Characterization of human IPS-MSCs and H9-MSCs. (A) Morphology of MSCs and iPS: H9-MSCs and IPS-MSCs were characterized by spindle-cell morphology (original magnification, 40×) and iPS-H9 colonies by ES-like morphology. (B) Expression of pluripotent markers in MSCs and iPS by flow cytometry. FACS analysis of Oct-4, Nanog, Lin 28 and Sox-2 were performed after permeabilization in PFA 3%/Saponin 0.1% in MSC-H9 before reprogramming, iPSC-H9 and iPS-differentiated MSCs. Marker expression is presented as histograms. Filled histograms indicate a negative isotype. (C) Immunohistochemical staining of surface antigens on human H9-MSCs and IPS-MSCs after differentiation toward adipogenic, osteogenic, and chondrogenic lineages (original magnification, 40×). Staining with alizarin red shows development of calcium accumulation under inductive osteogenic conditions, in vitro, for 4 weeks, whereas oil red O staining shows development of lipid accumulation under inductive adipogenic conditions, in vitro, for 4 weeks. Alcian blue stain was used to detect extracellular matrix proteoglycans under inductive chondrogenic conditions, in vitro, for 4 weeks.