MicroBeads (Miltenyi).
4. The suspension is depleted of leukocytes, using anti-CD45 MicroBeads (Miltenyi), following the manufacturer’s instructions using the VarioMACS column (Miltenyi).
5. The CD45-depleted fraction undergoes a second magnetic separation for CD133 (CD133 Cell Isolation Kit, containing the anti-CD133/1 mAb, clone AC133, also used for hematopoietic stem cell sorting). The separation is performed by magnetic cell sorting using LS columns (Miltenyi). The positive cell fractions usually consists of >97% of CD24+CD133+ cells.
Cells are cultured on dishes in EGM-MV 20% FBS. Cultures are checked for simultaneous expression of CD133 and CD24 by flow cytometry and routine cell passaging is performed. Medium is changed twice a week.
In addition, this population was largely characterized, the mechanisms at the basis of their homing toward injured kidneys were identified, and their beneficial effect in acute as well as chronic preclinical models of glomerular injury was addressed. In particular, we demonstrated that APEMP represent an heterogeneous and hierarchical population of progenitors with different commitment toward the podocyte or tubular lineage. We indeed demonstrated that APEMP can regenerate podocytes and improve proteinuria in in vivo models of glomerular injury, and we also demonstrated that APEMP can be distinguished in bipotent as well as tubular committed progenitors on the basis of their expression of the surface marker CD106. Consistently, both these populations could regenerate injured tubular tissue and improve renal function in mice modela of acute renal failure related to tubular injury. Finally, we investigated the mechanisms that mediate APEMP migration, homing, and repopulation, which are crucial for the success of a clinical strategy of SC-based kidney regeneration. To identify APEMP homing factors, the chemokine receptor expression pattern of these cells was investigated. APEMP exhibit high expression of the two receptors for SDF-1, CXCR4, and CXCR7. To assess whether the interaction of SDF-1 with CXCR4 and/or CXCR7 was involved in the regenerative capacity of APEMP, glycerol-treated SCID mice were injected with the red fluorescent dye PKH26 APEMP labeled into the tail vein after pretreatment with a neutralizing anti-CXCR4 antibody, or a neutralizing anti-CXCR7 antibody. BUN levels, as well as areas of necrosis found 4 d after injury, were significantly higher in mice injected with saline or with PKH26-stained APEMP pretreated with either an anti-CXCR4 or -CXCR7 antibody in comparison with those found in mice injected with PKH26-stained APEMP, which had been pretreated with the respective isotype-matched antibody with irrelevant specificity. Similar results were obtained on day 14 after injury. Accordingly, evaluation of the fibrosis score with Masson's trichrome staining in mice injected with APEMP pretreated with either an anti-CXCR4 or -CXCR7 antibody, demonstrated a higher fibrosis score in comparison with the respective control mice injected with PKH26-stained APEMP, which had been pretreated with isotype-matched control antibody with irrelevant specificity. Quantitation of the number of APEMP over the total number of renal cells demonstrated a significantly higher percentage of APEMP in the kidney of mice injected with cells pretreated with the isotype-matched control antibody with irrelevant specificity in comparison with mice injected with cells pretreated with an anti-CXCR4 antibody or with an anti-CXCR7 antibody, 4 d after injury as well as 14 d after injury. The role of interaction between SDF-1 and its receptors CXCR4 and CXCR7 was also investigated in in vitro culture of APEMP. SDF-1 –induced migration of renal progenitor cells was only abolished by an anti-CXCR4 antibody, whereas transendothelial migration required the activity of both CXCR4 and CXCR7. CXCR7 resulted essential for renal progenitor cell adhesion to endothelial cells and renal progenitor cell survival. Taken together, these results suggest that APEMP may represent a tool for treatment of acute kidney injury.
3. Identification of Sca-1+Lin- “like” adult renal resident stem cells in adult human kidneys and standardization of protocols for isolation of these cells for cell therapy of renal failure.
In mouse, we focused on two major sources of stem cells for kidney repair. One was described in 2006 in adult kidneys known as the SCA+ Lin- cells that seem to represent in mice a mesenchymal renal residing stem cells. We have now continued characterizing the attributes of SCA+ Lin- cells in adult and embryonic mouse kidneys. In particular we attempted to examine the niche of these putative stem cells, which as in the bone marrow might comprise innervating and endothelial neighbouring cells. Our new data clearly shows that indeed the SCA+ Lin- cells reside in the kidney in a typical 3 dimensional niche as previously shown for these cells in the bone marrow. One important marker is Nestin, the expression of which in mature adult tissues is restricted usually to areas of regeneration. Nestin has been shown to interact with other cytoskeleton proteins, suggesting a role in regulating cellular cytoskeletal structure. In developing kidney, nestin is expressed in vascular cleft of the S-shaped body and vascular tuft of capillary loop–stage glomerulus. The nestin-positive structures are also labeled by endothelial cell marker CD31 in immature glomeruli. Nestin is not detected in epithelial cells of immature glomeruli. Likewise we identified similar niches in human fetal kidney by staining for nestin and CD34, co-expressed in glomerular and tubular structures, which are negative for panepithelial markers. This results will help to better understand the mechanisms of kidney regeneration in rodents models of renal injury.
Identification of bone marrow or kidney origin of Sca-1+Lin- cells
We have previously characterised a multipotent organ stem/progenitor cells in the adult mouse kidney (Dekel et al, 2006). This cell population is stem cell antigen-1 (Sca-1) positive and lacks CD45 and lineage markers and resides in the renal interstitial spaces. Since the same phenotype can also be found in the BM it was of interest to define whether these unique cells arrive in the kidney from the BM or develop endogenously in the kidney.
We now addressed this question by using BM radiation chimera derived by transplantation of GFP+ BM into lethally irradiated (10Gy TBI) recipients. Thus, when kidneys were harvested from these mice in which the blood cells were predominantly of donor type, we found that the majority (93%)of the SCA+CD45- CD31-CD326- population was of host origin ( GFP-) as opposed to almost 70 % of the hematopoietic SCA+CD45+ CDD31-CD326- cell population found to be GFP+ (i.e. originating from BM cells) . Taken together, these results suggest that the renal Sca1+Lin- stem cells likely represent an endogenous population developing in the kidney in-situ and not arriving from the BM, which has important implications to understand the regenerative mechanisms of the kidney.
4. Identification of novel, unknown types of renal stem cells in mouse/rat and adult human kidneys:
Unknown types of stem cells in kidney have been identified on the basis of their clonogenic properties. The presence of clonal cells in kidney tissue homogenates has been demonstrated by using colony-forming assay. In this in vitro culture system, only single renal progenitor forms colonies. Colonies have been analyzed for their multidifferentiation capacity and self-renewal potential. In a second strategy, cells expressing marker of metanephric mesenchyme which are down-regulated during the differentiation or expressing markers of stem cell derived from different tissues have been isolated using FACS. Their clonogenicity, multidifferentiation and self-renewal potential have been analyzed by limiting dilution assay and culture in differentiating medium.
Recent studies reported the existence of scattered CD133+CD24+ cells distributed within the proximal tubules in adult human kidneys, and suggested they may represent a subset of progenitors destined to tubular regeneration. However, a phenotypic and functional characterization of tubular CD133+CD24+ cells was not reported, because specific markers allowing distinction from the CD133+CD24+ cells of Bowman’s capsule are unknown. To perform phenotypic and functional studies separately on CD133+CD24+ cells of Bowman’s capsule as well as on CD133+CD24+ tubular cells, we sought to identify a surface marker that allowed to distinguish these two populations. To this aim, CD133+CD24+ cells were prepared from outgrowth of capsulated glomeruli or from the tubular portion of renal tissue and screened for mRNA expression of 279 genes, using TaqMan low-density array (TLDA) technology. More than 95% of the investigated genes showed similar expression in CD133+CD24+ cells obtained from glomerular outgrowths compared with CD133+CD24+ cells obtained from tubular tissue. However, the expression of Vascular cell adhesion molecule-1 (VCAM-1), also known as CD106, was about 300-fold higher in CD133+CD24+ cells obtained from glomerular outgrowths than in CD133+CD24+ cells obtained from tubular tissue.
In the human kidneys, CD133+CD24+CD106+ cells were localized at the urinary pole of Bowman’s capsule, while a distinct population of CD133+CD24+CD106- scattered cells was localized in the proximal tubule, as well as in the distal convoluted tubule.
CD133+CD24+CD106+ cells exhibited a high proliferative rate and could differentiate toward the podocyte as well as the tubular lineage. By contrast, CD133+CD24+CD106- cells showed a lower proliferative capacity and displayed a committed phenotype toward the tubular lineage. Interestingly, both CD133+CD24+CD106+ and CD133+CD24+CD106- cells showed higher resistance to injurious agents in comparison to all other differentiated cells of the kidney. Finally, once injected in SCID mice affected by acute tubular injury, both these populations displayed the capacity to engraft within the kidney, generated novel tubular cells and significantly improved renal function, a property that was not shared by other cell types of the adult kidney.
Isolation of “untouched” cell populations from human whole blood.
The magnetic bead-mediated isolation of rare cells is challenging because of the very rare nature of target cells, e.g. CD271+ mesenchymal stromal cells show a frequency of 0.02% in human bone marrow aspirate. The isolation protocol of a certain stem cell population from human or mouse tissue using specific monoclonal antibodies conjugated to superparamagnetic nanoparticles was optimized with regard to the following points:
1. Optimization of magnetic beads focused on the reduction of unspecific binding:
Unspecific binding is always a challenge when utilizing magnetic particles for the isolation of certain stem cell populations within heterogeneous cell suspensions. This background labeling is thought to occur as a result of non-specific antibody binding to endogenous Fc receptors, ionic effects, and hydrophobic interactions.
2. Optimization of a protocol for isolation of cell populations using whole blood:
Whole blood is an attractive source for lymphocytes and progenitor stem cells due to it's availability. Current technology requires isolation of leukocytes from whole blood, e.g. by a Ficoll density gradient separation, before specific cell populations can be further purified. We previously developed a novel cell separation technology to isolate target cells directly from whole blood without proceeding bulk separation steps such as density gradient centrifugation. This technology removes unwanted non-target cells. Monoclonal antibodies have been covalently conjugated to novel magnetic beads (200nm - 500 nm). Antibody-bead conjugates have been evaluated for depletion performance when combined with an erythrocyte aggregation reagent. Both components are added to diluted human whole blood, incubated for 10 minutes and unwanted components of blood (erythrocytes, thrombocytes, lineage positive cells) are allowed to sediment in a magnetic field generated by a specifically designed permanent magnet for 10 minutes.
