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. 2013 Mar 4;110(12):4598–4603. doi: 10.1073/pnas.1218682110

Fig. 2.

Fig. 2.

Assessment of R1 for endodermal and mesodermal lineage potential. (A) Immunostaining of R1 cells for PDX1, SOX17, and NKX6.1 after 12 d of pancreatic differentiation: 40% cells were SOX17+, almost all SOX17+ cells also expressed PDX1 and NKX6.1. (B) Oil Red O staining of R1 cells and MSCs (experimental control) after 9 d of adipogenic differentiation. Transcript levels of fatty acid binding protein 4 (FABP4), LEPTIN, and PPARγ (normalized to glucuronidase B) (GUSB) in directly sorted R1, R1 in expansion medium (R1-ctrl), R1 in adipogenic differentiation medium (R1-diff), MSC in expansion medium (MSC-ctrl), MSC in adipogenic differentiation medium (MSC-diff), and directly sorted R4. R4 cells fail to grow under R1 expansion conditions. Expression differences were validated at the protein level for FABP4 by immunostaining. (C) Analysis of expression of the endothelial marker CD31 in R1 cells after 7 d of endothelial differentiation by FACS. Positive control: human umbilical vein endothelial cells (HUVEC) cells. (D) Cord formation capacity evaluated after 24 h in endothelial Matrigel differentiation assay by phase-contrast microscopy for (a) CD31+ R1 cells from C, (b) CD31+ HUVEC from C, (c) primary human mammary epithelial cells, or (d) primary mammary epithelial cells after 24 h of culture in mammary epithelial cell growth medium (MEGM) as a negative control. (Scale bars, 100 μm.) These results were obtained in five of five analyses.