OSR1 and SIX2 Drive Divergent Transcrip7onal Programs in Human Kidney Cells: Implica7ons for Regenera7on and Tumorigenesis

Naomi Pode-Shakked^1,2,3Osnat Cohen-Zontag^2,4Dorit Omer^2,4Einav Vax^4,5Orit Harari-Steinberg^2,6Oren Pleniceanu^2,7Benjamin Dekel^2,4,8*

• ¹Pediatric Nephrology, Dana Dwek Children's Hospital, Tel Aviv Medical Center, Israel, TEL AVIV, Israel
• ²Gray Faculty of Medical & Health Sciences, Tel Aviv University, Israel, TEL AVIV, Israel
• ³Laboratory for Human Kidney Development Research under the Center Of Regeneration And Longevity (CORAL), Tel Aviv Medical Center, Israel, TEL AVIV, Israel
• ⁴Pediatric Stem Cell Research Institute and Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel Hashomer, Israel, RAMAT GAN, Israel
• ⁵Chaim Sheba Academic Medical Center, RAMAT GAN, Israel
• ⁶Tissue Engineering Research Laboratory, Sheba Medical Center, Tel Hashomer, Ramat-Gan, Israel., RAMAT GAN, Israel
• ⁷Kidney Research Lab, The Institute of Nephrology and Hypertension, Sheba Medical Center, Tel-Hashomer, Israel, RAMAT GAN, Israel
• ⁸Sagol Center for Regenerative Medicin, Tel Aviv University, Israel, TEL AVIV, Israel

Background: During human nephrogenesis, nephron progenitor cells (NPCs) generate approximately one million nephrons. Postnatally, the silencing of key kidney progenitor genes results in the depleJon of this progenitor pool, limiJng the mature kidney's capacity for regeneraJon. Concurrently, the rising incidence of end-stage kidney disease underscores the urgent need for innovaJve regeneraJve strategies. Methods: We employed lenJviral vectors to ectopically induce key kidney progenitor genes—OSR1 and SIX2, individually or together—in primary adult human kidney (hAK) cells. We then analyzed the cellular and molecular consequences through morphological assessment, funcJonal assays, in vivo transplantaJon studies, and comprehensive transcripJonal profiling. Results: OSR1 and SIX2 induced disJnct reprogramming programs with differenJal funcJonal outcomes. SIX2 overexpression maintained epithelial morphology while significantly enhancing proliferaJon and clonogenic efficiency. TranscripJonally, SIX2 established epithelializaJon and cell cycle networks, downregulaJng proximal tubule markers, while upregulaJng distal nephron markers and proliferaJon genes. In vivo, SIX2-expressing cells formed organized tubular structures with disJnct luminal architecture in a proof-of-concept model.. In contrast, OSR1 overexpression induced morphological changes and acJvated developmental morphogeneJc pathways including epithelial tube morphogenesis and canonical Wnt signaling, but did not enhance proliferaJon and showed minimal tubulogenic capacity in vivo. Unexpectedly, OSR1 overexpression led to malignant transformaJon in one clone, exhibiJng Wilms' tumor-like features including expression of kidney developmental markers (i.e. SIX2, NCAM1, WT1) and mesenchymal phenotype. Conclusion: Our findings suggest that SIX2 overexpression in primary human adult kidney cells funcJonally confers enhanced self-renewal and tubulogenic capacity while transcripJonally Inducing a proximal to distal tubular cell diversion with maintained proliferaJve programs. In contrast, OSR1 acJvates broader developmental morphogeneJc networks but poses potenJal oncogenic risks. The malignant transformaJon observed with OSR1 overexpression provides insight into potenJal cellular origins of Wilms' tumor and raises important safety consideraJons for regeneraJve medicine approaches involving developmental gene inducJon in adult kidney cells.