Achievements:
a.) Several magnet designs have been evaluated and developed. A design of magnet yoke, magnet size and shape and plastics housing could be selected to use 30 ml of human whole blood for cell separation in a 50 ml centrifuge tube.
b.) Several lineage markers were evaluated for depletion performance of non-stem cells, including CD3, CD19, CD56, CD14, CD15, CD16, CD2, CD7, CD36, CD4, CD8. Most of the conjugates allowed removing 99% of the cells positive for a lineage marker from whole blood.
c.) A method has been developed for further reducing erythrocyte content in the final stem cell preparation. Especially for rare target cell populations such as progenitor cells the 99.6% removal of erythrocytes in the single step cell separation method results in a huge excess of erythrocytes over target cells. An additional removal of 99% of erythrocytes can be achieved by use of an erythrocyte specific antibody conjugated to magnetic beads.
d.) Combinations of lineage markers have been evaluated for optimized depletion of unwanted lineage positive cells.
e.) A lyophilisation process has been established to stabilize cocktails of cell separation reagents.
5. Preparation of protocols for the characterization of human adult stem cells
1. Characterization of the cell surface expression of human mesenchymal stromal cells (MSCs)
The state of the art marker panel for the characterization of in vitro expanded human MSCs was published by Dominici et al (2006). They defined the markers CD73, CD90 and CD105 to be expressed on MSCs and lack of expression of hematopoietic markers like CD14, CD19, CD34 and CD45. With regard to this minimal criteria we optimized a fast and reliable FACS based assay. It consists of a MSC staining cocktail including CD73-APC, CD90-FITC, CD105-PE, CD14-PerCP, CD20-PerCP, CD34-PerCP, CD45-PerCP and a MSC control cocktail including corresponding isotype controls.
MSCs show a bright auto-fluorescence and the fluorochrome cocktails include four different fluorochrome dyes. Hence, the analysis requires a proper compensation of the used flow cytometer. Therefore, we optimized a procedure for a fast and accurate compensation procedure using single staining of each fluorochrome.
2. Functional analysis of the immunosuppressive potential of human mesenchymal stromal cells (MSCs)
MSCs show immunosuppressive capacity when co-cultured with proliferating Tcells. This in vitro observation may give a certain insight into the interaction of MSCs and immune cells in vivo within several clinical settings and can be used as a functional quality control assay for MSCs used in the clinics. There is a demand for a standardized and optimized assay and therefore we optimized a protocol for the examination of the immunosuppressive effect of MSCs on proliferating T cells. The protocol is based on MACSiBead particles pre-loaded with biotinylated CD2, CD3, and CD28 antibodies. We optimized the loading conditions of MACSiBead particles with antibodies and titrated the MACSiBeads for an optimal T cell stimulation. This process was embedded into a protocol including the optimized concentration of MACSiBeads, T-Cells and MSCs within a 96 well platform for an accurate high throughput analysis.
WP2 Isolation of fetal renal progenitors/stem cells and assessment of their ex-novo kidney organogenesis potential
1 Characterization of CD24+CD133+ human fetal renal resident stem cells and standardization of protocols for isolation and expansion of these cells for cell therapy of renal failure.
To obtain CD24+CD133+fetal renal resident stem cells, the embryonic kidneys were minced and digested with collagenase IV (500U/ml) for 20 minutes at 37°C. The renal suspensions were collected and checked for simultaneous expression of CD133 and CD24 by flow cytometry. CD133 was evidenced by using a PE-conjugated anti CD133/2 mAb combined with an anti CD24 mAb (IgG1) followed by an Alexa Fluor® 633-labelled goat anti-mouse IgG1. Since CD133+ cells represented a subset of whole CD24+ cells, the CD24+CD133+cells were isolated by using the CD133 Cell Isolation Kit (Miltenyi Biotec GmbH). The positive cell fractions consisted of >99% of CD24+CD133+ cells; the recovered cells were plated in EGM-MV (Cambrex Bio Science, East Rutherford, NJ) 20% FBS (Hyclone, Logan, UT) and then used for characterization. FACS analysis showed that such a population homogeneously exhibited not only the presence of CD24 and CD133, but also expressed CD106, CD44, CD54 and CD29, while haematopoietic markers (CD34, CD45) were not detectable. The effect of CD133+CD24+ cells obtained from adult human kidneys was compared with that of cells obtained from fetal human kidneys. When injected into mice with ARF related to tubulointerstitial injury (mice models of glycerol-induced acute kidney injury in SCID mice), both cell types regenerated cells of different portions of the nephron, reduced tissue necrosis and fibrosis and significantly improved renal function with similar efficiency, regenerating tubular structures.
2 Identification of novel, unknown types of renal stem cells in mouse, rat and human fetal kidneys.
a. through phenotypic and functional screening. The lab of partner 8 has focused during 2009 to develop assay systems to analyze reliable the capacities of specific cell types, especially their potential to reconstitute specific structures in the developing kidney. For this purpose the mouse embryonic kidney has been prepared at midgestation and the ureteric bud has been mechanically separated from the adjacent metanephric mesenchyme. Thereafter the kidney mesenchymal cells have been dissociated to single cells. Followed by this the cells have been incubated in a variety of mixtures consisting of specific factors that are known to be important in the control of kidney development. The aim here has been to maintain the competence of the kidney mesenchymal cells to form the nephrons. During 2009 the so called kidney mesenchyme dissociation and reaggregation model has been established. We can now efficiently dissociate the kidney mesenchyme to single cells and maintain the competence in these cells at least until 48 hrs prior to experimental induction of nephrogenesis in these cells when reaggregated. Such system has enabled us to screen the potential of a variety of cells in their capacity to integrate efficiently to the structures that are derived from the kidney mesenchyme, namely the nephrons with is numerous cell types and the stromal cells. We have also screened the reconstitution potential of such reaggregates by analyzing to what degree the reaggregated and induced kidney mesenchyme can indeed form a segmented nephron. As it seems based on the data, the induced nephron developing in such reaggregates can form the major segments of the nephron. We have used this system to address the potential of variety of pluripotent cells such as genetically labeled embryonic stem (ES) cells and even human embryonic stem cells. These studies are starting to point that the genetically indicator labeled cells can be directed towards the cell lineages of the pretubular cells. We are using this system to assay the requirements of the ES cells or other the pluripotent cells to be induced towards the structures of the nephron. We are also developing methodology to infect the dissociated mesenchymal cells with a variety of viruses to introduce specific components to the kidney mesenchymal cells or the pluripotent cells. We are using the reaggregation system and genetically GFP labeled cells to address the potential of the individual cells of the kidney to reconstitute the kidney structures. Finally we also develop the same methodologies for the epithelial ureteric bud to address its potential for differentiation and viral infection. This work is in progress.
In addition, partner 7 further developed a system for making fine-grained chimaeric kidneys, into which stem cells can be introduced to test their potential when placed in an appropriate environment. Specifically, while screening potential stem cells from mouse and human, partner 7 has identified an AFSC line that incorporates very well into this system and is currently assessing the degree of renal differentiation using human-specific RT-PCR for renal genes. In addition, partner 7 is also using this system to test the potential of cells isolated from embryos of different developmental stages to contribute to renal tissue, to map the range of cell types into which we could reprogram stem cells and reasonably expect them to produce renal tissue.
b. Identification of novel nephron stem cells with the aid of the Wnt-4 GFP Cre knock in mouse.
In order to fate map the cells that have expressed the Wnt-4 gene we have targeted the CreGFP cassette under the control of the endogenous Wnt-4 promoter. The constructs have been made and successfully targeted to the Wnt-4 locus. The ES cells have been injected to the blastocyst and the mouse line has been established. We have used the Wnt-4GFPCre mouse line to verify that the Wnt-4 promoter drives correctly the GFP and Cre. Indeed the GFP becomes expressed by the cells that normally express Wnt-4 gene. Given this success we have used a floxed indicator mouse line, namely the floxed Rosa26 where LacZ becomes permanently activated upon the Cre reaction. These studies have allowed us to map the fate of the cells that have expressed the Wnt-4 gene. The in vivo fate mapping studies have indicated that the cells that have expressed the Wnt-4 gene generate the nephrons. However on top of that we also found some LacZ positive cells in the stroma suggesting that the Wnt-4+ cells may have also other fates. To address this possibility in detail we established an organ culture based system where the fate of the cells was analyzed by a time-lapse set up. This set up allows almost real-time monitoring of the fates generated by those cells that have expressed the Wnt-4. The time-lapse studies revealed the dynamic nature of the cells that express the Wnt-4 gene. The stem cells become motile. Also the dynamics how the nephrons are generated from the nephron stem cells can be visualized. Interestingly we also could show that the first few nephrons are transient and that these cells likely transform to generate the medullary stromal cells. These medullary stromal cells are however transient in their nature but are likely essential for further development of the smooth muscle actin positive stromal cells induced by the signals that are expressed by the transient stromal precursor cells. Fate mapping with other Cre lines should be done to obtain additional evidence for this possibility.
