Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells
Isolation and characterization of calvaria-derived osteoblasts from newborn mice carrying both Osx1-Cre::GFP and Oct4-EGFP transgenes. (a) Genotyping analysis of 8 neonatal mice carrying Osx1-GFP::Cre and Oct4-EGFP transgenes for coding regions of Oct4-EGFP and Cre. (b) Bright field images of the newborn calvaria. No GFP expression was detected in Oct4-EGFP calvaria, whereas GFP expression was observed in calvaria isolated from a transgenic mouse carrying both Osx1-GFP::Cre and Oct4-EGFP. Representative FACS plot of isolating osteoblasts expressing GFP from calvarial bone. Sorted osteoblasts express GFP in culture. Inset shows phase image of sorted osteoblasts. Scale bars: 20 μm for calvarial bone; 5 μm for GFP+ cells. (c) qPCR analysis of the indicated transcript levels in MSCs, ESCs, unsorted osteoblasts, and sorted GFP+ osteoblasts. Individual mRNA expression levels were normalized to Gapdh with fold change relative to dermal fibroblasts, which is arbitrary set to 1. (d) From left to right: phase image of sorted osteoblasts. GFP+ osteoblasts were positive for Alizarin Red S staining. Scale bar: 20 μm. Three independent experiments are represented in (c). Data are expressed as means ± SEM. ;;.