Identification, homing and monitoring of therapeutic cells for regenerative medicine – Identify, Enrich, Accelerate | IDEA | Project | News & Multimedia | FP7 | CORDIS

Final Report Summary - IDEA (Identification, homing and monitoring of therapeutic cells for regenerative medicine – Identify, Enrich, Accelerate)

Executive Summary:
IDEA, “Identification, Enrichment and Acceleration”, is a multidisciplinary collaborative project aiming to develop new tools for regenerative medicine.
Establishing regenerative medicine in the clinic is currently hampered by several limitations that are associated with the complex process of the therapeutic intervention characterizing this innovative medical approach: harvesting of progenitor cells from donors, identify, separate and clone them under GMP condition, transfer them to the recipient and monitor the therapeutic and potential side effects.
During the IDEA project new tools for regenerative medicine were developed addressing current needs: identification, homing and monitoring of therapeutic cells. Specifically, a new photonic method that allow a contact and marker-free identification and selection of cells prior transplantation, devices and tracer for homing, capturing and monitoring of such cells in vivo and a new imaging platform to guide therapeutic interventions of regenerative medicine. The latter is a prerequisite to any meaningful performance of clinical studies.
To achieve the ambiguous aims of the IDEA project the consortium brought together academic, clinical and industrial expertise to comprehensively tackle the complex parameters required for development and evaluation of new tools, technologies and devices.

The results generated during the IDEA project will contribute substantially to pave the way towards meaningful new therapeutic procedures in regenerative medicine and will take regenerative medicine to the next level of testing in clinical studies.

Project Context and Objectives:

Project Context

Regenerative medicine is the process of creating living, functional tissues to repair or replace tissue or organ function lost due to damage, or congenital defects. It refers to a group of biomedical approaches to clinical therapies that may involve the use of stem cells or progenitor cells. However, it is unknown what kind of cell has the appropriate healing capacity for repairing damaged tissue.

Stem cells are cells found in all multicellular organisms. They are characterized by the ability to renew themselves through mitotic cell division and differentiation into a diverse range of specialized cell types. Research in the stem cell field grew out of findings by Ernest A. McCulloch and James E. Till at the University of Toronto in the 1960s.

The two broad types of mammalian stem cells are: embryonic stem cells (ESCs) that are isolated from the inner cell mass of blastocysts and adult stem cells (ASCs) that are found in different tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenished in adult tissues. In a developing embryo, stem cells can differentiate into all of the specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues. The most important difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, whereas progenitor cells can only divide a limited number of times. Controversy about the exact definition remains and the concept is still evolving.

Stem cells can be grown and transformed into specialized cell types with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture. Highly plastic adult stem cells, so called mesenchymal stem cells (MSCs), can be generated from a variety of sources, including umbilical cord blood and bone arrow. They are routinely used in medical therapies. Embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies. In 2006 Yamanaka and co-workers surprised the scientific community when they demonstrated that both mouse embryonic fibroblasts and tail tip fibroblasts could be reprogrammed into a pluripotent state similar to that observed in ESCs. This was achieved by the retroviral transduction of Oct4, Sox2, Klf4, and c-Myc genes. These reprogrammed cells were named induced pluripotent stem cells (iPSCs). An ongoing debate is the identification of the most optimal cell type. The advantage of using human ESCs is their ability to self-renew and to differentiate into any cell; thus, these cells can potentially regenerate the entire organ. Their disadvantage is a potential for immunologic rejection and need for immunosuppressive therapy. Use of cells from embryos has also met with several regulatory hurdles and ethical concerns. Only recently has the FDA approved the first phase 1 clinical trial using human ESCs for the neural regeneration in patients with severe spinal cord injury.

Adult progenitor cells, on the other hand, are limited to differentiating into cell types from the tissue of origin. Adult progenitor cells are rare in mature organs and can be difficult to expand in culture. However, they are considered less immunogenic than human ESCs and their therapeutic application has been less controversial. Thus, the majority of clinical trials have used adult progenitor cells. Results, however, have been mixed, leading many to question their regeneration potential and ability to differentiate, survive and engraft. The establishment of tissue and stem cell banks worldwide does and will make the embryonic and foetal stem cell lines available to all researchers so the creation of new stem cell lines will not be necessary. Another option with less controversial ethical issues is the use of adult neural stem/progenitor cells. Although these are a very appealing alternative, they have proven to be difficult to isolate and expand, and their differentiation potential is more restricted. An alternative, especially for the nervous system, has been shown to be the use of MSCs from umbilical cord, bone marrow and adipose tissue.

A very recent development that can overcome some of the ethical and availability issues, as well as immunosuppression problems is the generation of fibroblast-derived induced pluripotent stem cells (iPSCs) cells by expressing four ‘pluripotentiality’ genes in the cell. The patient will be both the host and donor of fibroblasts which can then be genetically reprogrammed to resemble “stem cells” and create any needed cell type. These cells can be cultured, expanded and differentiated in vitro and transplanted into injured tissue. However, the efficiency of reprogramming and differentiation remains low. Whether these cells truly differentiate into target tissue or retain some features of their tissue of origin has also been questioned. Finally, efficient derivation of iPSCs still requires viral transfection for reprogramming, presenting obstacles for regulatory approval. A clinical trial has not yet been initiated using iPSCs.

Despite the various sources for cells with healing capacities, any one has to be characterized. Differentiation dramatically changes a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are largely due to highly controlled modifications in gene expression and rarely involve changes to the actual genome. Thus, different cell types can have very different characteristics despite having the same DNA sequence. Whether stem cells differentiate in vivo after transplantation (e.g. adult progenitor cells) or in vitro prior to transplantation (e.g. ESCs and iPSCs), it is important to confirm cell specific differentiation and lineage commitment. If not performed adequately, cells incapable of providing the appropriate function will be given to patients. For example, although early attempts to derive β-islet cells directly from ESCs appeared successful in generating insulin-containing cells in mice with streptozotocin-induced diabetes, further studies revealed that these cells did not produce insulin or respond to glucagon or glucose in vitro. The confirmation of stem cell differentiation into the target cell type can be performed by demonstrating appropriate 1) cell morphology, 2) lineage specific markers, 3) gene expression, and 4) cell function.

