Article

Identification of the critical extracellular matrix proteins that promote human embryonic stem cell assembly

Division of Research Immunology and Bone Marrow Transplantation, Childrens Hospital of Los Angeles, Los Angeles, CA 90095-1732, USA.
Stem cells and development (Impact Factor: 4.2). 12/2008; 18(6):919-28. DOI: 10.1089/scd.2008.0293
Source: PubMed

ABSTRACT Human embryonic stem cells (hESC) exist as large colonies containing tightly adherent, undifferentiated cells. Disaggregation of hESC as single cells significantly affects their survival and differentiation, suggesting that adhesion mechanisms are critical for the assembly and maintenance of hESC colonies. The goal of these studies was to determine the key extracellular matrix (ECM) components that regulate assembly and growth of hESC. Our studies demonstrate that undifferentiated hESC express a specific subtype of laminin (laminin-511) and nidogen-1. The addition of a purified protein complex comprised of human laminin-511 and nidogen-1 to single-cell suspensions of hESC is sufficient to restore hESC assembly in the absence of murine embryonic fibroblasts or exogenous chemicals. The mechanism of hESC aggregation is through binding of the alpha6beta1 integrin receptor highly expressed in the membranes of undifferentiated hESC; aggregation can be inhibited by an antibody against alpha6 and almost completely blocked by an antibody against the beta1 subunit. Reassembly of defined numbers of purified hESC with the laminin-nidogen complex allows consistent production of uniform embryoid bodies (EBs) ("LN-EBs") that differentiate into endodermal, ectodermal, and mesodermal derivatives, and are highly efficient in generating hematoendothelial progenitors. These data reveal for the first time the crucial role of the ECM proteins laminin-511 and nidogen-1 in hESC assembly, and provide a novel practical tool to investigate hESC differentiation in a xenogen-free microenvironment.

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    • "*P<0.05 Cell Tissue Res (2012) 350:289–303 299 (Evseenko et al. 2009). CXCL12 enhances the survival of mESCs (Guo et al. 2005). "
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    Cell and Tissue Research 08/2012; 350(2):289-303. DOI:10.1007/s00441-012-1476-7 · 3.33 Impact Factor
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    • "Taken together with the heterogeneity of laminin expression, this generates the hypothesis that each source may contain different laminin trimers, each of which may have different functions. In keeping with this, the laminin a1 chain is present on blood vessels but not fractones (Kerever et al., 2007), while laminin 511 has been shown to promote epidermal and pancreatic stem cell maintenance (Otonkoski et al., 2008; Paquet-Fifield et al., 2009) and a5 laminins used as a culture substrate are more effective than other laminins in maintaining embryonic stem (ES) cells in an undifferentiated state (Domogatskaya et al., 2008; Miyazaki et al., 2008; Evseenko et al., 2009; Vuoristo et al., 2009). However, the lack of purified laminins of each trimer combination, and the lack of trimer specific antibodies, has meant that at present the contribution of this heterogeneity to the function of the SEZ niche (or in any other stem cell niche) remains undefined. "
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    • "This dichotomy is likely created by the presence of GROα (and perhaps other growth factors) in our novel culture medium and differences in the microenvironment provided by the serum vs. matrigel substrate (Ilic, 2006). One possible interpretation of this result is that dissociation of colonies into single cell suspensions destroys the tight and adherent junction complexes by removing the basement membrane components that hESCs synthesize (Evseenko et al., 2009). Consequently, when single cells are plated on substrates that are not enriched in basement membrane components, they may not be able to reestablish polarity, which is one of the essential characteristics of undifferentiated hESCs (Krtolica et al., 2007). "
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