The work has been published during 2009 (Shan J, Jokela T, Skovorodkin I, Vainio S. Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development. Differentiation. 2010 Jan;79(1):57-64). To address the role of the Wnt-4 protein itself in the control of the nephron stem cells we have also generated a floxed allele of the Wnt-4 gene. This allows detailed studies of the role of Wnt-4 in specification of the different cell lineages of the nephron (Shan J, Jokela T, Peltoketo H, Vainio S. Generation of an allele to inactivate Wnt4 gene function conditionally in the mouse. Genesis. 2009 Nov;47(11):782-8).
c. Purification and replacement of novel fetal nephron stem cells through Avidin-LDL fusion protein, Lodavin approach. In order to purify and replace the nephron stem cells we have generated and applied two different mouse lines in the laboratory. We have established the constructs to target Lodavin to the Rosa26 locus. This has been achieved during 2009. We have also generated the Lodavin mouse like and confirmed that Lodavin expression can be activated by the Cre mediated approach. We have successfully obtained activation and expression of Lodavin by using several Cre lines including the Wnt-4 Cre to target the nephron stem cells. Lodavin expression is indentified with a specific antibody against avidin. We are in a process of testing if Lodavin can be used as a vehicle to also purify the nephron stem cells. We have recently obtained a FACS machine to our university. We will use this in trials to try to purify the Wnt-4+ cells from the embryonic kidney by using Lodavin or the GFP that is expressed from the genetically manipulated Wnt-4 locus. In conjunction with the aims to purify the nephron stem cells we have also applied a mouse line where human diphtheria toxin receptor can be activated and directed to those cells that express the Cre recombinase. This mouse line called iDTR has been targeted to the Rosa26 locus. Indeed we have used the organ culture setting to assay if the iDTR expression and thus susceptibility to diphtheria toxin (DT) can be directed to the nephron stem cells to ablate them specifically. Here iDTR expression has been activated to the nephron stem cells with the aid of Wnt-4 Cre. The embryonic kidneys have been prepared from the compound positive embryos thus expressing iDTR and Wnt-4Cre and DT has been applied in organ culture. In this setting we can successfully delete the nephron stem cells. We are currently using the set up to develop in vivo models for renal cell replacement therapies based on the use of the nephron stem cells.
Assessment of the developmental potential of renal stem cells using a novel and rapid in vitro system.
We have tested the ability of a number of different stem cell types to produce renal tissue in chimaeric engineered kidneys, made from suspensions of mouse foetal kidney stem cells (direct from the foetus) and labeled test cells. These test cells have included embryonic stem (ES) cells, ES cells treated in various ways, for example with BMPs, to try to encourage them towards the renal lineage, human amniotic fluid stem cells (AFSCs), mouse AFSCs, and putative stem cells isolated from mature kidneys. We have found (and published) that human AFSCs seem to show a strong ability to do this, and further have found that TOR signaling is important to that. We have found the ability of the other stem cells to be disappointing. In an effort to find out why, we have gone back in developmental time, testing the ability of cells from successively earlier stages of mouse development, in the lineage that will eventually give rise to kidney, to contribute to renal tissue in this assay. This work has identified a critical ‘switch’ – everything from ES cells to embryonic day 8 intermediate mesoderm has a weak ability to contribute to kidney formation, while everything later performs well. We will now (in other projects) concentrate on understanding what mediates this change, to see if we can being ES cells to the capable state ‘manually’ by treatment with defined molecules.
3 De novo kidney organogenesis in vitro.
A novel organ culture set up was developed that allows 3D imaging of the stem cell behaviour to be monitored close to real time by time lapse approach. Here the kidney is cultured under a certain pressure and this pressure improves the exchange of gases and allows also good visualization of the developing kidney rudiment. The set up is excellent to follow the fate and developmental capacity of the locally injected stem cells that are either derived from the embryonic kidney or inserted from other sources. Such flattened culture set up is beneficial likely due to reduction of hypoxia. These aspects will be published as a result of the project. Moreover it has been developed and published a novel ‘low volume’ technique for culturing intact mouse fetal kidneys that allows proper formation of cortico-medullary zonation and loops of Henle. Second, The development of techniques for engineering fetal kidneys from simple suspensions of cells has been continued: now it is possible to build kidneys properly arranged around a single branched collecting duct system, and kidneys that can attract blood vessels from living tissue and form glomeruli.
Routine in vitro kidney cultures are hampered by the absence of a normal vascular network. As a result glomeruli do not develop beyond the late S-shaped body stage and filtration never occurs. To circumvent this problem, we catheterized the dorsal aorta of embryos at various stages and perfused the developing kidneys with oxygenated growth medium (micropump-controlled flow rate and pressure). Control kidneys showed a high rate of necrosis and apoptosis due to the absence of oxygen in deeper tissue layers. By contrast, perfused kidneys displayed only very few apoptotic cells and appeared to develop glomeruli with an intact podocyte/endothelial interface. In conclusion, this approach may represent a significant improvement over existing techniques of kidney organ culture and may in future pave the way for the growth of human kidneys in vitro.
4 De novo kidney organogenesis in vivo.
During this project, we set up a technique to dissociate the separated constituent parts of the early embryonic kidney rudiment and recombine the separate tissue and obtain de novo kidney organogenesis. Similarly we can use the set up to introduce differentiation directed originally pluripotent cells to the system and obtain directed differentiation towards the cell lineages of the kidney. By such mechanism and the developed diphtheria toxin system we are heading towards the ultimate goal of being able to obtain a complete kidney from originally pluripotent cells. The objective to obtain de novo kidney organogenesis was approached by using a specific toxin based approach. The diphtheria toxin (DT) system was selected as the compound since normally the mouse cells are not susceptible for the DT but can be converted responsive if a human DT receptor has been introduced to the kidney stem cells. Novel mouse lines and crosses were generated and the system tested. Indeed evidence was provided that the nephron stem cells can be specifically ablated by the DT mediated approach. The developed protocols and systems serve as the essential basics to obtain de novo kidney organogenesis in vivo.
Significant results:
The Wnt4+ cells indeed serve as the stem cells to generate the whole nephron. The first few epithelialized embryonic kidney mesenchymal cells undergo a second cell transition and form a transient interstitial stromal cell population. This population is important as an inductive signalling source to contribute to secondary stroma development. The evidence for this is that if Wnt-4 gene is inactivated the secondary stroma cell differentiation fails completely.
WP3 Comparison of in vivo renal regenerative potential of stem cells obtained from different sources in animal models of acute and chronic renal failure
1 Comparison of the in vivo renal regenerative potential of stem cells obtained from different sources in animal models of acute kidney injury with tubulointerstitial damage.
The ability of CD133+CD24+CD106+ and CD133+CD24+CD106- cells to regenerate injured renal cells was then assessed in a model of rhabdomyolysis-induced acute kidney injury in SCID mice, generated by intramuscular injection of glycerol. To this end, CD133+CD24+CD106+, CD133+CD24+CD106- cells or saline were injected into the tail vein of SCID mice with rhabdomyolysis-induced acute kidney injury 4 and 20 h after glycerol injection. As an additional control, glycerol-treated SCID mice were injected with a mixture of CD133−CD24− renal cells, which represent 90-95% of the total cells of the kidney. Measurement of blood urea nitrogen (BUN) levels demonstrated that mice injected with CD133+CD24+CD106+ cells or CD133+CD24+CD106- cells showed a significantly reduced severity of ARF at day 5, 8, 11 and 15 that was not observed in mice treated with saline or with CD133−CD24− renal cells. Interestingly, only injection of CD133+CD24+CD106+ cells resulted in a significant reduction of the severity of ARF at day 3, as revealed by the lower BUN levels in comparison to mice treated with CD133+CD24+CD106- cells or saline. Treatment with CD133+CD24+CD106+ cells or CD133+CD24+CD106- cells was also associated with a better preservation of renal structure and a reduction of renal fibrosis in comparison to mice treated with CD24-CD133- cells or saline at day 15 after induction of ARF, as assessed with Masson's trichrome and scoring for the presence of renal scarring. To further investigate their ability to regenerate injured tubular cells, CD133+CD24+CD106+, CD133+CD24+CD106- or CD133−CD24− renal cells were labeled with the red fluorescent dye PKH26 before their injection into glycerol-treated SCID mice. Labeled CD133+CD24+CD106+ cells engrafted within the tissue and generated novel tubular cells, acquiring the proximal tubule specific marker LTA or the distal nephron marker Dolichos Biflorus Agglutinin (DBA). Identical results were obtained when CD133+CD24+CD106- cells were used. Quantitation of the number of PKH26-labeled cells over the total number of proximal tubular cells was performed on sections that were stained with LTA and demonstrated that in mice injected with CD133+CD24+CD106+ cells, 7.81±3.1% of PKH26-labeled/LTA-stained and 4.92±1.8% of PKH26-labeled/DBA-stained tubular cells were observed, while in mice injected with CD133+CD24+CD106- cells, 6.7±2.2% of PKH26-labeled/LTA-stained and 5.23±2.13% of PKH26-labeled/DBA-stained tubular cells were observed at day 15 after injury. By contrast, red labeling was never observed in mice injected with CD133−CD24− renal cells or with saline solution. Furthermore, human HLA-I antigen was detected in mice that were administered an injection of CD133+CD24+CD106+ cells or CD133+CD24+CD106- cells, whereas in SCID mice that received CD133-CD24- cells, HLA-I human antigen expression was never found, as demonstrated by double-label immunohistochemistry for human HLA-I and pan-cytokeratin. Taken together, these results suggest that only CD133+CD24+ cells display the property to differentiate into tubular cells of distinct portions of the nephron.
Taken together, the results of this study provide evidence that the tubular compartment of the nephron contains scattered renal progenitors that display functional regenerating capacity for injured renal tubular cells, as well as increased resistance to apoptotic stimuli.
The existence of tubular progenitors suggests that novel treatments aimed at promoting the regenerative capacities of the kidney could conceivably be possible, and be used to prevent and treat tubular injury.
A model of Acute Kidney Injury (AKI) induced by the nephrotoxic drug cisplatin in immunodeficient NOD-SCID mice was set up to test regenerative effects of human mesenchymal stem cells (MSCs) isolated from different sources including bone marrow (BM), adipose tissue and cord blood (CB). Infusion of CB-MSCs in immunodeficient mice with cisplatin-induced AKI ameliorated both renal function and tubular cell injury, and remarkably prolonged survival in respect to mice injected with BM-MSC. By contrast, infusion of adipose tissue-derived MSC failed to ameliorate renal function and structure in mice with AKI. Transplanted CB-MSCs localized in peritubular areas, limited capillary alterations and neutrophil infiltration. Apoptosis was reduced and tubular cell proliferation increased by virtue of stem cell capacity to produce growth factors. The reno-protective effect of CB-MSCs was further confirmed by their ability to inhibit oxidative damage and to induce the pro-survival factor Akt in tubular cells. The evidence that CB-MSCs in vitro increased the production of growth factors and inhibited IL-1b and TNFa synthesis when co-cultured with damaged-proximal tubular cells indicates a regenerative and anti-inflammatory action of stem cell treatment. Altogether these results highlight the potential of human CB-MSCs as future cell therapy for testing in human AKI.