Stem cells from virtually every source other than blood-derived hematopoietic stem cells are maintained in tissue culture for some defined period of time. This is necessary to obtain a sufficient number of cells for use in clinical studies involving transplantation. Culturing human stem cells requires the use of formulated liquid media supplemented with growth factors and other chemical substances that promote cellular replication and govern the differentiation of the cultured human stem cells. Since human stem cells are a dynamic, biological entity, failure to standardize procedures for maintaining and expanding cells in culture could result in unintended alterations in the intrinsic properties of the cells. The initial seeding density of the cells, the frequency with which the culture medium is replenished, and the density cells are permitted to achieve before subdividing will all affect the characteristics of human stem cells maintained in culture. Altering the concentrations of supplemental growth factors and chemical substances, even switching from one supplier to another, may lead to changes in cell growth rate, expression of defining cell markers, and differentiation potential. Alterations in stem cell properties caused by the use of non-standardized culture practices are likely to affect the behavior and effectiveness of the cells once transplanted.

Objectives of the project

The scientific and technological objectives of IDEA are encouraged by the established clinical practice for hematopoietic stem cell transplantation in patients with life-threatening malignant and non-malignant diseases. An estimated 45,000 to 50,000 hematopoietic cell transplants (bone marrow, PBSC, or cord blood transplants) are performed annually worldwide and has become a standard of care for some diseases as well as a newer treatment option for others. Clinical experiences with hematopoietic cell transplantation revealed the needs before expanding cell transplantation beyond the borders of current clinical practice: (1) a better identification of appropriate cells with healing potential, (2) more efficient homing of these cells to the injured tissue, and (3) a meaningful non invasive monitoring of the therapeutic effects.

IDEA established a multidisciplinary approach that combined academic, clinical and industrial expertise to comprehensively tackle the complex parameters required for development and evaluation of new tools, technologies and devices for regenerative medicine. To achieve this ambitious aim the project has been developed in three parallel R&D-sections and three cross-over sections that supported the others.

One R&D objective of IDEA focused on the development and testing of photonic methods that allowed a contact and marker-free identification and selection of cells with healing capacity for transplantation into animal models of altered vascular, musculoskeletal and neuronal function. The second R&D objective was dedicated to developing a magnetic cell select devices in conjunction with specific magnetic nanoparticles that capture and transport cells with healing potential through the circulatory system to damaged tissue and organs improving homing and elimination of unnecessary cells to reduce patient risk. The third R&D objective addressed non invasive monitoring of the clinical interventions in regenerative medicine. New tracer and imaging technologies were utilized to measure the therapeutic effects by showing anatomic structure applying state of the art Magnetic Resonance Imaging (MRI) and developing Magnetic Particle Imaging (MPI) as new tool to localize and visualize cellular function in vivo.

The cross-over sections supported all others. One of these sections addressed regulatory affairs, guaranteed scientific coordination and quality assurance to realize economic goals. Another cross-over section was dedicated to public affairs including dissemination and exploitation, ethical issues and intellectual properties. Finally one of the cross-over sections has been set up to perform best management practice.

Project Results:
The main science and technologic results/foreground of the IDEA project are descripted below. In a first section key findings were ordered by the work packages of IDEA (WP). In a second section major achievements were given for the IDEA consortium partners.

First section: Key findings ordered by IDEA Work Packages (WP)

WP 1: The IDEA consortium used state of the art methods to characterize potential cell types with healing capacity during the course of harvesting, expanding, and transplantation. The standardized practices and procedures between all IDEA partners enabled them to identify with high certainty the most optimal cell type for transplantation.

WP 2: The IDEA consortium developed a non-contact and marker-free cell analyzing and sorting system based on pure photonic technologies for identification and separation of stem/progenitor cells and subsequent cloning and transplantation of these cells in animal models of vascular injury, Alzheimer’s and Parkinson’s disease.

WP 3: IDEA provided prototypes of magnetic cell select devices that allow a better homing of therapeutic cells in damaged tissue and help to reduce the potential hazard of non-engrafted cells through their elimination. Preclinical testing has been performed and exploratory clinical applications evaluated after initiated, conducted and accomplished medical device registration (MDR).

WP 4: IDEA produced and tested specific iron oxide nanoparticles for the delivery of stem cells and for subsequent monitoring in vivo. The IDEA consortium knows initial toxicology data and the pharmacokinetic profile of these particles and after supplementary preclinical studies scale up under GMP standards could be initiated for an investigational new drug application (IND) and submitted to regulatory authorities in the near future.

WP 5: IDEA performed cell transplantation in established animal models of vascular injury, Alzheimer’s and Parkinson’s disease using different types of stem/progenitor cells. Applying Raman micro-spectroscopy, cell select devices and magnetic particles for homing and noninvasive monitoring of the therapeutic intervention could be anticipated to perform exploratory clinical trials after the project period of the IDEA program.

WP 6: IDEA used dedicated functionalized nanoparticles for MPI allowing evaluation of the location, distribution and long-term viability of cells having specific regenerative potential in a non-invasive and repeatable manner. In addition to MPI, MRI delivered anatomical information. Thus, in this project MPI has been combined with MRI to monitor the therapeutic effects of interventional regenerative medicine. This combined technological innovation delivers an imaging platform for monitoring of regenerative therapy for many of the world's major diseases, such as heart disease, stroke and cancer.

WP 7: IDEA aimed to comply with current regulatory rules and directives. During the lifespan of the IDEA project the Directive 536/2014/EC that regulate clinical trials on medicinal products for human use, became in force since May 2016 and repealing Directive 2001/20/EC. These issues needed implementation into the transitional goals of IDEA to apply for medical device registration, submit an investigational new drug application and path the way for clinical studies in regenerative medicine in the near future.

WP 8: Dissemination, exploitation and ethics in IDEA revealed major scientific publication in reviewed international journals, new tracer for imaging using MRI and MPI, a new marker-free cell analyzing and sorting platform based on Raman spectroscopy and advanced the boarder of current knowledge and know how concerning MPI tremendously. The ethical issues were addressed throughout the project and supervised by an external expert.

WP 9: Best management practice was carried out throughout the IDEA project.

Second section: major scientific achievements ordered by IDEA consortium partners

King’s College London (KCL)

KCL was leading work package (WP) 1, “Source, expand and characterize stem/progenitor cells from various proveniences”.

