Regulation of endodermal differentiation of human embryonic stem cells through integrin-ECM interactions

Department of Cellular and Molecular Medicine, Stem Cell Program, University of California, La Jolla, CA, USA.
Cell death and differentiation (Impact Factor: 8.18). 11/2012; 20(3). DOI: 10.1038/cdd.2012.138
Source: PubMed


Many cellular responses during development are regulated by interactions between integrin receptors and extracellular matrix proteins (ECMPs). Although the majority of recent studies in human embryonic stem cell (hESC) differentiation have focused on the role of growth factors, such as FGF, TGFβ, and WNT, relatively little is known about the role of ECMP-integrin signaling in this process. Moreover, current strategies to direct hESC differentiation into various lineages are inefficient and have yet to produce functionally mature cells in vitro. This suggests that additional factors, such as ECMPs, are required for the efficient differentiation of hESCs. Using a high-throughput multifactorial cellular array technology, we investigated the effect of hundreds of ECMP combinations and concentrations on differentiation of several hPSC lines to definitive endoderm (DE), an early embryonic cell population fated to give rise to internal organs such as the lung, liver, pancreas, stomach, and intestine. From this screen we identified fibronectin (FN) and vitronectin (VTN) as ECMP components that promoted DE differentiation. Analysis of integrin expression revealed that differentiation toward DE led to an increase in FN-binding integrin α5 (ITGA5) and VTN-binding integrin αV (ITGAV). Conditional short hairpin RNA-mediated knockdown of ITGA5 and ITGAV disrupted hESC differentiation toward DE. Finally, fluorescence-based cell sorting for ITGA5 and ITGAV significantly enriched cells with gene expression signatures associated with DE, demonstrating that these cell surface proteins permit isolation and enrichment of DE from hESCs. These data provide evidence that FN and VTN promote endoderm differentiation of hESCs through interaction with ITGA5 and ITGAV, and that ECMP-integrin interactions are required for hESC differentiation into functionally mature cells.Cell Death and Differentiation advance online publication, 16 November 2012; doi:10.1038/cdd.2012.138.

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    • "Induction of DE formation is the first and critical step of development in endodermal organs (Noguchi, 2009; Brafman et al., 2013; Hoveizi et al., 2014a). This germ layer gives rise to the intestinal epithelium and various other cell types, such as pancreas, hepatocytes, derived from the gut (D'Amour et al., 2005; Kim et al., 2010; Brafman et al., 2013; Jiang et al., 2013). The importance of the Activin A, Nodal, Wnt3a and TGFb pathway in DE induction has been demonstrated (Hosoya et al., 2012). "
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    ABSTRACT: First step in the formation of hepatocytes and beta cellsis the generation of definitive endoderm (DE) which involves a central issue in developmental biology. Human induced pluripotent stem cells (hiPSCs) have the pluripotencyto differentiate into all three germ layers in vitro and have been considered potent candidates for regenerative medicine as an unlimited source of cells for therapeutic applications. In this study, we investigated the differentiating potential of hiPSCs on poly (-caprolactone) (PCL)nanofibrous scaffoldito DE cells. Here, we demonstrate directed differentiation of hiPSCs by factors such as Activin A and Wnt3a. The differentiation was determined by immunofluoresence staining with Sox17, FoxA2 and Goosecoid (Gsc) and also by qRT-PCR analysis. The results of this study showed that hiPSCs, as a new cell source, have ability to differentiate into DE cells with a high capacity and also demonstrate that three dimension (3D) culture provides a suitable nanoenviroment for growth, proliferation and differentiation of hiPSCs. PCL nanofibrou sscaffold with essential supplements, stimulating factors and EB-derived cells is able to provide a novel method for enhancing functional differentiation of hiPSCs into DE cells.
    Cell Biology International 01/2015; 39(5). DOI:10.1002/cbin.10430/abstract · 1.93 Impact Factor
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    • "Moreover, small amounts of foreign atoms or molecules on the surface can dramatically alter the surface reactivity [42]. As ECM is crucial to mediating cell adhesion/attachment onto the surface of implants [43]. And the cellular response to material involves a chain of complex biological reactions including protein adsorption, receptor-ligand binding and signal transduction. "
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    ABSTRACT: Medical device implants are drawing increasing amounts of interest from modern medical practitioners. However, this attention is not evenly spread across all such devices; most of these implantable devices can cause adverse reactions such as inflammation, fibrosis, thrombosis, and infection. In this work, the biocompatibility of silicone rubber (SR) was improved through carbon (C) ion implantation. Scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) results confirmed that these newly generated carbon-implanted silicone rubbers (C-SRs) had large, irregular peaks and deep valleys on their surfaces. The water contact angle of the SR surface decreased significantly after C ion implantation. C ion implantation also changed the surface charge distribution, silicone oxygen rate, and chemical-element distribution of SR to favor cell attachment. The dermal fibroblasts cultured on the surface C-SR grew faster and showed more typical fibroblastic shapes. The expression levels of major adhesion proteins, including talin-1, zyxin, and vinculin, were significantly higher in dermal fibroblasts cultured on C-SR coated plates than in dermal fibroblasts cultured on SR. Those same dermal fibroblasts on C-SRs showed more pronounced adhesion and migration abilities. Osteopontin (OPN), a critical extracellular matrix (ECM) protein, was up-regulated and secreted from dermal fibroblasts cultured on C-SR. Matrix metalloproteinase-9 (MMP-9) activity was also increased. These cells were highly mobile and were able to adhere to surfaces, but these abilities were inhibited by the monoclonal antibody against OPN, or by shRNA-mediated MMP-9 knockdown. Together, these results suggest that C ion implantation significantly improves SR biocompatibility, and that OPN is important to promote cell adhesion to the C-SR surface.
    PLoS ONE 06/2014; 9(6):e98320. DOI:10.1371/journal.pone.0098320 · 3.23 Impact Factor
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    • "Figure 2 Growth models of hPSCs under various growth conditions. (A) Growth models of hPSC colonies in hESC medium that contains growth factors FGF2 and TGFβ on Matrigel-based extracellular matrices with deduced anatomical structures and molecular complexes: Laminin (LM), fibronectin (FN), and vitronectin (VNT) mediate their effect via the α6β1, α5β1, and αVβ5 integrins respectively (Braam et al., 2008; Brafman et al., 2012). Different zones of out-growing hPSC colonies have differential epithelial-to-mesenchymal transition (EMT) states, in which the cells in the center of the colonies possess more epithelial characteristics and cells at the periphery develop mesenchymal-like phenotypes. "
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    ABSTRACT: Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-based therapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are free from harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields, unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. Here, we shed light on the structural relationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methods based on the insights from developmental biology. We further highlight core signaling pathways that underlie multiple epithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyze emerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth models for hPSC expansion and differentiation. Furthermore, based on the influence of cell-cell interactions and signaling pathways, we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and miniorganoids, which are pivotal steps needed for future clinical applications.
    Stem Cell Research 05/2014; 12(3). DOI:10.1016/j.scr.2014.02.002 · 3.69 Impact Factor
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