Monitoring and maintaining the differentiation status of human ipsc derived kidney organoids

Abstract

Background & Aim Human induced pluripotent stem cell (iPSC) derived kidney organoids resemble first trimester fetal kidney and offer a great model for studying kidney development and disease. During early renal development, the inductive interaction between two intermediate mesoderm (IM)-derived progenitor cell populations, the ureteric bud and the kidney mesenchyme, promotes nephron and collecting duct formation. Protocols are developed to in vitro differentiate human iPSC into ureteric epithelium and metanephric mesenchyme simultaneously, and then aggregate them to generate a complex multicellular kidney organoids. However, this is a complex procedure with unpredictable outcomes. Furthermore, after 25 days of culture, kidney organoids start to degenerate and form fibrotic and cartilage tissue. We investigated how kidney organoid differentiation can be monitored and how the survival of kidney organoids can be prolonged. Methods, Results & Conclusion We treated five human iPSC with CHIR99021 and followed by FGF9 to induce IM for 7 days. These IM like cells were formed into cell pellets and transferred to air-liquid interface for a further 18 up to 32 days. Successfully differentiated organoids demonstrate glomerular and tubular structures in H&E section, while failed differentiation results in amorphous fibrotic tissue. The same iPSC line had variable results among batch to batch. Along the differentiation process, LDH activity was measured to monitor organoid metabolism, which reached a peak on days 7∼9. LDH peaks higher than 50 milliunits/ml indicated excessive necrosis and resulted in abnormal organoid formation. We observed that iPSC with elevated OCT4 expression showed induced capacity to differentiate into kidney organoids. Furthermore high TGFβ expression at day 7 was associated with fibrosis in the kidney organoids. As shortage of oxygen supply to kidney organoids due to lack of functional vasculature limits their long term stability, we split the organoids on day 8 and day 21 and placed the organoids on a shaker on day 21 to allow for better oxygen supply. Using this method, it was possible to preserve the kidney organoids for 2 additional weeks with maintenance of glomerular and tubular structures. Taken together, these data will help us to monitor the differentiation and prolong the culture time of kidney organoids.