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Ivestigating the ability of embryonic stem cell derivatives to improve renal function in a murine model of kidney disease

Final Report Summary - KIDREGEN (Ivestigating the ability of embryonic stem cell derivatives to improve renal function in a murine model of kidney disease)

Overview and objectives

The overall aim of my project was to evaluate the efficacy and safety of stem cell therapies for kidney disease, particularly focussing on the potential of embryonic stem cells (ESCs) and their derivatives to treat chronic kidney disease (CKD). The long-term goal would be to administer stem cell therapies to a patient in the early stages of their chronic disease, in order to prevent progression to end stage renal disease, thus obviating the requirement for either transplantation or dialysis. However, before such therapies can be applied to humans, we need to know that they are effective and safe in animal models.

My project had three main objectives:

(1) to investigate if ESCs and/or their mesodermal derivatives had any detrimental effects on host tissues, and if they were able to generate functional renal cells;
(2) to establish a system for tracking the fate of the cells that did not affect their viability or phenotype;
(3) to establish a mouse CKD model in my lab.

Methodological approach and results

Objective 1:

To evaluate the ability of ESCs and their mesodermal and non-mesodermal derivatives to generate renal cells, and identify any possible adverse effects, the cells were incorporated into mouse embryo kidneys in vitro, and after a three to five day culture period, I performed a range of assays to answer the following questions: did the ESCs, their mesodermal, or non-mesodermal derivatives:

(i) have any negative effect on kidney growth or development;
(ii) form inappropriate cell types;
(iii) generate functional renal cells?

I found that the non-mesodermal derivatives had a marked detrimental effect on kidney growth and development. Undifferentiated ESCs also had a detrimental effect on kidney development, as they resulted in kidneys with more numerous, but smaller nephrons. On the other hand, the mesodermal derivatives caused no adverse effects. Further analysis showed that over time, the ESCs gave rise to large colonies of undifferentiated ESCs within the developing kidneys, whereas the mesodermal cells did not. Finally, in contrast to the ESCs, the mesodermal cells were able to generate podocytes, proximal tubule cells (PTCs) and collecting tubule cells within the kidney. Importantly, the mesoderm-derived PTCs displayed secretory function, similar to their in vivo counterparts.

Objective 2:

I undertook some preliminary studies to evaluate the potential of a range of cell labelling methods to track the ESCs and their derivatives. These studies indicated that quantum dots (QDs) had most potential because they allowed the cells to be tracked unambiguously for the longest period. I therefore undertook a comprehensive study to thoroughly evaluate the effectiveness of QDs as a tracking agent. I performed a range of assays to answer the following questions:

(i) what is the labelling efficiency of the QDs;
(ii) do QDs affect viability or the ability of stem cells to differentiate;
(ii) for how long can the QD signal be detected in the cells;
(iii) do QDs transfer to host cells?

I found that QDs had a high labelling efficiency and had no effect on the viability or differentiation potential of either mouse ESCs or mouse kidney-derived stem cells, but were rapidly depleted from both stem cell types when cultured under self-renewal conditions in 2D culture, indicating that they are only suitable for short-term tracking in rapidly proliferating cell types. However, when QD-labelled cells were cultured in 3D kidney organ culture, conditions in which the stem cells are expected to differentiate, the degree of QD depletion was minimal; this was likely due to the lower proliferation rate of the cells following differentiation. Cell division was the main cause of QD depletion in KSCs and ESCs. QDs were not released into the extracellular environment through excretion, or due to cell death, and were not readily transferred to neighbouring cells. Furthermore, the QDs had no effect on the incidence of fusion. Taken together, my results showed that the QDs used in this study (655 nm CdSe/ZnS dots coated with positively charged peptides) were suitable for short-term tracking of mouse ESCs and KSCs.

Objective 3:

I decided to set-up the adriamycin (ADR) CKD model because ADR causes an acute kidney injury, which partially resolves, but then progressed to CKD, as evidenced by the presence of glomerulosclerosis. My main questions were as follows:

(i) determine which parameter(s) of renal injury I should measure in order to demonstrate a beneficial effect of stem cell therapy;
(ii) determine the best time point for administering the stem cells.

I found that the most robust parameters for measuring kidney injury were the extent of albuminurea/24h period (from week 2), and the extent of glomerulosclersosis at week 6. However, given that the extent of albuminuria was the parameter that was most different between the control and treatment group, I will use this to calculate the number of animals I should use in each experiment to establish if there is a beneficial effect of cell therapy. To determine the best time point for administering the stem cells, I have been collabourating with the group of Norbert Gretz, Heidelberg, who has engineered a novel device for measuring the glomerular filtration rate (GFR) in live, and awake, mice. GFR is the gold standard for indicating kidney function. I will therefore use this device to establish the peak of injury in the ADR model, as my hypothesis is that this will be the best time point to administer the stem cells.

Conclusions and impact

My work has shown that:

(i) ESC-derived mesoderm could have potential to treat CKD, whereas ESCs and their non-mesodermal derivatives do not; that QDs are effective tracking agents for following cell fate over the short-term;
(iii) that the extent of albuminuria is the optimum parameter for measuring the efficacy of stem cell therapy, and that a novel device for measuring GFR could be useful for determining the peak of injury in the ADR model; this information could be used to establish the best time point for administering the cell therapy.

In the longer term, I hope to acquire quantitative efficacy data for cell therapy in the ADR model, and combine this with a thorough safety evaluation, in order to establish the risk:benefit ratio of such therapies. The impact to this will be to inform if these type of cell therapies could be suitable to translate to the clinic.