Derivation and characterization of induced pluripotent stem cells derived from human granulosa cells. (A) Reprogramming of induced pluripotent stem cell (iPSC) lines: (a) parental granulosa cells at early passage (P1), (b) early reprogrammed cell colonies, and (c) human embryonic stem cell (hESC)-like colonies prior to being manually picked. (B) Genetic analysis of reprogrammed cell lines: (a) genomic PCR was used to reveal integration of all four retroviral transgenes, and (b) RT-PCR was used to demonstrate inactivation of exogenous viral transgenes and (c) expression of endogenous pluripotency genes. H9 hESCs (H9) and human granulosa cells (G) were used as controls for PCR and RT-PCR reactions. (C) Immunofluorescence analysis of pluripotency hESC markers (OCT4, SSEA4, TRA-1-60, TG30, HESCA-1, and HESCA-1) in a representative human granulosa cell-derived iPSC line (iGRA1). Scale bars = 200 μm (A,a,b,c) and 50 μm (C).