The effect of stem cell therapy was evaluated on renal functional parameters and on renal cell proliferation, apoptosis and animal survival. Quantification of the engraftment of the different human MSC populations in damaged renal tissue and in other organs as lung, liver, brain, heart was performed. Results have evidenced cord blood-derived MSCs as the most powerful population for cell therapy in the process of kidney regeneration in experimenthal AKI.
These results suggest that both MSC and renal stem cells can be used to treat AKI, although they exert their beneficial effect through different mechanisms.
MSC’s in animal models of acute kidney injury
Kidney ischemia-reperfusion injury was induced in a model of bilateral warm renal ischemia in mice followed by three days of observation.
Mouse model of renal I/R injury
Normal male C57BL6 mice of 10 weeks old were used in this study. Isoflurane anaesthesia was used. A baseline blood sample was obtained from the tail vein. Via a midline laparotomy the renal pedicles were identified and bilaterally clamped for 30 minutes using microvascular clamps (S&T, Neuhausen, Switzerland). In the sham group identical surgical procedures were used, except that clips were not applied. After the renal pedicle clamps were removed, the kidneys were observed for color change indicating reflow. One milliliter of warm saline was left in the abdominal cavity before the incision was sutured in two layers and the mice were allowed to recover from anesthesia. As analgesic 0.05mg/kg buprenorfine was administered s.c. After 72 hours the mice were anesthetized again. The final blood sample was obtained using cardiac puncture. Immediately after blood collection, the kidneys were removed and sagitally cut. One halve was snap frozen in liquid nitrogen and the other halve kept on formalin. Blood samples were collected in heparin coated capillary tubes. Plasma urea concentration was measured using the reflotron system (Roche diagnostics, Almere, The Netherlands) as indicator of kidney function at baseline and 72 hours after reperfusion. Throughout the whole experimental period the mice were maintained on standard diet and given water ad libitum. The study was approved by the veterinary authorities of the LUMC.
Murine MSC’s combined with ARA290 in IR injury
The beneficial effects of MSCs in renal IR have repeatedly been shown, but recently it appeared that these effects may even be increased by culturing MSCs under hypoxic conditions. This effect is most probabaly mediated by an increase in hypoxia inducible factor (HIF)-1 that regulates secretion of erythropoietine (EPO) from MSC's. When EPO or EPO receptor is inhibited this positive effect on MSC’s is diminished and in myocardial I/R it is already shown that the combination of EPO and MSCs worked synergistically. Here we used ARA290, a synthetic EPO analogue that does not have the hematopoietic characteristics of EPO.
Mice were divided into the following four groups: 1) I/R treated with ARA290 only (n=6); 2) I/R treated MSC’s combined with ARA290 (n=6); 3) I/R treated with PBS (n=4) and 4) sham treated with PBS (n=2). Bilateral kidney ischemia was induced by clamping the renal vessels for 30 minutes, followed by reperfusion. Six hours after reperfusion the treatment (ie MSC and/or ARA290) was administered by i.p injection. Kidney function was measured by plasma urea concentration. Kidneys were harvested at 72 hours after reperfusion. There were no significant differences in kidney function between mice treated with ARA290 alone and combined ARA290 and MSC’s.
2 Comparison of the in vivo renal regenerative potential of stem cells obtained from different sources in animal models of acute kidney injury with glomerular damage.
The rat model of adriamycin (ADR) induced nephropathy was employed as a model of kidney injury with glomerular damage, serving as a tool to evaluate the efficacy of cell therapy with bone marrow-derived mesenchymal stem cells (MSCs). The regenerative potential of treatment with bone marrow-derived mesenchymal stem cells (MSCs) on glomerular resident cells of distinct compartments was evaluated. Nephrosis was induced in male Lewis rats by ADR (5mg/kg i.v.). Rat MSCs (2x106 cells) were injected into the tail vein at 36, 60 hours, 3, 5, 7, 14 and 21 days after ADR. Rats were sacrificed at 3, 9, 16 and 30 days. MSC treatment did not affect the development of proteinuria nor serum creatinine levels of nephrotic rats. Morphometric analysis revealed an early decrease of WT1+podocytes in response to ADR in respect to control rats associated with a decreased glomerular expression of nephrin and CD2AP. Increased rate of apoptotic podocytes and a decreased expression of glomerular VEGF expression were additional features of the model. As a consequence of ADR-induced podocyte injury, glomerular adhesions of the capillary tuft to the Bowman’s capsule (synechiae) were observed from 16 days, followed by crescents-like lesions and glomerulosclerosis. Cellular components of sinechiae were either NCAM+parietal progenitor cells or nestin+podocytes. Engraftment of MSCs in the damaged kidney, documented by the presence of PKH-26-labeled cells in glomeruli of ADR-rats, limited podocyte loss and apoptosis, and partially preserved nephrin and CD2AP. Stem cell infusion attenuated the formation of glomerular podocyte-PEC bridges and normalized the distribution of NCAM+progenitor cells along the Bowman’s capsule. Consistent with the restoration of Bowman’s capsule architecture, a decrease of glomerulosclerosis was observed. Finding that MSCs increased glomerular VEGF expression and preserved microvascular rarefaction may explain the pro-survival effect displayed by stem cell therapy. MSC infusion also lessened the inflammation with lower glomerular and interstitial accumulation of ED1-positive monocytes/macrophages.
These findings suggest that bone marrow-derived MSC by virtue of their tropism for damaged kidney and their ability to provide a local pro-survival environment may represent a useful strategy to preserve podocyte viability and reduce glomerular inflammation and sclerosis.
3 Comparison of the in vivo renal regenerative potential of stem cells obtained from different sources in animal models of chronic renal failure with tubulointerstitial injury.
Glomerular and tubular interstitial damage was assessed during time in the experimental model of 5/6 nephrectomy in the rat to establish the optimal timing of cell therapy. While studying models of chronic renal failure available in the lab, we identified a new renal progenitor cell population in the Bowman’s capsule of Munich Wistar Frömter (MWF) adult rats, which develop progressive glomerular injury with time. In this model, we previously reported that angiotensin-converting enzyme inhibitor (ACEi) renoprotection was associated with podocyte repopulation and preservation of glomerular architecture. Here, we studied the time course of the lesions, their cellular components and the effect of ACEi. Early glomerular lesions were synechiae followed by extracapillary crescents and glomerulosclerosis later on. Majority of cells forming crescents were claudin1+ parietal epithelial cells and to a lesser extent WT1+ podocytes, both in active proliferation. In crescents, cells expressing the metanephric mesenchyme marker NCAM, were also found. Three distinct populations of parietal epithelial cells were identified in the rat Bowman’s capsule at different stages of differentiation from immature progenitor cells to differentiated parietal podocytes. A large population expressing stemness markes including NCAM and CD24, transitional cells expressing NCAM+and the podocyte marker WT1+ and NCAM-WT1+cells, the latter population representing parietal podocytes were found. After exposure to inductive medium, cultured parietal epithelial cells obtained by capsulated glomeruli generated podocytes, documenting their progenitor nature. Mitotic activity of cultured renal progenitors was induced by Angiotensin II through the downregulation of cell-cycle inhibitor C/EBPδ expression. In vivo, chaotic migration and proliferation of the Bowman’s capsule progenitor cells paved the way to crescent formation and subsequent sclerosis. An unprecedented result of this study was that ACEi, beside their effect of lowering blood pressure and proteinuria, reduced the formation of crescents and the evolution toward glomerulosclerosis in MWF rats. A new mechanism of renoprotection by ACEi through the limitation of NCAM+ progenitor proliferation and migration via the modulation of C/EBPδ has been disclosed leading to restoration of glomerular architecture. These data provide a clue for designing specific molecules targeted to novel players of renal repair that can possibly foster the intrinsic capacity of the kidney to regenerate. These observations were further supported by results obtained through injection of APEMP in mice models of chronic glomerular injury improved proteinuria and generated novel podocytes within injured glomeruli. This means that strategies aimed at controlling the growth and differentiation of APEMP can lead to glomerular and tubulointerstitial regenration in mice models of chronic renal failure.
At the university of Aachen, three different protocols of MSC treatment were tested in 5/6 nephrectomized rats: Daily i.v. injection of 2 ml of MSC conditioned medium for 40 consecutive days, weekly i.v. injection of 1 mio MSC for 5 weeks or for 11 weeks vs. respective controls. None of the treatments resulted in significant amelioration of disease course, nevertheless a beneficial trend was noted in the most intensive treatment group (11 weekly injections). The group of Prof. Floege and Prof. U. Kunter assessed the kidneys for adverse, i.e. profibrotic effects of MSC treatment but found no differences in collagen accumulation between the groups
In the renal IR model in mice at LUMC, a longer follow-up time of 3 weeks was chosen to assess chronic kidney injury after IR. Consecutive urea measurements during the follow up period showed regain of kidney function with time. Within three weeks of follow up, a considerable amount of kidney fibrosis developed, which was quantified by Sirius red (SR) staining. Histological tissue injury was scored on the PAS staining with semi-quantitative score on amount of proximal tubule necrosis (score 0-3), band necrosis of cortex and medulla (score 0-1) and protein casts in the tubules (score 0-1).
Murine MSC’s administered late after IR injury
It is unclear whether MSCs act more efficiently when applied after induction of tissue damage, as compared to administration before induction of damage. Therefore we used the same model of murine MSCs that were administered to Bl6 mice undergoing renal IR, with the modification that MSCs were administered 7 days after renal IR. No ARA290 was administered. No difference between untreated mice and mice treated with MSC’s in terms of renal function could be demonstrated.
