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.
"Many examples of this approach are available in literatures. For example, the coating of a substrate with single or multiple ECM proteins has been explored for hESC maintenance , , . While the coating of a substrate with one or two ECM proteins has been successful, it is not ideal because the production of recombinant ECM proteins is still very expensive. "
[Show abstract][Hide abstract] ABSTRACT: Human induced pluripotent stem cells have the potential to become an unlimited cell source for cell replacement therapy. The realization of this potential, however, depends on the availability of culture methods that are robust, scalable, and use chemically defined materials. Despite significant advances in hiPSC technologies, the expansion of hiPSCs relies upon the use of animal-derived extracellular matrix extracts, such as Matrigel, which raises safety concerns over the use of these products. In this work, we investigated the feasibility of expanding and differentiating hiPSCs on a chemically defined, xeno-free synthetic peptide substrate, i.e. Corning Synthemax® Surface. We demonstrated that the Synthemax Surface supports the attachment, spreading, and proliferation of hiPSCs, as well as hiPSCs' lineage-specific differentiation. hiPSCs colonies grown on Synthemax Surfaces exhibit less spread and more compact morphology compared to cells grown on Matrigel™. The cytoskeleton characterization of hiPSCs grown on the Synthemax Surface revealed formation of denser actin filaments in the cell-cell interface. The down-regulation of vinculin and up-regulation of zyxin expression were also observed in hiPSCs grown on the Synthemax Surface. Further examination of cell-ECM interaction revealed that hiPSCs grown on the Synthemax Surface primarily utilize α(v)β(5) integrins to mediate attachment to the substrate, whereas multiple integrins are involved in cell attachment to Matrigel. Finally, hiPSCs can be maintained undifferentiated on the Synthemax Surface for more than ten passages. These studies provide a novel approach for expansion of hiPSCs using synthetic peptide engineered surface as a substrate to avoid a potential risk of contamination and lot-to-lot variability with animal derived materials.
PLoS ONE 11/2012; 7(11):e50880. DOI:10.1371/journal.pone.0050880 · 3.23 Impact Factor
[Show abstract][Hide abstract] 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 · 3.57 Impact Factor
"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. "
[Show abstract][Hide abstract] 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.
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