The first major theme of the work was to identify, characterize and optimize cells suitable for transplantation into disease models. Once these cells had been identified studies were undertaken to determine which of the iron particles being developed by other partners were the most suitable for transfection into the chosen cells. The final part was the transplantation of the cells into a model of a neurodegenerative disease, in this case Alzheimer’s disease.

KCL was fortunate to have access to a cell line (CTX0E03) that had already undergone human trials for stroke (PICES). In the event that these studies proved successful this would have allowed easy translation from preclinical to clinical studies. Early studies have shown that these cells were more resistant than other neural stem cells to toxic insults relevant to Alzheimer’s disease and demonstrated higher levels of expression of trophic factors associated with neurorepair than other cell lines. KCL was also able to establish that CT0XE03 cells would take up the iron oxide particles (M4A) that would be required for identification and homing.

At this stage the cells were ready for testing in vivo in a mouse model of Alzheimer’s disease. The chosen model, 3xTG, had been extensively used in research and in particular had shown benefit in a range of outcomes following transplantation of mouse neural stem cells. Unfortunately, the pathology of this mouse strain proved unreliable and no behavioral changes were identified during and after preliminary transplantation studies. There was some limited evidence that transplantation of CT0XE03 cells increased expression of trophic factors and synaptic markers in the mice. However, as these mice did not meet the criteria required we therefore moved to another model with good evidence of pathology and reliable behavioral changes, 5XFAD. These mice showed clear evidence of Alzheimer’s disease related pathology at 8 months of age but as yet no in the range of behavioral tests applied. Beyond the tenue of the award, the mice were allowed to age for a further 4 months and have been recently culled. Immediately prior to culling the range of behavioral tests was conducted again. On this occasion, there was evidence of deficits in the 5XFAD mice but no impact of cell transplantation. Biochemical and neuropathological assessment of the impact of cell transplantation on the 5XFAD mice will be conducted over the next 3-6 months beyond the lifetime of the IDEA project under our own funding.

CellTool GmbH (CELL)

CELL was leading WP 2 “Develop and test Raman micro-spectroscopy and laser teasers for cell characterization”.

Thanks to the IDEA funding, CELL has developed an innovative Raman-microscope platform - the BioRam® - that can intuitively be handled by biologists and physicians – as easy as a light microscope – to make Raman spectroscopy available for biomedical applications. The inverse Raman microscope is the ideal platform for novel cell analyses, advanced medical diagnosis and quality assurance of cell based products. It provides necessary conditions for direct measurements of native cells, 3D-tissues, grafts and fluids. Due to its sophisticated setup, BioRam® includes an optical trap, which ensures that specimen in solution are captured within the laser focus during Raman analysis - resulting in most reliable spectra even of motile cells. In addition, cells and particles can be moved and positioned - and possibly sorted - using this trapping effect in combination with microfluidic chips.

During the project the project partners learned a lot about the benefits of Raman spectroscopy to identify and analyze biological specimen. Applications for Raman spectroscopy are very versatile. Whenever only small sample amounts are available or problems arise with antibody-based markers, Raman spectroscopy is the tool-of-choice as it enables non-invasive investigation of cells and tissue under physiological conditions. With this, the analyzed cells remain vital and intact for further cultivation and downstream analyses. This is especially important when working with tedious stem cells that easily change their characteristics, or if cells are designated for therapeutic use, as here antibodies, fluorescent labels or magnetic beads are not allowed.

Together with the project partners CELL tested various stem cells in different states and analyzed how cells react on nanoparticles of different fabrication and with various coatings. They also examined the sensitivity of Raman spectroscopy to find out which changes of the cells’ metabolome can be detected.

Important steps were the findings of optimal substrates and optimized cell handling for Raman analysis. Protocols have been developed that allow analysis of even fixed cells - but there are some limitations. To detect small differences between cells or minor changes within the metabolome, Raman analysis should be performed on living cells. Another important step was the successive built-up of expertise for biomedical Raman spectra analysis and meaningful data interpretation, including development of specific algorithms for data evaluation.

During the project CELL has established a professional Service-Lab with an attached cell culture laboratory for system demonstrations, proof-of-concept measurements, research collaboration and contract work. During development of the Raman microscope, several re-adjustments were necessary. For ‘calibration’ of the device an own test system has been set up – the comparison of control and necrotic cells of the tumor cell line 4T1. In addition, Raman difference analyses of those samples were used as standard to test new substrates and scaffolds.

Up until now, about one hundred customers and interested parties from academia and industry have visited the CELL lab or sent samples for analysis. CELL has analyzed living and fixed cells in 2D culture or within histological tissue sections, but also within 3D-cell tissues or scaffolds. In addition, solid material and fluids, like cell supernatants, vaccines, etc. have been measured.

So far, it became clear that Raman spectral analysis can identify (1) bacterial strains and cell types, (2) characterize diseased or infected cells, (3) discriminate tumor cells from non-tumor cells and (4) follow cell development such as stem cell differentiation or cell decay (apoptosis, necrosis). Furthermore, it was possible to (5) determine tumor aggressiveness, (6) monitor quality of cell based products and (7) follow cell reaction upon drugs or toxins.

In many cases the data obtained with BioRam® have been confirmed by common methods like FACS, MACS, immuno-cytochemical procedures, patch-camp, DNA or RNA arrays. These methods, however, require larger amounts of cell material and are more time- and cost intensive. Furthermore, most of the methods are endpoint analyses impairing cell viability.

Contract Medical International (CMI)

CMI was leading WP 3 “Develop and test a magnetic “cell select device” for capturing and homing of therapeutic cells in vivo”.

CMI focused on developing of a magnetic device suitable for manipulation of stem cells. The second prototype of a Cell Select Device designed as a structure of NdFeB magnets and ferromagnetic metal rings coated with gold to provide surface resistance and biocompatibility was constructed and tested using an artificial circulatory system. The system of magnets and ferromagnetic rings was simulated by Finite Element Method with the aim to optimize shaping and positions of magnets with respect to the requested shape of the device and generated gradient magnetic field necessary for iron nanoparticles attraction. The designed shape was optimized to obtain periodic magnetic circuits with focused magnetic flux on each segment metal-magnet-metal to provide a maximal attraction for labeled cells or similar magnetically active objects.

