Differentation of human induced pluripotent stem cells( IPSC) into kidney organdies for use in kidney regeneration

A.S. Shankar, S.S. Korevaar, M.C. Clahsen-van Groningen, M. Ghazvini, J. Gribnau, C.C. Baan, E.J. Hoorn, M.J. Hoogduijn

Thursday 15 march 2018

17:05 - 17:15h at Van Weelde Zaal

Categories: Basic / translational research, Session (parallel)

Parallel session: Parallel session 7: Basic / translational research

Introduction

The available treatments for end-stage kidney disease patients are not sufficient. Therefore there is an interest in iPSC-based therapies for kidney regeneration. In this study, we generated iPSC-derived kidney organoids and performed in-depth analysis of the differentiation status of the organoids. We also evaluated the risk of teratoma formation of iPSC and iPSC-derived kidney organoids in an immune-deficient mouse model.

Materials and methods

Two human iPSC lines were grown on Mouse Embryonic Fibroblasts (MEFs). The iPSC were pre-treated with CHIR99201, a glycogen synthase kinase inhibitor, for four days and fibroblast growth factor 9 (FGF9) for three days after which cell pellets were formed and placed on a Transwell membrane. The organoids were harvested at day 7, 14 and 18 for analysis by RT-PCR and immunohistochemistry. For the teratoma assays, ~0,5x10e6 iPSC or cells of iPSC-derived organoids were subcutaneously injected into four locations in four immune-deficient mice. Five weeks later the mice were sacrificed for histopathological analysis of teratoma formation.

Results

Compared to iPSC, there was a 40-fold increase of renal markers WT1 and HOXD11, while a 10-fold increase of GDNF could be observed. Moreover, there was a 10-fold increase in the expression of the kidney transporters OAT1 and OAT3 in the organoids at day 18. In contrast, iPSC marker NANOG decreased 15-fold compared to iPSC, while other iPSC markers cMYC, REX-1 and Klf4 remained around initial levels. Immunostaining revealed that organoids contained proximal and distal tubuli, glomerular and endothelial cells. A high expression of cMYC was present diffusely in a low number of cells in the organoids. In two mice that received iPSC there was tumor growth in seven out of eight locations. Histopathology confirmed the presence of teratoma formation. On the other hand, none of the injected kidney organoids resulted in visible tumor growth and histopathology indicated the presence of only small diffused tubular structures.

Conclusion

These findings demonstrate that iPSC can successfully be differentiated into kidney organoids. Even though there is an indication of residual presence of iPSC in the kidney organoids, this does not result in teratoma formation in an immune-deficient mouse model. This implies that iPSC-derived kidney organoids do not carry the same tumorigenic risk compared to iPSC and that future application in a transplantation model may be safe.