The group at INSERM has previously shown that bone marrow MSCs (BMMSCs) may inhibit fibroblast activation through the secretion of paracrine factors (Mias C and al, Stem Cells. 2009 Nov;27(11):2734-43). Based on these results, we aimed to investigate the effects of bone marrow MSCs on progression of post-ischemia reperfusion renal remodeling and dysfunction and the mechanisms potentially involved. With this scope, we used an original rodent model that we have recently designed (Chaaya R and al, Nephrol. Dial. Transplant. 2011 Feb;26(2):489-98) mimicking the development of chronic renal disease occurring in clinic after ischemia-reperfusion and immunosuppressive therapy.
Using this model, we investigated (i) whether cell-based therapy using bone marrow MSCs directly injected in the kidney prevents or reduces the development of fibrosis; (ii) the impact of paracrine factors secreted by bone marrow MSCs on epithelio-mesenchymal transition of kidney proximal tubular cells in vitro.
In order to mimic better the clinical frame of post ischemia-reperfusion chronic renal disease occurring in humans, we used uninephrectomized rats submitted to ischemia-reperfusion and chronic cyclosporine treatment. The use of cyclosporine has a double interest: firstly, as observed for immunosuppressive therapy in human, cyclosporin aggravate the outcome of renal disease and increases interstitial fibrosis and tubular damage; second, its immunosuppressive properties minimize the previously described immunomodulation activity of grafted MSCs. Using this original model we have been able to determine the mechanisms of prevention of renal fibrosis by BMMSCs in chronic renal failure.
4 Establishment of which stem cell type displays the best regenerative properties in mice models of chronic renal failure with glomerular injury
Among bone marrow derived stem cells, we tested rat mesenchymal stem cells. We tested different MSC preparations in the model of Thy1.1 nephritis, a model of mesangioproliferative glomerulonephritis. MSC were isolated from healthy rats (H-MSC), healthy trackable hPLAP transgenic rats (TG-MSC) and from CKD donors (CKD-MSC). In addition, given our negative outcomes in the remnant kidney model, we tested healthy MSC after 7 days incubation in a novel “uremic medium” (UM-MSC). Only healthy donor MSC (H-MSC, TG-MSC) significantly improved renal repair (reduction in mesangiolysis score and proteinuria, increase in glomerular mitoses) whereas CKD-MSC and UM-MSC did not. These findings suggest that heterologous MSC from healthy donors might be more beneficial than autologous MSC in patients with progressive CKD. In addition, the regenerative potential of APEMP at different stages of differentiation was evaluated in mouse model with adriamycin-induced nephropathy. Adriamycin nephropathy was induced in female SCID mice at the age of 6 wk by a single intravenous injection of adriamycin on day 0 in the tail vein. On day 1, and again on days 4, 9, 18, and 25 after adriamycin injection, two groups of mice received intravenous administration of saline or of PKH26-labeled CD133+CD24+PDX- cells. Additional groups of mice were treated with saline, CD133-CD24-, CD133+CD24+PDX+, CD133+CD24+PDX-, or with clonally expanded PKH26-labeled CD133+CD24+PDX- cells. CD133+CD24+PDX- cells, but not CD133+CD24+PDX+ or CD133-CD24- cells, into mice with adriamycin-induced nephropathy reduced proteinuria and improved chronic glomerular damage, suggesting that CD133+CD24+PDX- cells could potentially treat glomerular disorders
5 Models of chronic renal failure related to genetically inheritable diseases:
Frasier animals carrying point mutations in the Wilms’ tumor suppressor gene WT1 and which mimic the human Frasier syndrome have been better characterized. To generate this mouse model and to clarify the function of the + and −KTS proteins in development, a specific mutations has been introduced into the endogenous Wt1 locus, designed to interfere with the production of one of the two variants. Heterozygous mice with a reduction of +KTS levels develop glomerulosclerosis and represent a model for Frasier syndrome. To better characterize the Wt1-Frasier (Wt1 (+KTS)-/+) mice phenotype on a molecular level we carried out exome array analysis and identified a set of genes deregulated in Frasier syndrome. We reasoned this was important information that needed to be obtained for any kind of approaches in cell replacement therapies. Incidentally, the identified target genes represent excellent candidates for genes involved in glomerular disease. To further establish the ideal genetic background to test out replacement therapies, we crossed the Frasier mutant mice with a variety of different genetic strains. While heterozygous mice on a CBA or Bl6 background survived up to 15 months, crosses on the FVBN background developed fulminant proteinuria and died as early as 6 weeks after birth. We therefore have established a colony of Wt1 (+KTS)-/+ mice, which will now be suitable for transplantation experiments of stem cells, and that can potentially be useful to evaluate the effects of cell therapy using MSC or APEMP in genetically inheritable renal diseases.
6 Functional and histomorphometric analysis of stem cells regenerative potential Stem cell therapy
Both CD133+CD24+ cells obtained from glomerular and tubular fraction were tested for their effect in inducing renal function improvement and tubular regeneration in glycerol-induced acute renal failure, which mimics rhabdomyolisis-induced acute tubular necrosis. Rhabdomyolysis-induced ARF was studied in 6 week old female SCID mice (Harlan, S. Pietro al Natisone, Italy), by intramuscular injection with hypertonic glycerol (8 ml/kg body weight of a 50% glycerol solution; Sigma-Aldrich) into the inferior hind limbs. Animal experiments were performed in accordance with institutional, regional, and state guidelines and in adherence with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. On day 0, mice received an i.v. injection into the tail vein of CD133+CD24+CD106+ cells (0.75 × 106 cells 4 h after glycerol injection and 0.75 × 106 cells 20 h after glycerol injection) and CD133+CD24+CD106- cells (0.75 × 106 cells 4 h after glycerol injection and 0.75 × 106 cells 20 h after glycerol injection). The cells, obtained from five different human donors (2 men and 3 women) were labeled with the PKH26 Red Fluorescence Cell Linker kit (Sigma-Aldrich) immediately before injection.
Blood samples were obtained from the submandibular venous sinus at day 3, 5, 8, 11 or 15, and BUN levels were measured by Reflotron System (Roche Diagnostics, Rotkreuz, Swiss). Normal range in our experiments was between 30 and 37 mg/dl, as calculated in 10 additional untreated mice (day 0). BUN levels that exceeded 40 mg/dl were considered abnormal.
For histomorphometric analysis kidney sections of mice were stained with a Masson-Goldner trichromic kit (Bio-Optica). Nonoverlapping fields of the entire section (20 fields for each mouse) were independently analyzed using a 20X objective by two observers. The severity of renal scarring was assessed as follows: normal tubulointerstitium scored 0; mild tubular atrophy with interstitial edema or fibrosis affecting up to 25% of the field scored 1; moderate tubular atrophy with interstitial edema or fibrosis affecting 25–50% of the field scored 2; and severe tubular atrophy with interstitial edema or fibrosis affecting > 50% of the field scored 3.
Using the model of chronic renal failure (nephrectomy/ischemia-reperfusion/cyclosporine), we showed that a single intrarenal administration of bone marrow MSCs 7 days after ischemia-reperfusion significantly improved renal function and modified renal remodelling. The improvement of renal function was associated to reduced extracellular matrix accumulation as shown by the decrease in collagen staining and mRNA expression measured by histomorphological analysis and semiquantitative RT-PCR, respectively. In addition, MSCs administration also reduced tubular dilation, which is a classical feature of progressive renal failure.
WP4 Evaluation of mechanisms of in vivo regenerative potential as well as safety, potential side effects and immunogenicity of stem cells obtained from different sources in animal models of acute and chronic renal failure
1. Evaluation of the mechanisms at the basis of stem cell-mediated regenerative potential.
A large effort was devoted to understand the mechanisms at the basis of stem- cell mediated regenerative potential. First of all we analysed the mechanisms at the basis of MSC-induced beneficial effects in mice models of glomerular and tubular injury. In view of the podocyte protective effect of bone marrow-derived mesenchymal stem cells (BM-MSC) in adriamycin (ADR)-treated rats, vascular endothelial growth factor (VEGF), highly produced by rat MSC and known to exert pro-survival and angiogenic activity was assessed. VEGF staining was significantly reduced in glomeruli of ADR-treated rats as compared to control rats. Indeed, mice treated with adriamycin develop a nephrotic syndrome related to podocyte injury. Infusions with MSC enhanced glomerular VEGF expression in rats receiving MSC were significantly higher than those of rats given saline at days 9 and 16. Furthermore, a marked reduction in volume density of endothelial cells in ADR-treated rats given saline compared with control rats was found. The degree of microvessel rarefaction was significantly limited in glomeruli of ADR-rats infused with MSC versus glomeruli of nephrotic rats on saline. In addition, an increased number of ED1-positive monocytes/macrophages was found in the glomeruli of ADR-treated rats given saline as compared to control rats. Infusion of MSC resulted in an important anti-inflammatory effect as shown by a significant reduction of glomerular cell infiltrates with respect to ADR-rats on saline. In addition, treatment with repeated injections of MSC limited early podocyte depletion, as indicated by the significantly higher number of podocytes per glomerulus in the group of ADR-treated rats receiving MSC as compared to saline. MSC displayed a marked anti-apoptotic effect as indicated by the lower number of apoptotic podocytes in renal tissue of MSC-treated rats with respect to ADR-rats on saline at the corresponding times. Consistent with the ability of infused MSC to reduce podocyte dysfunction, MSC-treatment also significantly limited the presence of glomerular podocyte-APEMP bridges. Specifically, in ADR rats receiving MSC the percentage of glomeruli with area occupied by >50% adhesion of the tuft to the Bowman’s capsule was significantly lower than that observed in rats given saline. In control animals, no glomeruli with adhesions more than 50% were found. Following MSC therapy, the distribution of APEMP was restored along the Bowman’s capsule to a pattern similar to controls. MSC treatment decreased the extension of sclerotic lesions at day 30 as indicated by a significant reduction of the glomerulosclerosis index compared to ADR-rats receiving saline.