CMI also developed metal plating technique applied to the metal rings whilst neodymium magnets were purchased from the supplier as already plated. The device consists of NdFeB ring magnets in diameter of 0.8 mm and golden plated iron rings, strung on NiTiNol guide-wire. Tip and ends are sealed with UV glue to provide stability and to eliminate sharp edges. The guide wire manufactured from Nickel-Titanium alloy provides stability of metal rings and brittle neodymium magnets. It also provides excellent mechanical safety and biocompatibility to be tested within biological samples. For a better biocompatibility was surface sealed with biocompatible resin.

With the help from partner UNIWUE, KI and TOPASS, the function of the prototype could be clearly demonstrated in vitro. However, large animal studies are needed to further test the prototypes before medical device registration. This will be an issue for the future.

NanoPET Pharma GmbH (NANO)

NANO was leading WP4, “Develop and test magnetic nanoparticles”.

Monitoring and homing of mesenchymal stem cells (hMSCs) in vivo is essential for improved understanding of their interactions within the organism and their therapeutic effects. In collaboration with our project partners MICRO, UNIWUE and MRB we developed highly effective and biocompatible iron oxide nanoparticles which are optimized for efficient stem cell labeling (homing) and possess optimal contrast properties for MRI as well as MPI (monitoring). The N7B particles were identified as the most promising NANO prototype.

The manufacturing process is scalable and highly reproducible which lead to fixed particle parameters summarized in a specification sheet. In order to classify the results the particle were compared to the particles of the gold standard Resovist®. Due to the negative surface potential of the particles a labelling concentration of 100 µg / mL iron is already sufficient to obtain an almost double as high uptake in 2D cell culture conditions compared to Resovist®, in 3D cell culture even an much higher uptake. The particles were formulated according to physiological conditions and finally autoclaved. As a result, the particles do not impact hMSC viability and growth and show no cytotoxic effects at all (tests performed at UNIWUE).

The contrast properties, determined at MRB, were found to be highly improved. The MR relaxivity was twice as higher (R2/R1 = 22) compared to Resovist® (R2/R1 = 11) and the MPS data show an increase up to a factor of three for the amplitude of the third harmonic with a less decay at higher harmonics. In order to demonstrate the superior contrast properties, MRI and MPS gel phantoms of labelled cells were prepared. A MPS cell phantom with 50000 cells was still able to be measured with sufficient amplitudes. T2*-weighted images of the MR phantom show the typical darkening originated by the iron oxide nanoparticles. A phantom with only 5000 labeled cells was able to be visualized and offers the possibility to monitor therapeutic effects in vitro as well as in vivo.

The particle characteristics allow a direct labeling without the use of any transfection agents or other additional treatments, which is favorable for the future development of clinical applications. The optimal iron uptake characteristics enable the use of external magnetic fields to influence the labelled cells, allowing not only their homing to a specific target but also capturing of the labeled stem cells in vivo.

Micromod Partikeltechnologie GmbH (MICRO)

MICRO developed another prototype for cell labeling during the IDEA project.

Magnetic nanoparticles (MNPs) are interesting tools for the tracking and homing of stem cells. The tracking of stem cells with MNPs by MRI is known for more than 15 years (e.g. Bulte, J.W. et al., Nature biotechnology, 2001. 19(12): p. 1141-1147). Within the last 10 years Magnetic Particle Imaging (MPI) was developed as technique with a higher imaging sensitivity in comparison to MRI. Characteristic for MPI is the highly non-linear MNP response to moderate magnetic fields up to µ0H= 20 mT. A rich harmonic spectrum with high amplitudes Ak (k= 1 ... 69) of MNP magnetization (magnetic particle spectroscopy developed at MRB) was one of the main targets of particle development. To exploit the advantages of MPI in combination with MRI for stem cell tracking MICRO has developed a new type of MNPs with 2 – 3 fold higher signal intensities in comparison to Resovist®, the gold standard for these methods. The M4 particles were the most promising micromod prototype with highest contrast efficacy as well for MPI (A3 > 0.8 Am2/mol Fe) as for MRI (R2/R1 > 60).

Dextran iron oxide NPs are established contrast agents for in vivo applications. Thus MICRO has developed the dextran iron oxide composite particles M4 as prototype for the stem cell labeling. In collaboration with UNIWUE the optimal surface potential of M4 particles was identified that allows a direct stem cell labeling without the use of transfection agents. M4E particles with a moderate positive zeta potential of +15 mV were obtained by introduction of amino groups on the particle surface. At UNIWUE it was found that M4E did not affect the viability, growth and proliferation of hMSC and did not cause chromosomal aberrations. The protocol for labeling of hMSC with M4E particles from UNIWUE was successfully implemented by other partners (KCL, KI, TOPASS). MICRO has established the technical specification of M4E, validated the synthesis of M4E according to this specification and implemented the essential production techniques in the clean room. The final filtration through 0.2 µm filters was evaluated as possible sterilization method for in vivo application of the particles in the future.

Karolinska Institutet (KI)

KI was leading WP 5: “Perform cell transplantation in vivo”.

The first goal of IDEA project towards a clinical study was to develop cellular techniques that can help the healing process of injured vessels after surgical procedures. During the process the cells and techniques for transplantation should be investigated. Moreover this should be done with application of nanoparticles technologies for homing of the cells, removing the cells and monitoring of function.

KI found that both mesenchymal stem cells and endothelial progenitor cells can contribute to healing process. Our results were published in ATVB 2013 and Transpl. Proc. 2017. Moreover we found that special procedures such as controlling of angiogenesis by VEGF can improve organ functions such brain in Alzheimer disease. The study using magnetic iron oxide nanoparticles indicated that this technology could be used for cell labelling. This process was confirmed by staining. Planed technologies on particle imaging using MPI were not available for us at the end of project.

The preliminary data using MPI spectroscopy were negative. KI tried to use the magnetic cell select device for removing transplanted cells labelled with iron oxide nanoparticles. This study can be proceeded further to clinical application. Since MPI was not available for us, KI used high resolutions MRI and video recording to develop novel techniques for studying blood flow in vessels and vascular function. The results of this final part during the IDEA project are pblished in the near future and will help to study clinical application.

