Efficient differentiation of human pluripotent stem cells into functional CD34+ progenitor cells by combined modulation of the MEK/ERK and BMP4 signaling pathways

Abstract

Differentiation of human pluripotent stem cells (hPSCs) into functional cell types is IntroductionHuman embryonic stem cells (hESCs) derived from an early embryo can self-renew indefinitely and differentiate into a variety of cell types. 1 It has been reported that the "stemness" of hESCs is likely maintained through the harmonious actions of signaling pathway networks. 2 Basic fibroblast growth factor (bFGF) is essential for maintaining the stemness of hESCs by highly activating mitogen-activated protein kinase (MAPK) extracellular signalregulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling, which plays an important role in the stemness of hESCs. 3 Stemness of hESCs is also supported by bFGF-mediated regulation of transforming growth factor-␤ (TGF-␤) signaling 4 ; activation of the TGF-␤/activin/nodal signaling pathway is required to maintain stemness in cooperation with the FGF signaling pathway, whereas its inhibition results in differentiation of hESCs. 5,6 The effect of Wnt signaling on stemness of hESCs is still controversial. Activation of the Wnt pathway by 6-bromoindirubin-3-oxmie, a specific inhibitor of glycogen synthase-3, sustains the undifferentiated status of hESCs. 7 However, activation of Wnt signaling is insufficient to maintain the undifferentiated status of hESCs, because a canonical Wnt signaling is highly activated during differentiation. 8 The stemness of human induced pluripotent stem cells (hiPSCs), like that of hESCs, seems to be maintained by coordinated networks of signaling molecules, although few differences are observed in the gene expression profile. 9,10 Thus, the stemness and differentiation of hESCs and hiPSCs is regulated by complex networks of signaling pathways. It is likely that the modulation of these signaling pathways can induce differentiation of hESCs into a specialized cell type. In fact, bone morphogenic protein 2/4 (BMP2/4), a member of the TGF␤ superfamily, could differentiate hESCs into trophoblasts, primitive endoderm cells, and mesodermal cells. [11][12][13] However, it has been reported that BMP4 is required for sustaining stemness of mouse ESCs by blocking neural differentiation. 14 hESCs could be differentiated to definitive endoderm cells by activation of activin/nodal signaling and suppression of phosphoinositide 3-kinase (PI3K) signaling. 15 Dual inhibition of SMAD signaling by treatment with Noggin and SB431542 resulted in differentiation of hESCs and hiPSCs into neural cells. 16 Many studies have tried to isolate specialized cell types from spontaneously differentiated cells via formation of hESC-derived embryoid bodies using antibodies against cell-typespecific surface markers. 17,18 However, spontaneous differentiation remains inefficient, in that hPSCs cannot be guided toward a specialized lineage at the initial commitment step.The hPSCs provide a possibility that degenerative or damaged tissues can be replaced with hPSC-derived functional cells. A paucity of number and activity of endothelial progenitor cells is correla...

Angiopoietin-1 promotes endothelial differentiation from embryonic stem cells and induced pluripotent stem cells

“…Endothelial differentiation of human ESCs (CHA4-hES) 22,23 was also performed as described previously. 23 Briefly, undifferentiated hESCs were cultured for 3 days in ESC medium (ESCM; DMEM/F12 medium supplemented with 20% knockout serum replacement, 1% nonessential amino acids, 0.1mM ␤-mercaptoethanol; all from Invitrogen) with MEK/ERK inhibitor (50M, PD98059; Calbiochem) and BMP4 (20 ng/mL; Peprotech), and cultured for another 6 days in ESCM with VEGF-A (100 ng/mL) and b-FGF (100 ng/mL; R&D Systems). CD34 ϩ progenitor cells were purified by MACS using anti-human CD34 microbeads, replated, and cultured in EC basal medium-2 with growth supplements (EGM-2; Clonetics).…”

“…Recently, iPSCs have become a conceptual but promising source of pluripotent cells. 23,45,46 Therefore, it would be useful to establish a method to generate specific target cells from iPSCs. We have demonstrated herein that the effect of Ang1 supplementation was recapitulated in iPSCs and even in human ESCs.…”

Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and reendothelialization in tissue-engineered vessels

“…iPS cells have displayed the potential to differentiate into a number of cell lineages, such as CD34 + progenitor cells (14), cardiomyocytes (15,16), and ECs (17). However, the main limitation for iPS cell application is the risk of tumor development, because these cells are reprogrammed to a fully pluripotent state.…”

Novel Stem Cell–Based Drug Discovery Platforms for Cardiovascular Disease

“…In general, this was accomplished either through the formation of differentiating heterogeneous 3D embryoid bodies from which a cell type may be isolated, as exemplified within Figure 2, or though addition of various factors to encourage pluripotent colonies to differentiate along a specific lineage. In particular, human iPSCs within various in vitro contexts have been differentiated into endothelium, [29][30][31][32] smooth muscle cells, 30,[33][34][35] monocytes, 36 and neutrophils. [37][38][39] Despite these reports, which describe the emergence of cell populations largely bearing molecular markers of the desired cell type, there is a paucity of functional characterization of the differentiated cell types.…”