Figure 1.

Hypoxic Preconditioning Stimulates the Differentiation of mESC-Derived EBs to the Myogenic Lineage

(A) EBs were formed from C57 mESCs by the hanging drop method for 3 days, cultured under normoxic or hypoxic conditions for 16 h, and allowed to attach to a 0.3% gelatin-coated plate in DMEM/10% FBS for 1 day; the medium was then changed to fresh media and cells were further differentiated for up to 10 days. (B) The pluripotency marker Oct4 was significantly downregulated compared to expression under conditions of normoxia, and the myogenic marker MyoD was significantly upregulated in hypoxic cells compared to expression in normoxic cells based on real-time PCR analysis. Graphs show the relative percent change (n = 4); *p < 0.05, **p < 0.01, ***p < 0.001 versus the normoxic group. (C) Immunofluorescence staining for MyoD (red) and MyHC (red, arrows) 10 days after EB reattachment. Nor-EB, normoxic-EBs; Hyp-EB, hypoxia-primed EBs. Nuclear DNA was counterstained with DAPI (blue). Representative confocal microscopic photographs are shown. Magnification, 200×; scale bars, 50 μm.