Expression levels and stoichiometry of Hnf1β, Emx2, Pax8 and Hnf4 influence direct reprogramming of induced renal tubular epithelial cells

Generation of induced renal epithelial cells (iRECs) from fibroblasts offers great opportunities for renal disease modeling and kidney regeneration. However, the low reprogramming efficiency of the current approach to generate iRECs has hindered potential therapeutic application and regenerative approach. This could be in part attributed to heterogeneous and unbalanced expression of reprogramming factors (RFs) Hnf1β (H1), Emx2 (E), Pax8 (P), and Hnf4α (H4) in transduced fibroblasts. Here, we establish an advanced retroviral vector system that expresses H1, E, P, and H4 in high levels and distinct ratios from bicistronic transcripts separated by P2A. Mouse embryonic fibroblasts (MEFs) harboring Cdh16-Cre; mT/mG allele are utilized to conduct iREC reprogramming via directly monitoring single cell fate conversion. Three sets of bicistronic RF combinations including H1E/H4P, H1H4/EP, and H1P/H4E have been generated to induce iREC reprogramming. Each of the RF combinations gives rise to distinct H1, E, P, and H4 expression levels and different reprogramming efficiencies. The desired H1E/H4P combination that results in high expression levels of RFs with balanced stoichiometry. substantially enhances the efficiency and quality of iRECs compared with transduction of separate H1, E, P, and H4 lentiviruses. We find that H1E/H4P-induced iRECs exhibit the superior features of renal tubular epithelial cells, as evidenced by expressing renal tubular-specific genes, possessing endocytotic arrogation activity and assembling into tubules along decellularized kidney scaffolds. This study establishes H1E/H4P cassette as a valuable platform for future iREC studies and regenerative medicine.