Previously utilization of a stochastic genetic-labeling strategy to permanently mark a single cell and its progeny, showed growth to occur by the clonal proliferation of distinct kidney epithelial cells which act as local unipotent progenitors, giving rise to region-specific nephron epithelia. Herein, we established culture conditions enabling robust single-cell clonal proliferation of dissociated human kidney tissues and analyses of clonal progeny. Human adult kidney single-cell derived clonal cultures maintained self-renewal allowing steady growth kinetics and preservation of epithelial cell morphology so that a single cell generated approx. 5 million cells. In contrast, bulk cultures grown under the same conditions showed enhanced proliferation, loss of epithelial morphology with advent of senescence in early culture passage. Comparative next-generation sequencing of 9 clonal cultures revealed a reduction in proliferation-related, cell cycle, pro-apoptotic and epithelial-mesenchymal transition genes whereas the PI3K-AKT-mTOR and glycolysis-related metabolic pathways were upregulated. Concomitantly, genes involved in tissue-specific mature kidney cell functions were maintained. Analysis of the tissue-specific genes, which mark different kidney regions, revealed segment-specificity and lineage restriction in each clonal culture rather than encompassing multiple adult nephron lineages. These data suggest in vitro single cell growth of the human adult kidney may phenocopy and even outperform in-vivo characteristics whereby mature cells are endowed with clonogenic/self renewal capacity akin to unipotential tissue stem cells. Moreover, this long term in-vitro culturing method of human adult kidney cells that maintains quiescence-proliferation balance and preserves renal identity provides a unique source to study replicating kidney cells.
