Background: Human induced pluripotent stem cell (iPSC) derived kidney organoids are a tool for studying kidney development, physiology and disease. The kidney plays an important endocrine role, including the secretion of erythropoietin (EPO) under hypoxia. We aimed to generate stable EPO-overexpressing kidney organoids by using DNA vectors to study the effects of EPO on kidney development and to examine the physiological effects of EPO-producing organoids.

Methods: A scaffold matrix attachment region (S/MAR) DNA vector was used to genetically modify iPSC, which induced robust and persistent expression of EPO at all stages of differentiation. The EPO-overexpressing (EPO-oe) organoids were characterized using markers including PODXL (podocyte), Villin-1 (proximal tubule) and E-cadherin (distal tubule). To study whether EPO-oe organoids had physiological effects, EPO-oe kidney organoids were subcutaneously implanted in immunodeficient mice and mouse hematocrit levels were measured after 1 month.

Results: In control organoids, EPO was undetectable at the transcript and protein levels. After 24 hours in hypoxia (1% O2), EPO was detected at 0.43 (interquartile range [IQR] 0.34-0.59) mIU/ml in the supernatant. This low level of EPO was inadequate for functional studies. EPO-oe kidney organoids produced constitutive high levels of EPO mRNA expression for over 25 days. EPO levels in the supernatant were 933 mIU/ day per EPO-oe kidney organoid. Immunohistochemistry staining showed that EPO overexpressing kidney organoids formed similar kidney structures compared to control organoids and the expression of the markers PODXL, Villin-1 and E-cadherin were not changed. One month after implantation of organoids in mice, hematocrit levels were significantly increased in mice that received EPO-oe organoids (0.48, IQR 0.44-0.55) compared to mice that received control organoids (0.40, IQR 0.40-0.41) (p=0.0092, n=9). Meanwhile, implanted EPO-oe organoids showed 6.5-fold higher CD31 expression than control organoids and displayed endothelial structures with an elongated morphology.

Conclusions: S/MAR DNA vectors can be used to generate robust and stable transgene expression in iPSC throughout their differentiation into kidney organoids. EPO-oe human kidney organoids showed high levels of sustained and functional EPO release. The implantation of EPO-oe organoids illustrates the potential of using human gene-edited iPSC-derived kidney organoids for cell therapy.