Figure 1.

Spontaneous Differentiation of hESCs without the Feeder Cell Layer

(A) Alkaline phosphatase staining and quantitative alkaline phosphatase activity assay at 0, 2, 4, and 7 days. Scale bar indicates 200 μm.

(B) qRT-PCR results of pluripotent markers (NANOG, OCT4, POU5F1) at 0, 2, 4, and 7 days.

(C) qRT-PCR results of an ectodermal gene (PAX6), mesodermal genes (PDGFR-α and BRACHYURY), and an endodermal gene (FOXA2) at 0, 2, 4, and 7 days.

(D) Flow cytometry analysis of cells expressing MSC surface markers.

(E) Four-color flow cytometry analysis for CD45, CD90, CD73, and CD146 expression was used to isolate CD73⁺CD90⁺CD146⁺CD45⁻ MSCs.

(F) Western blot of p65 and phosphorylated p65 at 0, 2, 4, and 7 days of hESC differentiation.

(G) Western blot of IκBα and phosphorylated IκBα at 0, 2, 4, and 7 days of H1 hESC differentiation. Three independent experiments were performed.

^∗p < 0.05, ^∗∗p < 0.001.