Clonal Cultures Unravel Precursor Functions of the Human Adult Kidney

נעמי פודה-שקד ^1,2,3,7,8 Rotem Gershon ^1,2 Osnat Cohen-Zontag ^1,2 Itamar Kanter ⁴ Dorit Omer ^1,2,7 Oren Pleniceanu ^1,2,7 Gal Tam ⁴ Sarit Oriel ⁴ Herzl Ben-Hur ⁶ Guy Katz ^2,5,7 Tomer Kaliski ⁴ Benjamin Dekel ^1,2,7,8

¹Pediatric Stem Cell Research Institute, Sheba Medical Center, ישראל
²Sheba Centers for Regenerative Medicine and Cancer Research,, Sheba Medical Center, ישראל
³Pediatric Department A, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, ישראל
⁴Faculty of Engineering and Bar-Ilan Institute of Nanotechnology and Advanced Materials (Bina), Bar-Ilan University, ישראל
⁵The Joseph Buchman Gynecology and Maternity Center, Sheba Medical Center, ישראל
⁶Department of Obstet and Gynecology, Assaf Harofeh, ישראל
⁷Sackler Faculty of Medicine, Tel-Aviv University, ישראל
⁸Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, ישראל

Primary adult stem cell-derived cultures from the human kidney hold great potential to serve as models for genetic kidney disease, drug screens and for regenerative medicine. Basic insight is needed to understand principles of ex-vivo adult kidney cell growth. We therefore studied clonal behavior of adult human kidney epithelial cells by establishing conditions for single-cell derived clonal cultures.
A proportion of these clones showed cuboidal, epithelial-like (EL) cell phenotype, maintained a steady doubling time and could be serially passaged. In striking contrast, a spindle shaped fibroblast-like (FL) epithelial clones were also generated but, failed to serially propagate. The latter de-differentiated and underwent senescence at an early passage.
Global transcriptome analysis unveiled several fundamental differences. First, FL-clones clustered together with bulk kidney cultures suggesting a phenotypic-transcriptomic correlation. Second, EL-clones showed distal tubule segment-specific marker expression (i.e. high CD24, MUC1, ECAD, EpCAM), whereas FL-clones were characterized by a proximal-tubule (i.e. CD13, ENPEP) combined with embryonal/de-differentiation (i.e. NCAM) expression signature. Third, JAK/STAT, TGF-β/BMP and NOTCH signal transduction pathways were activated only in EL-clones putting these forward as potential drivers for epithelial kidney clonal expansion. Finally, transcriptome analysis of EL-clones growth over three months revealed preservation of cell cycle/division genes concomitant with a less restricted segment identity and elevation of embryonic stem cell transcriptional signature.
Thus, clonal cultures reveal adult stem cell functions of the human kidney. The ability to function as adult stem cells, clonally proliferate and propagate is related to the cell epithelial differentiation status favoring distal over proximal renal tubule identity.