A further mechanism of protection was identified in mice models of cisplatin-induced Acute kidney injury (AKI). Oxidative damage caused by reactive oxygen species has been implicated in the pathogenesis of cisplatin-induced renal failure. Peroxynitrite, the reaction product of nitric oxide with superoxide anion, has been taken as key oxidant species involved in the direct nitration of tyrosine residues causing protein oxidation. In cisplatin-mice with AKI, the expression of nitrotyrosine was significantly increased at 4 days in tubules of both the cortex and the medulla in respect to control mice. A marked reduction of nitrotyrosine staining at tubular levels was found when cisplatin-mice were treated with human MSC, indicating that human stem cells protect renal tissues from oxidative damage. Since in vitro and in vivo models of cisplatin–induced toxicity have shown that apoptosis represents one of the major causes of tubular cell loss, we studied whether human MSC treatment could exert an anti-apoptotic action on renal cells in mice with AKI. A significant increase in the number of apoptotic cells was observed in renal tissue of mice treated with cisplatin with respect to control mice. Human MSC infusion resulted in a significant reduction of apoptotic cells as compared to cisplatin-mice given saline, thereby suggesting a pro-survival effect of the stem cell therapy in mice with AKI. Tubular cell proliferation as an index of renal regeneration in the context of acute renal failure was also studied. Tubular cell proliferation slightly increased in cisplatin-mice given saline as compared to controls, indicating a spontaneous weak induction of the reparative process. Importantly, the treatment with human MSC strongly induced cell division, thus strengthening the meaning of a powerful induction of tissue recovery.
Taken together, these results suggest that MSC exert their beneficial effects through different mechanisms that involve, paracrine and anti-inflammatory effects, but do not exhibit the capacity to differentiate into renal tubular or glomerular cells. By contrast, APEMP could differentiate into tubular epithelial cells, as well as into glomerular epithelial cells. Once injected in SCID mice affected by ADR-induced nephropathy or glycerol-induced acute kidney injury, APEMP displayed the capacity to engraft within the kidney, generated novel podocytes as well as tubular cells and significantly improved renal function, a property that was not shared by other cell types of the adult kidney. Blocking APEMP engraftment through anti-CXCR4 and anti-CXCR7 antibodies, completely abolished APEMP-mediated beneficial effects on kidney function, suggesting that cell therapy using APEMP may help induce regeneration of injured resident renal cells through differentiation into podocytes or tubular cells. In addition, we analysed the mechanisms that regulate the growth and differentiation of APEMP, and we demonstrated that a regulated activation of the Notch pathway in APEMP during their phases of differentiation toward the podocyte lineage is critical to balance injury and regeneration in glomerular disorders and may be modulated through inhibitors of the Notch pathway. Taken together, these results suggest that MSC may represent a good tool for cell therapy of renal injury because of their paracrine and anti-inflammatory effects, while APEMP growth and differentiation may be pahrmacologically modulated to enhance the endogenous regenerative capacities of the kidney. These results have important implications for treatment of patients affected by acute or chronic renal disorders.
2 Evaluation of the potential risk of stem cell maldifferentiation.
We planned initially to follow-up our MSC-treated remnant kidney rats for histological signs of maldifferentiated MSC, a phenomenon we first described in 2007. In 5/6 nephrectomized rats that had received intraarterial MSC injection, kidneys at timepoint of kill exhibited frequent intraglomerular accumulation of finely dispersed lipids, mostly in the mesangium, that did not resemble the large lipid vacuoles that we observed in 2007 after intrarenal MSC injection. Rather, these lipid accumulations occurred in non-MSC treated control kidneys as well and thus cannot be linked to MSC treatment. The lipids were so ubiquitious that additional underlying rare “true maldifferentiation” could not be distinguished in light microscopy. In retrospective, it could be speculated whether our observation of intraglomerular maldifferentiation (exclusively observed in MSC treated rats with chronic Thy1 nephritis) in 2007 was caused by intraglomerular MSC that had undergone senescence due to the CKD environment, thus possibly explaining why no further maldifferentiation was reported by other groups who mainly inject MSC in acute renal failure, which does not cause CKD.
It has been known that a high inflammatory environment, acute or chronic, promotes adipogenic differentiation from MSC, confirmed by the fact that inflammatory states are associated with bone loss. As uremia implicates chronic inflammation, increased adipogenesis in our CKD-MSC seems well in line.
Impairment of stem/progenitor cells during pathological as well as physiological (aging) conditions has been acknowledged before). Senescence means that dividing cells upon extended cell culture passaging ultimately start to enlarge, become more granular, show reduced proliferation until they ultimately stop to divide although remaining metabolically active. They can be maintained in this state for years. Senescence occurs in vivo as well and increases with aging, it is considered a tumor-suppressing mechanism. We conclude that theoretically, all conditions that increase DNA damage (e.g. reactive oxygen species) can lead to stem/progenitor cell senescence and thus to increased spontaneous adipogenesis in MSC.
In the kidneys of cisplatin-treated mice infused with CB-MSCs, no sign of maldifferentiation of human CB-MSCs into other cell types of non-renal origin or adipocytes was observed in animals which survived 40 days after cell injection.
In summary, our current data suggest progressive CKD to be a risk factor for unwanted MSC differentiation events, be it osteogenic (Kramann et al. ATVB 2011) or adipogenic maldifferentiation.
Human renal Progenitors (APEMP) APEMP displayed the potential to generate tubular cells when injected into SCID mice models of acute tubular necrosis induced by glycerol injection. In these models differentiation toward glomerular cells or extrarenal cell types was not observed. Nevertheless, when injected into SCID mice affected by adriamycin nephropathy, which displayed tubular as well as podocyte injury, APEMP generated novel tubular cells as well as novel podocytes, as demonstrated through different methods: (i)l abelling before injection of different types of stem cells with the red fluorescent dye PKH26 (ii) evaluation of the presence of the Y chromosome by using fluorescence in situ hybridization (FISH) following injection of stem cells derived from male subjects in female mice (iii) staining by immunohistochemistry of the HLA-I human antigen performed on kidney sections.
However, abnormal regenerative responses of APEMP may occur during chronic inflammatory processes. Indeed, using confocal microscopy, laser capture microdissection, and real-time quantitative reverse transcriptase–PCR we also demonstrated that hypercellular lesions of different podocytopathies and crescentic glomerulonephritis consist of these three distinct populations: CD133+CD24+PDX- or nestin- (renal progenitors), CD133+CD24+PDX+ or nestin+ (transitional cells), and CD133-CD24-PDX+ or nestin+ (differentiated podocytes). These results suggest that glomerular hyperplastic lesions derive from the proliferation of renal progenitors at different stages of their differentiation toward mature podocytes, providing an explanation for the pathogenesis of hyperplastic lesions in podocytopathies and crescentic glomerulonephritis.
3 Prevention of maldifferentiation by particular experimental conditions, in vitro pretreatment, or in vivo pharmacological interventions
The importance of reactive oxygen species (ROS) in death of grafted cells has been supported by studies showing that prevention of oxidative stress through pharmacological approaches or genetic modifications allowed increasing MSCs survival after graft. At present, the intracellular sources of ROS and their involvement in MSCs death have not been clearly identified. We have shown that the generation of H2O2 in MSC after a treatment with serotonin, 5-HT (through its monoamine oxidase-dependant degradation) leads to an apoptosis of these cells. These results indicate that the exposure of MSCs to en environment susceptible of producing ROS may be deleterious for the efficiency of the cell therapy (Trouche E and al. Stem Cells Dev. 2010 Oct;19(10):1571-8). In order to improve cell survival after intraparenchymal graft, we have developed a pharmacological strategy based on melatonin pre-treatment. Exposure of MSCs to this pineal hormone induced an overexpression of anti-oxidant defences such as catalase and superoxide dismutase (SOD-1). This effect is associated with an improvement in the functional recovery after a renal injury and an increase in grafted-cells survival (Mias C and al. Stem Cells. 2008 Jul;26(7):1749-57).
4 Evaluation of Tumorigenicity.
We evaluate the tumorigenicity of human APEMP preparations by injection in NOD/SCID mice subcutaneously and observed no tumorigenic potential.
We also evaluated the tumorigenicity of human MSC preparations by injection in NOD/SCID mice subcutaneously and follow potential tumour growth for 4 months.
During the course of the project data was obtained showing that simple subcutaneous injection of MSCs in mice leads to scattering of the injected MSCs throughout the mice. Using luciferase transfected MSCs in numbers equalling those of cell numbers used in clinical trial and 10x this amount, we observed that one week after injection MSCs could not be traced back. These findings prompted us to initiate studies on the use of MSCs encapsulated in matrigel to analyse whether encapsulated MSCs placed subcutaneously would persist long enough to reliably exclude tumorigenicity. So far we have tested in matrigel encapsulated luciferase transfected mouse MSC’s in a syngeneic mouse model. MSC’s were traceable up to 8 weeks after injection without signs of tumorigenicity.
To establish the magnitude of the problems that might arise using the clinical products we performed karyotyping on MSC preparations prepared for clinical use. In over 50 MSC preparations derived from healthy individuals karyotypic abnormalities have not been observed. To avoid HLA-reactivity the use of autologous MSCs is preferable in the setting of kidney transplantation. When analysed bone-marrow derived MSC preparations from end stage renal disease (ESRD) patients show similar expansion potential and immunosuppressive potential compared to MSC preparations from healthy individuals. However, karyotypic anomalies were observed in 2 out of 12 expanded MSC preparations of ESRD patients. MSC preparations showing abnormalities in karyotype were omitted and not used in clinical trials. This observation requires further investigation.
Future research will focus on the tumorigenic potential of MSC’s derived from ESRD patients. The aim is to prepare encapsulate MSCs from these patients in matrigel and test these in NOC/SCID mouse models.
5 Evaluation of immunogenicity
When rats were treated with acute Thy1.1 nephritis with intrarenal injection of MSC preparations, it was assessed all kidneys for consecutive influx of monocytes/macrophages. It was not observed significant influx of inflammatory cells in the treated vs. control kidneys, although MSC secrete many cytokines that induce chemotaxis. One has to keep in mind that injured intrinsic renal cells express many inflammatory chemokines themselves providing a strong “background” signal. Interestingly, even kidneys that were treated with TG-MSC did not show increased inflammatory cell influx although the TG-MSC carry the human transgene on the outer cell membrane.
Pre-transplant infusion of syngeneic MSC significantly prolongs graft survival in a murine kidney transplant model.
