The SHB Adapter Protein Is Required for Normal Maturation of Mesoderm during in Vitro Differentiation of Embryonic Stem Cells
Journal of Biological Chemistry
Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in ␤-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB ؉/؊ ) or both SHB alleles (SHB ؊/؊ ). Differentiating embryoid bodies (EBs)
... ed from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB ؊/؊ EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB ؊/؊ EBs. SHB ؊/؊ EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB ؊/؊ ES cells formed fewer blood cell colonies than SHB ؉/؉ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context. Downloaded from FIGURE 6. Colony formation assay after 7 days of EB differentiation followed by a 7-10-day colony formation period. A, morphology of myeloid-like colonies. B, quantification of formation of blood colonies (means Ϯ S.E.; n ϭ 3-4; *** indicates p Ͻ 0.001).