Paracelsus Medizinische Universität Salzburg (PMU)

PMU was involved in finding the best cell type with healing capacity for musculoskeletal diseases such as spinal cord trauma and for Parkinson’s disease. As such they work close together with partner KCL and KI. In addition, PMU helped significantly to establish the Raman-microscope platform - the BioRam® - from CELL as a diagnostic tool for regenerative medicine, due to close geographic distance of the two partners.

Universitätsklinikum Würzburg (UNIWUE)

UNIWUE supported partners in the IDEA consortium with expertise in cell culture techniques and in vitro studies concerning toxicological impact of the medicinal products under development. In regenerative medicine the IDEA partners focused on repairing damaged tissues using appropriate cells for therapy, such as human mesenchymal stem cells (hMSCs). Tissue healing using stem cell therapy only works, if the cells reach their target and remain there. To track hMSCs they can be labeled with iron oxide nanoparticles for non-invasive monitoring. With the project partners NANO, MICRO and MRB, UNIWUE analyzed the suitability of different iron oxide nanoparticle prototypes for biocompatibility and safe hMSC labeling in vitro.

The iron oxide particle prototype M4E was identified as most promising. Applying a labeling concentration of 500 µg ml-1, M4E particles did not affect hMSC viability, growth and proliferation, did not cause chromosomal aberrations and could be identified using magnetic particle spectroscopy. Moreover, cell labeling maintenance was up to five times higher in three-dimensional cell culture conditions compared with 2D culture [Kilian et al. 2016]. Thus, M4E particles allowed safe hMSC labeling in vitro. Our hMSC-loaded, 3D tissue-engineered construct could serve as a graft for regenerative therapies, in which M4E-labeled hMSCs can migrate to their target.

Standard histological and molecular biological analyses to determine cell-particle interaction and particle-induced cytotoxicity are labor-intensive. Thus, a more rapid and non-invasive procedure to sort out promising from unsuitable particle prototypes with high accuracy would be helpful. Thus, in close collaboration with CELL, UNIWUE applied the combination of a micro-well-based single cell array and Raman spectroscopy to hMSCs that were labeled with the iron oxide nanoparticle types M4E and M4E4. Multivariate data analysis revealed differences in Raman signals that could be clearly assigned to the harmless interaction of M4E particles with hMSCs and a cytotoxic impact of M4E4 particles on hMSCs.

Using Raman spectroscopy UNIWUE could rapidly identify promising nanoparticle prototypes with an overall accuracy of 96.3% [manuscript in preparation].

MRB Forschungszentrum für Magnet-Resonanz-Bayern e.V. (MRB)

MRB was leading WP 6, “Develop and test imaging protocols for in vitro and in vivo application”.

Aim of this project was the development and testing of a new imaging tool for monitoring stem cells and their therapeutic effect in in vitro and in vivo applications in regenerative medicine based on the use of magnetic nanoparticles. Therefore, MRB (which later became project partner FRAUNHOFER) developed a MPS/MPI prototype for stem cell monitoring in vitro. MPS represents a technology allowing a much quicker analysis of 3D samples compared to a quite time-consuming histological work-up and cell counting. It can detect the magnetic particle signal background-free and its signal behaviour is highly linear to the particle concentration. Moreover, MPS data can provide information on cell-particle interaction and integrity of magnetically labelled hMSCs. Additionally, MPS has the potential to serve as tool for quick and non-destructive quality control of grafts loaded with magnetically labelled cells immediately before transplantation.

In parallel the development of dedicated functionalized nanoparticles for MPI/MPS was carried out allowing evaluation of the location, distribution and long-term viability of cells having specific regenerative potential in a non-invasive and repeatable manner. Together with IDEA project partners NANO, MICRO and UNIWUE various iron oxide nanoparticle prototypes with different coatings were tested regarding their potential for cell labelling and in vitro imaging. Two particles, M4E and N7B were found to have optimal characteristics to serve as a tracer for stem cell monitoring.

The MPS/MPI prototype allowed the project partner to assess stem cell vitality. In close collaboration with MICRO and UNIWUE it could be shown that M4E has the potential to provide information on vitality of transplanted cells using non-invasive imaging protocols.

For the use of the developed MPI-spectrometer in animal models MRB built a solenoidal receive coil with homogeneous response function. This coil with 65 mm in diameter and 30 mm penetration depth can be placed directly over the region of interest (e.g. hindlimb) for localization and to increase sensitivity. The developed transmit system was able to generate high field strengths up to 100 mT. Thus, the non-invasive new surface magnetic particle spectrometer is able to perform quantitative measurements with a high sensitivity in low concentrated particle systems like labelled stem cells. In vitro phantom experiments showed the potential that the surface magnetic particle spectrometer should be able to perform quantitative measurements in animal models with labelled stem cells.

At the end of the project, together with the M4E nanoparticle and the optimized cell culture and labelling protocols a new stem cell imaging tool with high potential for application in regenerative medicine is available.

TOPASS GmbH (TOPASS)

TOPASS was coordinating the IDEA project and leading WP 7, “Regulatory affairs, scientific coordination, data management”.

Promotool GmbH (PROMO)

PROMO was leading WP 8, “Dissemination, exploitation and ethics” and WP 9, “Management”.

Potential Impact:
Potential Impact

Key goals of the IDEA project included improving the health of European citizens and improving the translation of regenerative medicine approaches into clinical practice.

The WP led at KCL focused largely on Alzheimer’s disease. Alzheimer’s disease is a devastating and progressive neurodegenerative condition affecting more than 8 million citizens in Europe, resulting in a massive human and economic cost, and a substantial impact on the quality of life of many millions of European citizens. We have symptomatic treatments with modest benefits, but we still do not have any effective disease modifying therapies. A number of clinical trials have focused on treatments targeting beta amyloid, a key disease protein, but so far none of these trials have been successful and there is an urgent need to diversify treatment targets and to develop novel therapeutic approaches.

Regenerative medicine provides a key potential therapeutic opportunity, which so far has not been translated into clinical trials. The current program of work has evaluated different in vitro stem cell models, including two human grade neural stem cell lines, and demonstrated the importance of cell lines, which augment the production of key trophic factors. Critically KCL has also been able to demonstrate these positive characteristics in one of the human grade neural stem cell lines, and KCL has further been able to demonstrate biochemical benefits and a positive impact on trophic factors in two independent rodent transgenic models of Alzheimer’s disease, and furthermore demonstrate behavioral benefits in one of these animal studies.