We set up a severe and clinically relevant kidney transplant model. Recipient C57 mice (H-2b) were sensitized toward donor antigens by the infusion of donor Balb/c splenocytes (H-2d, 1x106 i.v. seven days before kidney transplantation). Transplantation of Balb/c kidneys into sensitized mice (group 1) resulted in a rapid increase in BUN levels (and acute graft rejection within 10 days in all transplanted mice. We next assessed the effect of pre-transplant MSC infusion in prolonging kidney graft survival. To this purpose donor-sensitized C57 recipient mice were given syngeneic MSC infusion (0.5x106 i.v.). Mice of group 2 and 3 received a single pre-transplant MSC infusion at day -1 (n=5) and at day -7 (n=5) respectively, and mice of group 4 received double pre-transplant MSC infusion at days -7 and -1 (n=5). All mice received a Balb/c kidney transplant at day 0.
A single (either day -7 or day -1) and double (at day -7 and at day -1) pre-transplant infusion of MSC significantly prolonged kidney graft survival as compared with untreated transplanted. In mice from groups 2, 3 and 4 surviving more than 20 days post-transplant, kidney graft function was well preserved during the 60 day-follow up.
Thus, pre-transplant syngeneic MSC represents a promising cell-based immunotherapy in solid organ transplantation.
We previously described in adult kidneys that the SCA+ Lin- cells represent in mice a mesenchymal renal residing stem cell. The second approach indicated already in 2004 in a study detailed in Nature Medicine is based on our ability to define optimal 'window'' of gestation ideally suited for harvest of embryonic pig or human embryonic tissue for transplantation. A major question related to these two stem cell sources is how immunogenic they are and how much immune suppression is required to enable long term engraftment of these cells in allogeneic recipients. Thus we found at the first part of the grant period that SCA+ Lin- mesenchymal stem cells although able to some degree to suppress immune response; they still exhibited significant immunogenicity upon transplantation into allogeneic recipients, inducing memory T cells that would reject promptly a second infusion of fibroblasts from the same donor. Thus, the prevailing arguments that MSC can evade the immune system and therefore it might be possible to use 'off-the-shelve' MSC for renal repair without immune suppression altogether, is highly unlikely in our opinion.
Unfortunately, we have reached similar conclusions also regarding the use of embryonic precursor tissues that are grown and develop within the recipients post transplant. Although they are capable to be predominantly vascularised by host blood vessels they can still be rejected through the indirect pathway (Cross priming). Thus for each source we might need some level of immune suppression if transplanted in allogeneic or in xenogeneic recipients. Otherwise, the best option will ber to use MSC from autologous sources, which is feasible in patients with kidney disorders, where the bone marrow is usually healthy.
WP5 Data Management and readouts to set up protocols for phase I/II clinical trials in humans
1 To set up protocols in view of phase I/II clinical trials in humans following standardization of results obtained from preclinical models, choosing the best type of stem cell for cell therapy of distinct clinical renal disorders.
A shared protocol for Good Manufacturing Practice (GMP)-expansion of human MSC preparation and use of human MSC was discussed among all the partners of the Consortium and developed in direct interaction with the European Group for Blood and Marrow Transplantation (EBMT)-MSC expansion consortium. and is reported below:
Set up of human MSC cultures
All the steps are carried out in circuit closed in a laminar flow hood located in a clean room of classroom ISO7. Cross contamination is prevented by separation of production rooms, one-way routing and double-layer clothing.
1. Bone marrow collection:
a) Bone marrow cells are obtained by suction of the medullar cavity of a flat bone (illium).
b) A numeration of the cellular starting suspension will be carried out by an automat of haematology before any manipulation.
c) 2–5 ml heparinized samples are separated by Ficoll-Hypaque gradients centrifugation (Lympholyte-H, Cedarlane, Ontario, Canada) to yield mononuclear cells. In the case of bag washouts, total nucleated cells from filter and bag residues are obtained after two washings with 250 ml saline and centrifugation at 2000 r.p.m. for 5 min.
d) The viability will be evaluated by the percentage of negative cells after staining by blue trypan.
e) A bacteriologic analysis will be carried out by sowing 0,5 ml of the suspension of bone marrow in each of 2 bottles of hemoculture (aerobic and anaerobic). Lastly, an evaluation of the CFU-F will be carried out (5x105 cells/25 cm² flask in complete medium).
2. Culture set-up:
a) The cells are put in culture at the concentration of 2x105 cells/cm2
b) Non-adherent cells are removed after 3–4 days and fresh medium is added.
• Medium: DMEM low glucose medium (Gibco Invitrogen,Carlbad, CA, USA) containing gentamicin 0.1mM (Mayne Pharma, Napoli, Italy) and heparin 1000 IU (Pfizer Italia, Latina, Italy) in the presence of 10% FBS (selected batch conform the EBMT MSC Expansion Consortium).
• Equipment to be used: Container of culture: T175 flaskor one floor culture chambers (surface area 636 cm2: CellSTACKTM, Corning) identified with the code of the donor, the date of culture initiation. To put in the incubator in wet atmosphere with 37°C with CO2 5%.
3. Medium exchange:
a) The medium is changed 2 times per week until the ending of the culture. The cell growth is appreciated by observation of the lower plateau under a microscope. The steps where the cellular layer is not covered any more with medium must be shortest as possible in order to avoid damaging the cells by desiccation.
b) Medium: DMEM low glucose medium (Gibco Invitrogen,Carlbad, CA, USA) containing gentamicin 0.1mM (Mayne Pharma, Napoli, Italy) and heparin 1000 IU (Pfizer Italia, Latina, Italy) in the presence of 10% FBS (selected batch conform the EBMT MSC Expansion Consortium).
4. Cell detachment and washing:
a) At subconfluency, cells are recovered after Trypsin-EDTA (StemCell Technologies, Vancouver, British Columbia, Canada) treatment and subsequently replated at approximately 3000 cells/cm2 in the same medium.
5 Products quality controls and release criteria
The final cell products undergo all the established quality control tests that are planned to adhere to specific criteria, including:
• Viability >80% measured by 7-amino-actinomycin D (7-AAD) vital dye staining and FACS analysis on a FacsScan (Becton Dickinson, San Jose`, CA, USA), according to standard procedure assays.
• Sterility of final product and culture supernatant performed by direct inoculation of at least 1% of the product or 5ml of pooled culture supernatant in BactAlert FA and FN media and automated measurement (Biomerieux, Marcy l’Etoile, France). Adequacy was reached when all cultures scored negative after a cultureof approximately 14 days (we use 7 days)
• Endotoxin content performed by the Limulus amebocyte lysate (LAL) test according to European Pharmacopeia (PBI S.p.A. Milano, Italy). Adequacy o2.5 EU/ml. This value is strictly dependent on the final volume of the product to be administrated, and therefore is subject to change according to the application.
• Mycoplasma contamination, detected with the MycoAlert test (Cambrex Corporation, Verviers, Belgium). Adequacy: no contamination detected.
• FACS analysis: hMSCs are detached with Trypsin-EDTA, washed and stained with the following conjugated monoclonal antibodies: CD45-FITC, CD34-PE, CD14-FITC, HLA-DRFITC, CD29-PE, CD90-FITC, CD73-PE, HLA-ABC-PE, CD105-PE or isotype-matched IgG-FITC and IgG-PE control antibodies. CD90 expression should be >80%, CD105 >80%, CD45 <10%.
• Genetic stability: We analyze the kariotype of the patient before the expansion, and the kariotype of the cells at the end of the culture. No genetic anomalies are detected after cell expansion.
• BSA: since FBS is used during expansion, we test BSA content in the final product using ELISA. Our release criterium is BSA < 75 EU/ml but, as for endotoxins, this value is dependent on the final volume of the product and it is subject to change according to the application (systemic or local delivery of the cells).
After incubation, cells are washed, resuspended in PBS and then analyzed by standard flow cytometry on a FacsScan following standard procedures.
Localization of infused MSC into the graft are associated with transient acute renal insufficiency in kidney transplant patients
We recently investigated safety and feasibility of MSC infusion in two living-related donor kidney recipients who were given ex-vivo expanded, autologous, bone marrow derived-MSC at day 7 post-transplant, after induction therapy with Basiliximab/low dose thymoglobulin. Few days after cell infusion, both MSC-treated patients developed acute renal insufficiency and we were able to perform a kidney biopsy of patient 2. Histologic and immunohistochemic analysis of graft infiltrating cells did exclude an acute cellular or humoral rejection. Indeed intragraft CD4+, CD8+ T cells, CD14+ monocytes, CD20+ B cells and CD68+ macrophages were very low as compared with those in control kidney graft biopsies from patients given the same immunosuppression who experienced acute cellular-rejection. We found a high number of granulocytes in the peritubular inflammatory infiltrate and increased complement C3 deposition in patient 2 than in grafts with early acute cellular rejection. MSC, identified as CD105-CD44 double positive cells, were found in the graft interstitium of patient 2, but not in naïve untransplanted kidneys nor in a renal graft with acute cellular-rejection.
Thus, autologous MSC given post-transplant in kidney transplant patients was associated with transient renal insufficiency associated with intragraft recruitment of neutrophils and complement C3 deposition. Despite the well known anti-inflammatory properties of MSCs, the various soluble factors produced by MSCs also include proinflammatory mediators, which eventually may have contributed to the intragraft recruitment of granulocytes and slow progressive deterioration of renal function. The safety concern of posttransplant MSCs anticipated the need to modify the study protocol moving cell infusion pretransplant, an approach being effective in experimental models of solid organ transplantation. We would also like to underline that after one year of follow-up the two patients are in good health and exhibit a good kidney function.
Preparation of a risk analysis table for the cellular expansion according to GMP guidelines
Risk management is an important aspect of the GMP pharmaceutical production, and its application is even more critical for products such as cell-based therapies that cannot be terminally sterilized after manufacturing.
Moreover, patient-specific cell therapies often start from a sample coming from a donor (the patient itself or an allogeneic source) that can transport viral or bacterial contamination. In a production facility where several batches of cells are manufactured at the same time, several other procedures must be put in place in order to prevent cross-contamination or sample mix-ups.
In order to facilitate the management and execution of clinical-grade expansions of the different cell populations identified in the project, we have performed a risk analysis of all the steps involved in the production process of a typical cell-based medicinal product, generated from a donor.starting sample.
