ABSTRACT: Recent demographic studies indicate that renal diseases are the leading cause of mortality globally. In the United States alone, approximately 37 million individuals are unaware that they suffer from chronic kidney disease (CKD). Podocytes, a primary cell type in the kidneys, play critical roles in blood filtration. These cells are terminally differentiated and incapable of division in vivo, making the establishment of primary cultures particularly challenging. The ability of cells to proliferate and avoid senescence is closely linked to telomere length, which shortens with each cell division. When telomere length becomes critically reduced, cells enter a state of proliferative arrest, triggering DNA damage responses and cellular senescence. In this study, we present the successful immortalization of a human nephron progenitor cell line derived from the urine of a 30-year-old African male (UM30-OSN). These cells demonstrate stable morphology and proliferation for over one year in culture (~100 passages). To achieve partial reprogramming, plasmids encoding the reprogramming factors OCT4, SOX2, NANOG, cMyc, and KLF4 were employed, resulting in successful reprogramming over multiple passages. Similar to the primary urine-derived renal progenitor cells (UdRPC) UM30, UM30-OSN expresses the pluripotency-associated marker SSEA4, as well as renal stem cell markers such as SIX2, CD133, CD24 and Vimentin, as determined by immunofluorescence, FACS and qPCR analyses. To verify the immortalization and rejuvenation potential of this cell line, the expression of key cell cycle regulators was analyzed by qPCR. We observed a downregulation of p21 and p53, alongside an upregulation of PCNA, KI67, and TERT, which supports the successful immortalization of UM30-OSN. Furthermore, we demonstrate the efficient differentiation of UM30-OSN into human podocytes, a process that was compared to the widely used immortalized human podocyte line (AB 8/13) (Moin Saleem et al.). UM30-OSN podocytes express specific podocyte markers, including NPHS2, NPHS1, CD2AP and SYNPO. In contrast, podocytes (AB 8/13) express NPHS1, NPHS2, CD2AP and SYNPO, which was further validated by immunofluorescence qPCR and Western blot. Given their robust proliferation capacity and ease of handling, UM30-OSN cells represent a valuable model for investigating nephrogenesis, kidney-related diseases, and drug screening. It is noteworthy that, in addition to the commonly used immortal podocyte (AB 8/13) cells, the UM30-OSN cells may serve as a potential alternative, exhibiting mature podocyte markers expression and a more stable karyotype.
