Pluripotent stem cells derived from patients, including embryonic stem (ES) cells and “induced pluripotent stem” (iPS) cells, are a promising area of regenerative medical research. A major roadblock toward human clinical therapies using ES cells or iPS cells is to define the factors that direct ES cell differentiation into lineage specific cells. We previously established a simple and efficient human embryonic stem cell (hESC) differentiation system to generate CD34+/CD31+ progenitor cells that gave rise to hematopoietic and endothelial cells (

Nat Biotech.
25
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317
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2007
). To advance potential clinical application and to define the effects of growth factors on hematopoietic and vascular differentiation, we assessed hESC differentiation on human feeder cells in serum-free condition without intermediate embryoid body (EB) formation. We investigated the roles of BMPs, TGFbeta, VEGF, and FGF2 in directing hESC differentiation. Growth factors were added into culture at different time points to test their stage specific roles. Our study demonstrated that BMP proteins, including BMP2, BMP4, and BMP7, but not BMP9, had synergic effects to VEGF and FGF-2 on hESC differentiation to CD34+/CD31+ progenitor cells. BMP4 was essential to initial CD34+/CD31+ cell development, whereas VEGF and FGF2 promoted the differentiation in later stage, suggesting the sequential roles of BMP4, VEGF and FGF2 in directing hESC differentiation to CD34+/CD31+ progenitor cells. TGFbeta or activin promoted hESC differentiation into CD34+/CD31− cells that were unable to give rise to hematopoietic, endothelial, and smooth muscle cells. Furthermore, TGFbeta or activin activated Smad2/3 signaling, and suppressed BMP4-induced CD34+/CD31+ cells. Microarray analysis revealed that BMP4-induced CD34+ cells expressed hematopoietic, endothelial and smooth muscle genes, including GATA2, gamma globins, VE-Cad, KDR, CD31, Tie2, and aortic smooth muscle actin, whereas TGFbeta-induced CD34+ cells expressed pluripotent markers and endoderm markers, including Oct3/4, Sox2, and Nanog, HHEX, GATA6, and FoxA2. Both canonical BMP signaling (Smad1/5/8-dependent) and non-canonical BMP signaling (p38 MAPK and p42 ERK pathway) were activated by BMP4 in hESCs. Dorsomorphin specifically inhibited BMP4-mediated phosphorylation of Smad1/5/8, and blocked hESC differentiation into CD34+/CD31+ cells. In summary, BMPs and TGFbeta regulate distinct populations of CD34+ cells in hESCs. BMP-Smad1/5/8 pathway is critical for hematopoietic and vascular progenitor development.

Topics:
embryonic stem cells, transforming growth factor beta, fibroblast growth factor 2, vascular endothelial growth factor a, activins, growth factor, idiopathic pneumonia syndrome, cd31 antigens, gamma-globins, mitogen-activated protein kinase p38

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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