As collaborators in an additional work plan, KCL has also been able to investigate iron oxide particles develop in another WP, and demonstrate that human grade neural stem cells can successfully be labelled with one of these particles in in vitro studies, work which will enable further commercial development by our SME collaborator.

The cooperation has contributed important understanding regarding the characteristics of stem cells that are likely to potentially confer therapeutic benefits, and will hence make an important contribution to the further development of future stem cell therapies for Alzheimer’s disease.

Even more importantly, the work has demonstrated the potential utility of a currently available human grade neural stem cell line as a potential therapeutic intervention for people with Alzheimer’s disease.

Raman trapping microscope system BioRam® from CELL provide significant advantages compared to traditional scientific techniques such as FACS, MACS or immunohistochemistry: (1) Faster results, as labeling is not required and sample preparation is negligible. (2) Cell and tissue sparing, due to non-destructive measurement. Only a few cells (60-500) are required for meaningful results. (3) Characterization of living cells, even in 3D-cultures and liquids. (4) Significantly improved quality assessment of cell-therapeutic products, blood products and vaccines. (5) Customers benefit from considerable time and money savings. (6) Early and reliable tumor cell analysis with additional therapeutic benefit becomes feasible and examination of histological tissue sections could support tumor diagnosis.

Out of numerous possible applications CELL has identified four main topics that the company is currently focusing on: (1) Sensitive in-process monitoring of cell development and differentiation (2) Efficient, cost-saving on-site Quality Control of cell-based products (3) Early diagnosis of Cancer and Disease and (4) Fast detection of cell reactions on drugs and toxins.

Within these areas CellTool participated at various congresses to identify possible customers. Interested parties usually started with measurements of their own samples in their Service Lab. The results have been published either as posters, lectures, scientific articles or as reports in various journals.

CELL collaborators and customers are mainly allocated in the fields of regenerative medicine and tissue engineering or dealing with quality assurance of cell based products such as autologous skin grafts (www.euroskingraft.eu) treatment of Macula degenerata (www.targetamd.eu) or quality control of vaccines and cell based therapeutics (Paul-Ehrlich-Institute, Langen). In parallel, CellTool is in process to achieve GMP approval by SwissMedic for the analysis of cell based skin products. The approval could subsequently be expanded onto various other kinds of ATMPs (Advanced Therapy Medicinal Products).

CELL has a German and an European patent for “Differential Raman Spectroscopy”, a German patent for “Prostate Tumor Diagnostics”, a German Patent for quality assurance in the production of cell based products prior to transplantation (skin graft, Tissue Engineering) as well as a German patent for the quality control of blood products. In addition, there are patent applications pending for compatibility tests of active agents, drugs and toxins as well as for determination of cell transfection rates.

For quality assurance of blood products immediately prior to transfusion, CELL has been granted subsidies from the German Federal Ministry of Education and Research “KMU-innovative: Medizintechnik: HämatoRam” (13GW0112A). Project and development target is the “HämatoRam“-System consisting of a Raman spectroscopy based detection unit and microfluidic chips for point-of-care analysis of stability and sterility of blood products immediately before transfusion. Potential customers are blood donation services, blood product industry and maximum care hospitals.

In summary, Raman-based cell and tissue analysis will sustainably impact cell-related research, development and manufacturing. Patients will benefit through advanced diagnosis and individualized therapy as well as through the feasibility of new innovative treatments with safe and well documented cell-based products. In the pharmaceutical, cosmetic and chemical industries Raman analysis of tissue models could lower R&D costs and may help reducing animal testing.
With its innovative Raman-trapping microscope, CellTool is the first manufacturer of an easy to handle and versatile tool for marker-free, non-invasive cell and tissue analysis on single cell level, including analysis of fluids, 2D cell cultures and 3D scaffolds.
CELL currently is installing quality management, finalizing its ISO 9001 and ISO 13485 certification (audit in November 2017) and enhancing its marketing and sales activities in Europe, USA and China.

CMI has performed several tests to verify the methods, principles and suitable materials for construction of the magnetic device. CMI has developed and built the second prototype of the magnetic catheter with a biocompatible surface suitable to be tested in biological samples as Cell Select Device. The provided intensity of magnetic field was simulated via FEM model and measured as 0.2 Tesla in the distance of 0.2 mm perpendicular to the surface which corresponds to the simulations and to the measurement method.

CMI provided Cell Select Devices to the partners TOPASS, UNIWUE, and KI. Despite initial tests using the artificial circulatory system at TOPASS, that didn’t demonstrate sufficient function on free dextran-coated particles as well as for Ferucarbotran-labeled cells, in the experiments performed at UNIWUE the team showed that the magnetic catheter assembly is capable of attracting M4E-labelled hMSC under static and dynamic conditions.

The test performed with pure nanoparticles suspension showed good interaction between nanoparticles and the magnetic device. Tests performed with labeled stem cells showed strong interaction between the Cell Select Device and M4E-labelled hMSC which opens the door for further experiments with the dynamic setup in vitro and in vivo.

CMI prepared necessary Master Validation Plan necessary for MDR of magnetic Cell Select Device for in vitro and in vivo tests, but the final design was not frozen yet due to unclear intended use and missing data from large animal studies.

During the IDEA project NANO developed new iron oxide based nanoparticles which can be used as imaging agent in MRI and tracer materials in MPI. The collaboration with the project partners enables the possibility of a comprehensive characterization including the in vitro characterization done by UNIWUE and the magnetic characterization done by MRB.

The excellent contrast properties of the particles, the standardized manufacturing process and the pharmaceutical formulation and sterilization of the final N7B particles is a requirement of a fast transfer on the market in the field of preclinical imaging agents. The new particle prototype will lead to essential improvement in diagnostic imaging for existing applications in MRI and will address new applications in MPI. Additionally, the set-up of a standard labelling protocol for hMSCs will help future scientist to standardize there labelling experiments and get more reliable results.

MICRO has established the trademark perimag® for the M4 particle type and offers these dextran iron oxide particles with a plain surface (M4A – product code: 102-00-132), with amino groups on the surface (M4E – product code: 102-01-132), with COOH groups on the surface (product code: 102-02-132) and with conjugated streptavidin (product code: 102-19-132) in micromod’s catalog (www.micromod.de). The perimag® particles are prepared according to ISO 13485 and can be produced under clean room conditions on customer’s request.

