Osteogenic Differentiation from Mouse Embryonic Stem Cells.

Embryonic stem cells (ESCs) are a unique model that allows the study of molecular pathways underlying commitment and differentiation. We have studied signaling pathways and their contributions to osteogenic differentiation. In addition to our previously published protocol where we recommended the addition of retinoic acid with later addition of dexamethasone to boost osteogenic lineage cells, here we describe an optimized protocol for osteogenic differentiation from R1 ESCs with suggestions for inhibition of Src activity.

c-Src kinase inhibits osteogenic differentiation via enhancing STAT1 stability.

The proto-oncogene Src is ubiquitously expressed and is involved in cellular differentiation. However, the role of Src in embryonic stem (ES) cell osteogenic differentiation is largely unknown. Using the small molecule inhibitor PP2, c-Src specific siRNAs, and tet-inducible lentiviral vectors overexpressing active c-Src, we delineated an inhibitory role of c-Src in osteogenic differentiation of mouse embryonic stem cells (mESCs) and mouse MC3T3-E1s preosteoblasts.

Calreticulin regulates Src kinase in osteogenic differentiation from embryonic stem cells.

Calreticulin, the major Ca buffer of the endoplasmic reticulum plays an important role in the choice of fate by embryonic stem cells. Using the embryoid body method of organogenesis, we showed impaired osteogenesis in crt cells vis-à-vis calreticulin-containing osteogenic WT cells. In the non-osteogenic crt cells, c-Src- a non-receptor tyrosine kinase- was activated and its inhibition rescued osteogenesis.

Calreticulin regulates a switch between osteoblast and chondrocyte lineages derived from murine embryonic stem cells.

Calreticulin is a highly conserved, ubiquitous Ca-buffering protein in the endoplasmic reticulum that controls transcriptional activity of various developmental programs and also of embryonic stem cell (ESC) differentiation. Calreticulin activates calcineurin, which dephosphorylates and induces the nuclear import of the osteogenic transcription regulator nuclear factor of activated T cells 1 (NFATC1). We investigated whether calreticulin controls a switch between osteogenesis and chondrogenesis in mouse ESCs through NFATC1.

Calreticulin Is Required for TGF-β-Induced Epithelial-to-Mesenchymal Transition during Cardiogenesis in Mouse Embryonic Stem Cells.

Calreticulin, a multifunctional endoplasmic reticulum resident protein, is required for TGF-β-induced epithelial-to-mesenchymal transition (EMT) and subsequent cardiomyogenesis. Using embryoid bodies (EBs) derived from calreticulin-null and wild-type (WT) embryonic stem cells (ESCs), we show that expression of EMT and cardiac differentiation markers is induced during differentiation of WT EBs. This induction is inhibited in the absence of calreticulin and can be mimicked by inhibiting TGF-β signaling in WT cells.