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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    ABSTRACT: Human embryonic stem cells (hESCs) have great potential for regenerative medicine as they have self-regenerative and pluripotent properties. Feeder cells or their conditioned medium are required for the maintenance of hESC in the undifferentiated state. Feeder cells have been postulated to produce growth factors and extracellular molecules for maintaining hESC in culture. The present study has aimed at identifying these molecules. The gene expression of supportive feeder cells, namely human foreskin fibroblast (hFF-1) and non-supportive human lung fibroblast (WI-38) was analyzed by microarray and 445 genes were found to be differentially expressed. Gene ontology analysis showed that 20.9% and 15.5% of the products of these genes belonged to the extracellular region and regulation of transcription activity, respectively. After validation of selected differentially expressed genes in both human and mouse feeder cells, transforming growth factor α (TGFα) was chosen for functional study. The results demonstrated that knockdown or protein neutralization of TGFα in hFF-1 led to increased expression of early differentiation markers and lower attachment rates of hESC. More importantly, TGFα maintained pluripotent gene expression levels, attachment rates and pluripotency by the in vitro differentiation of H9 under non-supportive conditions. TGFα treatment activated the p44/42 MAPK pathway but not the PI3K/Akt pathway. In addition, TGFα treatment increased the expression of pluripotent markers, NANOG and SSEA-3 but had no effects on the proliferation of hESCs. This study of the functional role of TGFα provides insights for the development of clinical grade hESCs for therapeutic applications. Electronic supplementary material The online version of this article (doi:10.1007/s00441-012-1476-7) contains supplementary material, which is available to authorized users.
    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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    ABSTRACT: Basal lamina is present in many stem cell niches, but we still have a poor understanding of the role of this and other extracellular matrix (ECM) components. Here, we review current knowledge regarding ECM expression and function in the neural stem cell niche, focusing on the subependymal zone of the adult CNS. An increasing complexity of ECM molecules has been described, and a number of receptors expressed on the stem cells identified. Experiments perturbing the niche using genetics or cytotoxic ablation of the rapidly dividing precursors, or using explant culture models to examine specific growth factors, have been influential in showing how changes in these ECM receptors might regulate neural stem cell behavior. However the role of changes in the matrix itself remains to be determined. The answers will be important, as they will point to the molecules required to engineer niches ex-vivo so as to provide tools for regenerative neuroscience.
    Developmental Neurobiology 11/2011; 71(11):1006-17. DOI:10.1002/dneu.20970 · 4.19 Impact Factor
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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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    ABSTRACT: Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ∼6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical-basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical-basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation-βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell-cell contacts can trigger adoption of a neuronal fate.
    Differentiation 03/2011; 81(4):222-32. DOI:10.1016/j.diff.2011.01.001 · 2.84 Impact Factor
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