Figure 1.

Conversion of Human Urine-Derived Stem Cells into Steroidogenic Cells

(A) Schematic illustrating our strategy for urine collection, processing, and reprogramming. Urine-derived cells (USCs) were cultured in specific media, and type-II colonies amplified and characterized through flow cytometry. Then they were either banked or expanded for experiments. USCs were infected at passage two with either a lentivirus encoding a transcription factor (TF) within an IRES-GFP vector, a combination of TFs, or mock infected (MOI = 200). Cells were treated with 8-br-cAMP (100 μM) unless stated otherwise and kept in culture for at least eight days before analyses.

(B) RT-PCR showing STAR expression on forced expression of each TF. The expression of exogenous SF1, PBX1, WT1, DAX1, and CITED2 was assessed by RT-PCR using primers encompassing the coding- and vector- specific regions. Human adrenal cDNA was used as a positive control for endogenous STAR expression and, along with non-template control (NTC), as a negative control for exogenous TF expression.

(C) qRT-PCR analyses of STAR expression on forced expression of SF1 with each TF (upper panel) and of SF1 with or without a combination of TFs (lower panel).

(D) Western blot analyses of PCNA and GAPDH expression in hiSCs and mock-reprogrammed USCs from four independent donors eight days after reprogramming (top left panels); cell counting (bottom left panels) and representative images (right panels) of hiSCs obtained from USCs and fibroblasts versus mock-reprogrammed cells. Scale bars, 50 μm.

(E) qRT-PCR analyses of STAR expression on forced expression of SF1 with or without the indicated treatments, started the day after infection for seven days. CNT, cells infected with empty control vector.

(F) qRT-PCR (upper panel) and RT-PCR (lower panels) analyses of STAR and SF1 expression after reprogramming USCs at different MOI of SF1 or empty control lentiviral vector (CNT).

(G) Morphological changes on SF1 overexpression in USCs eight days post-infection. Scale bars, 20 μm.

(H) Electron microscopy images of USCs and USCs eight days after reprogramming. Arrows point to mitochondria. Nu, nucleus.

Scale bars, 2 μm (left panels) and 1 μm (right panels). Data in (C)–(F) are represented as mean ± SEM, n ≥ 3. See also Figures S1 and S2.