Vascular progenitor cells contribute to repair of injured vasculature. In this study, KI aimed to investigate the role of bone marrow-derived cells in the intimal formation after arterial injury. The aim of this study was to examine the contribution of MSCs in intimal hyperplasia and to identify relevant factors that affect their recruitment. KI found that local inflammation in transplanted vessels exerts an effect on surrounding tissue and vessels that leads to phenotypic modulation of SMCs; however, those cells did not migrate to the intima and did not contribute to the intimal hyperplasia. Rather, as shown by transplantation of labeled cells, adventitial progenitor cells seemed to be a prominent source of host-derived cells in the lesion, and MCP-1 exhibits an important role in their recruitment and the pathological process of intimal hyperplasia.

Balloon injury of the femoral artery of wild-type mice was followed by local delivery of bone marrow-derived cells from GFP transgenic mice. The arteries were collected 1, 4, 7, and 14 days after injury and studied for morphology, localization, and phenotypes of delivered cells. Bone marrow-derived cells were present in the intima only at the early stages of arterial injury and expressed endothelial progenitor cell markers (CD31, CD34, and VEGFR-2). In the areas where intima was thicker, bone marrow-derived cells differentiated to intimal smooth muscle cells but they did not fuse with intimal cells. Delivery of CD34+ cells contributed to a 1.5-fold inhibition of intimal hyperplasia.

KI developed a system for monitoring blood flow in vessels. The phase-contrast MRI experiments enable the non-invasive measurement of flow velocities in all 3 directions. The scanning is carefully synchronized to the heartbeat through ECG-triggered data acquisition and results in obtaining 7-dimensional data (three spatial dimension, the cardiac phase, and the velocities in all three directions). The spatial resolution is typically better than 100x100x400 μm3 and the temporal resolution is 6.5 ms with the 9.4 T dedicated to animal MRI scanner at Karolinska Institute. This allows obtaining approximately 20 frames from each heart cycle in mice.

From the data acquired following described methods application, several relevant parameters which may influence the vessel can be calculated. Turbulent flow is revealed as a signal drop in the magnitude images. From the velocity profile across the vessel the wall shear stress can be calculated.
By these it’s possible to monitor both: vascular remodeling and vascular functions.

During the IDEA project MRB / FRAUNHOFER developed and built a prototype for hand-held MPI/MPS, a promising new imaging tool for monitoring stem cells and their therapeutic effect in in vitro and in vivo applications in regenerative medicine based on the use of magnetic nanoparticles.

In close cooperation with our consortium project partners it was possible to develop a new stem cell imaging tool consisting of dedicated iron-oxide nanoparticles (MICRO, NANO) and optimized cell culture and labelling protocols (UNIWUE). The M4E iron oxide nanoparticles are commercially available (perimag®) and can be used as a reporter for cell homing, migration and also for cell vitality. The in vitro measurements showed the potential that the surface magnetic particle spectrometer should be able to perform quantitative measurements with high sensitivity in animal models with labelled stem cells.

Main dissemination activities

In accordance with the dissemination strategy of the IDEA project, a series of conferences and workshops were organized. The management partner PROMO prepared these events as part of the annual meetings of all project partners and carried them out on site with the support of the respective hosts.

In 2013, for example, a workshop on Raman spectroscopy was held at the Paracelsus Medical University in Salzburg following the second annual IDEA meeting. In the context of the 2014 annual meeting, the general and specialist public was invited to discuss the contents and interim results of the IDEA project at King's College. In 2015, the IDEA meeting took place at the same time as the 4th International Conference "Strategies in tissue engineering", in which an IDEA partner played a mayor role. The Würzburg Initiative Tissue Engineering (WITE) invited IDEA to take over the organization of a lunch symposium, which took place on the second day of the conference.
At the end of the project the public was invited under the titel "New Tools for Regenerative Medicine - Identification, homing and monitoring of therapeutic cells for regenerative medicine: IDEA" for an international workshop. At the Campus Virchow Hospital, Charité Berlin, experts and specialists from various medical fields discussed with all project partners the different topics and aspects of the results achieved in the course of the project. A Presentation of the project partners and posters were presented and exhibited in addition to the program of the conference with lectures and discussions at the entrance hall of the Berlin-Brandenburg Center for Regenerative Therapies. All presentations of this final conference are available at the project website.

As dissemination activity, a part of the scientific results of the IDEA project were published with MRB as leading partner in collaboration with UNIWUE and MICRO [Fidler F, Steinke M, Kraupner A, Grüttner C, Hiller KH, Briel A, Westphal F, Walles H, Jakob PM. IEEE Transactions on Magnetics 51(2), 2015]. Furthermore, NANO presented the results obtained in the IDEA project at various conferences, e.g. at the WMIC (World Molecular Imaging Congress) annual meeting 2014 in Seoul (South Korea) with the project flyer directly at the nanoPET company booth. The company booth opens up the possibility to present the project more efficiently to the audience and to address a higher number of scientists and physicians. Additionally, specific promotion material of the project partners can be displayed at the booth (e.g. cooperation with MRB). 2015 NANO presented the results on a scientific poster at the WMIC in Honolulu (Hawaii, USA).

With UNIWUE as leading partner, MRB / FRAUNHOFER, NANO and MICRO co-authored another peer-reviewed publication [Kilian T, Fidler F, Kasten A, Nietzer S, Landgraf V, Weiß K, Walles H, Westphal F, Hackenberg S, Grüttner C, Steinke M. Nanomedicine (Lond) 11(15):1957-70, 2016]. Furthermore MRB / FRAUNHOFER presented the main findings at various national and international conferences.

MICRO co-authored together with MRB a paper from UNIWUE [Kilian T, Fidler F, Kasten A, Nietzer S, Landgraf V, Weiß K, Walles H, Westphal F, Hackenberg S, Grüttner C, Steinke M. Nanomedicine (Lond) 11(15):1957-70, 2016] and has presented posters with the results of the collaboration with UNIWUE, MRB and CMI at the International Biotechnica Fair in Hannover 2015 and at the conference "Frontiers in Biomagnetic Particles” in Telluride, USA, 2015 [Mueller, K, Steinke, M, Kilian, T, Fidler, F, Mathys, K, Grüttner, C.]. All these papers are referenced in micromod’s brochure on applications of magnetic particles for MPI at https://www.micromod.de/de/downloads-45.html.

