Figure 2.
Generation of human induced steroid-producing cells (hiSCs) from adrenal precursor-like cells
(A) Overview of the protocol to generate hiSCs.
(B) Generation of cell lines with different doses of NR5A1 (SF-1). Differentiated cells were infected with NR5A1-expressing lentivirus at an MOI of 0.5–8, selected with blasticidin, followed by analysis of gene expression (compared with adrenal at CS21) and cortisol secretion analyzed by ELISA.
(C) Endpoint PCR analysis of NR5A1, StAR, CYP11A1, CYP11B1, and MC2R.
(D) Immunocytochemistry for StAR, SF-1, and EGFP from lentivirus transduced NR5A1-IRES-tGFPnuc cells. DAPI (in blue) indicates nuclear staining. Scale bar, 200 μm.
(E) Schematic of different stages of adrenal, gonad, and kidney development.
(F) RT-qPCR analysis of undifferentiated hPSCs (d0), adrenal precursor-like cells (d6), or SF-1-transduced lines for NR5A1, WT1, and GATA4. Gene expression was compared with adrenal, kidney, and gonads at CS21.
(G) Immunocytochemistry for GATA4, WT1, and SF-1 from samples analyzed in F. DAPI (in blue) indicates nuclear staining. Scale bar, 200 μm. NTC, no template control. All data are represented as mean ± SEM. Green bars depict the column used for comparison to other bars. Statistical significance was determined using one-way ANOVA followed by Dunnet’s multiple comparison test correction. See also Figure S4.