The main productions steps were divided as follows:
1) Receipt and control of incoming sample
2) Sample handling during admission to the GMP facility
3) Sample handling during cellular expansion
4) Preparation and labelling of the finished product
For each of these passages we have identified the possible risks and proposed solutions that could decrease the chance of deviations and non-conformities, as well as mitigating the damage caused by unexpected events such as contaminations and leakages.
The table below summarizes all the aspects analyzed and the related risks and solutions:
Step / material Risk for the product Solution(s) Risk for operator/facility Solution(s)
1) Incoming patient sample
• Vial is broken
• Loss of traceability
• Check sample at receipt and register material integrity
• Check conformity on documents
• Vial is broken
• Contamination of facility
• Wear gloves
• Verify presence of Certificate of Analisys testing the absence of viruses
2) Sample handling during admission to the GMP facility
• Contamination during manipulation steps
• Cross-contaminations
• Work in GMP facility, perform environmental monitoring, train operators
• Use a dedicated incubator for each batch; clean surfaces after each manipulation
• Accidental contact with sample
• Leakage during storage at 4°C (quarantine)
• Wear gloves and general laboratory protection equipment; expose the product only under biohazard cabinet
• Keep sample in a sealed plastic bag inside the refrigerator
3) Sample handling during cellular expansion
• Contamination during manipulation steps
• Mycoplasma contamination
• Bacterial contamination
• Incubation at suboptimal temperature
• Work in GMP facility, perform environmental monitoring, train operators
• Check Mycoplasma contamination once a week
• Check medium for turbidity, changes in color, presence of molds; in case of contamination, isolate and identify microorganism and activate dedicated cleaning procedure
• Keep incubators under regular calibration schedule; keep incubators under remote alarm system
• Leakage of flask / bioreactor
• Bacterial contamination spread by sample
• Dispose of broken flask, clean biohazard cabinet or incubator with suitable detergent; sterilize incubator
• Identify microorganism and activate dedicated cleaning procedures
4) Preparation of the finished product
• Loss of traceability
• Wrong transport temperature to the clinic
• Labeling procedure is performed immediately after final filling. Procedure is monitored by QC personnel. A dedicated labelling form is filled during the operation and a copy of the label is kept together with the Batch Production Record.
• Use validated transport box; include datalogger in the package
WP6 Dissemination and Training
1 Dissemination of results
Web site: a project dedicated web site has been created and updated, in accordance to the project participants’ needs, in particular with the period reports provided to the EC and with the material prepared for the project meetings.
Publications: the project results have been described in several publication on peer reviewed journal with very high impact.
Meetings and conferences: PI, experienced researchers and young and experienced scientists involved in STAR-T REK project participated to national and international scientific courses, workshop and congresses. These events were important opportunities for the dissemination of project results and also useful tools of training for the young scientists.
2 Training activities will be substantially the following:
Exchange of key staff members from one lab to another (short-term fellowships) in order to enhance cohesion and implementation of project activities, to share skills, expertise and reagents and to standardise techniques used in the different laboratories.
Dr. Ilya Skovorodkin from Prof. Seppo Vainio laboratory has visited Dr. Paola Romagnani laboratory for a week during 2010.
During the Final meeting held in Milan, at Istituto di Ricerche Farmacologiche Mario Negri, a round table for young scientists involved in the project was organized together with the visit to the labs. The young scientists, that were the one involved in the experimental part of the project and were the main actors of the experiments.
WP7 Project management
The management activities have been conducted aiming at the successful progress of the project within the agreed time, cost and quality limits as defined by the project contract signed with the EU and the Consortium Agreement signed among the beneficiaries. The coordinating team worked also to establish effective communication among the consortium beneficiaries, to guarantee appropriate administrative and scientific management of the project and correct dealing of ethical issues and to maintain a good flow of information and data.
1 Set up of project organizational structure to establish a common baseline for task and responsibility allocation. The project responsibility were allocated at the beginning of the project.
Project coordinator- general coordination of the project
Non scientific Management – coordination of the non-scientific activities of the project (meeting, training, dissemination, daily management of the project, amendments, guidance on FP7 rules and duties)
List of WP leaders –for the coordination and supervision of the scientific activities.
2 Administrative Issues Management to ensure on-time provision of periodic management reports, and cost statements. Handle the EC project reviews, payment issues and Amendments to the GA. ALTA supported the project coordinator in the organization of the non-scientific activities: meetings; amendments to the consortium agreement; interim reports; in the payments (collecting the bank account, checking the distribution of the money); setting up and management of the web site; preparation and coordination of the EC project reporting.
Tracking: to guarantee control, validation and verification of project results, to ensure that plans are fulfilled and implement necessary corrective actions. The scientific coordinator constantly checked the work of each beneficiary and intervened in to solve problems causing delays.
Task 4. Intellectual property management
Achievements: A Consortium Agreement was prepared to define consortium members’ rights and duties. The final draft has been signed by all the project participants.
For protecting and safeguard the IPR of the participants, the abstract of the manuscripts, posters and of any dissemination document, together with sufficient information concerning the planned dissemination activity and the envisaged data of dissemination, were circulated within the consortium before the disseminating action. Any objection to the planned activity would have been made in accordance with the EC-GA in writing to the Coordinator and to any Party concerned within 30 days after receipt of the notice. If no objection was made within the time limit stated above, the publication was permitted.
Potential Impact:
The STAR-T REK project has given a fundamental contribution towards the expected impacts listed in the work programme HEALTH-2007-1.4. Innovative therapeutic approaches and intervention. The objectives of the topic were to research, consolidate and ensure further developments in advanced therapies and technologies with broad potential application. The focus of STAR-T REK project was on regenerative medicine. The specific topic of the call addressed was HEALTH-2007-1.4-8: Stem cells for kidney regeneration.
The project was aimed at increasing the developing European capability in regenerative medicine of injured organs in general and of damaged kidneys in particular.
This project has generate many data, new insights into important mechanism of cell based regenerative processes and created synergies among scientists working in the field of kidney repair for developing novel therapeutic strategies based on stem cell therapies for several types of acute and chronic kidney disorders, which affect about 11% of the adult population.
International leader groups joined their resources and efforts for setting up standardized protocols of SC isolation and administration in patients affected by renal failure. The information produced during the project is a very important baseline for further studies and will influence deeply the scientific community towards further improvements and new directions of regenerative medicine and cell therapy.
The novelty of this proposal is that it assesses the regenerative potential of stem cells derived from different sources and investigate the possible obstacles to their utilization, as well as their potential side effects in preclinical models of acute and chronic renal failure. Indeed, the clinical usefulness of the treatment, as well as the need to understand currently discrepant results, need comparative experimental studies that have never been performed before either in SC therapy of kidney injury, or in that of other organs.
The expected outcome of the studies has been the identification of which cell type (between bone marrow derived stem cells, adult renal stem cells fetal renal stem cells) is better suitable for beneficial effect in preclinical models of acute and chronic renal failure, and whether these effects are mediated directly by the transplanted cells or indirectly through involvement of other cell types.
In summary, the main project outcomes are the following:
- identification of which cell type is better suitable for beneficial effect in preclinical models of acute and chronic renal failure;
- assessment whether the beneficial (or not) effects are mediated directly by the transplanted cells or indirectly through involvement of other cell types;
- insights on the mechanisms of stem cells-mediated regenerative effects, which are essential to set up cell therapies that should be effective and safe;
- novel concept that renal re generation can be achieved through paharmacological modulation of renal stem cell growth and differentiation;
- possibility of tissue bioengineering a de novo replacement organ using fetal renal stem cells or other strategies of kidney tissue engineering, with an ultimate aim of being able to implant functional renal replacements in patients with end stage renal disease.
The contribution of the project to each of these single aims was critical. The first most direct impact of our work is the development of a method of engineering foetal kidneys from simple suspensions of foetal kidney stem cells. For the rest of the Star-t-rek group, this has provided a test system for assessing the abilities of different types of stem cells to contribute to kidneys. More broadly, the system suggests a means of practical tissue engineering for a range of tissues, and it has already been taken up by other researchers. The eventual societal impact, if all goes well, will be a practical clinical technique. The work has been presented at a number of Public Understanding events and has been covered by international newspapers, confirming that its potential impact is seen by public commentators and not just by the Star-t-rek researchers involved. This project finally lead to clarify that MSC can represent a helpful tool for treatment of acute kidney injury, although their main mechanisms of their beneficial effect is mostly related to their capacity to secrete benficial factors and to reduce inflammatory processes. We also did not observe tumor formation in our preclinical models of acute kidney injury, and we however observed that these cells can be immunogenic and thus their administration would probably require some immunosuppressive treatment. Based on the standardized protocol for human MSC preparation and injection that was set-up during the project, we also started a phase I/II clinical trial in patients who underwent kidney transplantation. We demonstrated for the first time that MSC can be safely administered to kidney-transplanted patients. We observed a mild, transient acute renal failure related to MSC administration about 20 days after their administration, which anticipated the need to modify the study protocol moving cell infusion pretransplant, an approach being effective in experimental models of solid organ transplantation. However, after long term (one year) follow-up, patients are in good health and exhibit a normal renal function, suggesting that MSC may indeed represent a source for induction of tolerogenic responses in kidney transplanted-patients. Thus, the STAR-TREK project has lead critical contribution to our knowledge of safety and clinical utility of cell therapy using MSC. Finally, the STAR-TREK Project dramaticaaly increased our knowledge of the regenerative capacities of the kidney. Indeed, a renal progenitor system was identified and largely characterized by the partners during the course of this project, that lead to the definition of a novel concept in kidney biology, the one of the existence of a “renopoietic system”. The discovery that adult kidney can be potentially regenerated by an endogenous progenitor system changes our view of kidney biology. Indeed, during the course of the Project we also demonstrated that the regenerative capacity of the kidney can be enhanced through pharmacological agents, which opens a novel perspective for innovative therapeutic tools for patients affected by kidney disorders. We thus believe that STAR-T REK will indirectly contribute to the establishment of Europe as a major player in the regenerative medicine of injured organs and tissue and in the set up of novel therapeutic strategies for treatement of acute and chronic kidney diseases due to the main and important data that generated.