Furthermore perimag® particles were advertised in the Proceedings of the International Workshop of Magnetic Particle Imaging (IWMPI) in Prague, Czech Republic, 2017 and of the 34th Annual Meeting of the Society for Thermal Medicine in Cancun, Mexico, 2017.

UNIWUE conducted the preclinical in vitro experiments to identify promising iron oxide nanoparticle prototypes. The data provide the basis for subsequent in vivo studies in small and large animal models. Moreover, the results support our partners to bring their most promising particles to the market (commercialisation).
With MRB as leading partner, UNIWUE, NANO and MICRO co-authored another peer-reviewed publication [Fidler F, Steinke M, Kraupner A, Grüttner C, Hiller KH, Briel A, Westphal F, Walles H, Jakob PM. IEEE Transactions on Magnetics 51(2), 2015].

UNIWUE supported CELL to demonstrate the reliability of the BioRam® system. For future studies, the combination of the BioRam® system and micro-well structures could allow repeated, even high throughput analysis of identical living cells, automated measuring, laser-based cell sorting and subsequent downstream analyses. Raman imaging of single cells has great potential to perform in-depth analysis, e.g. genotoxic effects of nanoparticles or stem cell differentiation potential. In this part of the project, UNIWUE published a peer-reviewed manuscript on Raman data for cell quality control in collaboration with CELL [Steinke M, Gross R, Walles H, Gangnus R, Schütze K, Walles T. Biomat 35:7355-7362, 2014].

UNIWUE presented the main findings at different national and international conferences. At the 4th International Conference “Strategies in Tissue Engineering”, which was held in Würzburg in 2015, UNIWUE organized an IDEA lunch symposium. Here, the IDEA partners gave talks on their data and intensely discussed with the audience.

Exploitation of results

The main outcome of the work has been to develop a commercial collaboration with an SME to enable a first in disease phase 1 clinical trial of human neural stem cells in people with Alzheimer’s disease. Work is currently ongoing to finalize the study protocol and to obtain regulatory approval. The results of our IDEA work package have made a critical contribution to the case for support for the trial and for the regulatory approval package. It is hoped that this phase 1 study will commence in June 2018, and will potentially herald a new wave of opportunity for stem cell and regenerative medicine interventions for people with Alzheimer’s disease.

IDEA has also provided a platform to enable other parallel funding for studies developing neuropsychological and blood biomarker studies focusing neurogenesis, which have in term enabled us to develop a more robust clinical trial protocol with improved target validation.

Raman spectra analysis with CELLs Raman trapping microscope system BioRam® provide significant advantages compared to traditional scientific techniques such as FACS, MACS or immunohistochemistry: (1) Faster results as labeling is not required and sample preparation is negligible. (2) Cell and tissue sparing due to non-destructive measurement. Only a few cells (60-500) are required for meaningful results. (3) Characterization of living cells, even in 3D-cultures and liquids. (4) Significantly improved quality assessment of cell-therapeutic products, blood products and vaccines. (5) Customers benefit from considerable time and money savings. (6) Early and reliable tumor cell analysis with additional therapeutic benefit becomes feasible and examination of histological tissue sections could support tumor diagnosis.

Out of numerous possible applications CELL has identified four main topics that the company is currently focusing on: (1) Sensitive in-process monitoring of cell development and differentiation (2) Efficient, cost-saving on-site Quality Control of cell-based products (3) Early diagnosis of Cancer and Disease and (4) Fast detection of cell reactions on drugs and toxins.

Within these areas CELL participated at various congresses to identify possible customers. Interested parties usually started with measurements of their own samples in our Service Lab. The results have been published either as posters, lectures, scientific articles or as reports in various journals.

CELLs collaborators and customers are mainly allocated in the fields of regenerative medicine and tissue engineering or dealing with quality assurance of cell based products such as autologous skin grafts (www.euroskingraft.eu) treatment of Macula degenerata (www.targetamd.eu) or quality control of vaccines and cell based therapeutics (Paul-Ehrlich-Institute, Langen). In parallel, CellTool is in process to achieve GMP approval by SwissMedic for the analysis of cell based skin products. The approval could subsequently be expanded onto various other kinds of ATMPs (Advanced Therapy Medicinal Products).

CELL has a German and an European patent for “Differential Raman Spectroscopy”, a German patent for “Prostate Tumor Diagnostics”, a German Patent for quality assurance in the production of cell based products prior to transplantation (skin graft, Tissue Engineering) as well as a German patent for the quality control of blood products. In addition, there are patent applications pending for compatibility tests of active agents, drugs and toxins as well as for determination of cell transfection rates.

For quality assurance of blood products immediately prior to transfusion, CellTool as been granted subsidies from the German Federal Ministry of Education and Research “KMU-innovative: Medizintechnik: HämatoRam” (13GW0112A). Project and development target is the “HämatoRam“-System consisting of a Raman spectroscopy based detection unit and microfluidic chips for point-of-care analysis of stability and sterility of blood products immediately before transfusion. Potential customers are blood donation services, blood product industry and maximum care hospitals.

In summary, Raman-based cell and tissue analysis will sustainably impact cell-related research, development and manufacturing. Patients will benefit through advanced diagnosis and individualized therapy as well as through the feasibility of new innovative treatments with safe and well documented cell-based products. In the pharmaceutical, cosmetic and chemical industries Raman analysis of tissue models could lower R&D costs and may help reducing animal testing.

With its innovative Raman-trapping microscope, CELL is the first manufacturer of an easy to handle and versatile tool for marker-free, non-invasive cell and tissue analysis on single cell level, including analysis of fluids, 2D cell cultures and 3D scaffolds.
CELL currently is installing quality management, finalizing its ISO 9001 and ISO 13485 certification (audit in November 2017) and enhancing its marketing and sales activities in Europe, USA and China.

List of Websites:
http://www.idea-cell-technologies.eu

Identification, homing and monitoring of therapeutic cells for regenerative medicine – Identify, Enrich, Accelerate

Final Report Summary - IDEA (Identification, homing and monitoring of therapeutic cells for regenerative medicine – Identify, Enrich, Accelerate)

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