Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media

ArticleinBiomaterials 31(32):8281-8 · November 2010with27 Reads
DOI: 10.1016/j.biomaterials.2010.07.037 · Source: PubMed
Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.
    • "Synthetic polymers [51] Human E-cadherin-Fc chimeric protein Recombinant protein [31, 52] serum and bone [54]. Several studies showed that vitronectin robustly supported long-term self-renewal in hPSCs for longterm culture [35][36][37][38]. Recombinant truncated vitronectin (VTN-N), which lacked N-terminal Somatomedin B domain, was designed for use with Essential 8 defined medium (Thermo Fisher Scientific or Stem Cell Technologies) and supported human pluripotent stem cell attachment and survival better than wild type vitronectin [39]. "
    [Show abstract] [Hide abstract] ABSTRACT: In recent years, as human pluripotent stem cells (hPSCs) have been commonly cultured in feeder-free conditions, a number of cell culture substrates have been applied or developed. However, the functional roles of these substrates in maintaining hPSC self-renewal remain unclear. Here in this review, we summarize the types of these substrates and their effect on maintaining hPSC self-renewal. Endogenous extracellular matrix (ECM) protein expression has been shown to be crucial in maintaining hPSC self-renewal. These ECM molecules interact with integrin cell-surface receptors and transmit their cellular signaling. We discuss the possible effect of integrin-mediated signaling pathways on maintaining hPSC self-renewal. Activation of integrin-linked kinase (ILK), which transmits ECM-integrin signaling to AKT (also known as protein kinase B), has been shown to be critical in maintaining hPSC self-renewal. Also, since naïve pluripotency has been widely recognized as an alternative pluripotent state of hPSCs, we discuss the possible effects of culture substrates and integrin signaling on naïve hPSCs based on the studies of mouse embryonic stem cells. Understanding the role of culture substrates in hPSC self-renewal and differentiation enables us to control hPSC behavior precisely and to establish scalable or microfabricated culture technologies for regenerative medicine and drug development.
    Full-text · Article · Aug 2016
    • "The ECM serves as an important component of all tissues, and its composition and mechanical properties play significant roles in the self-renewal or differentiation of cells. ECM composition and signalling in stem cell niches promotes the self-renewal of stem or progenitor cells and this knowledge has been utilized early on in embryonic stem cell culture for ESC maintenance in vitro using MEFs secreting ECM [42], ECM-based substrates such as Matrigel 1 [43], specific ECM proteins such as laminins, collagen type I, or vitronectin444546. ECM proteins have also been utilized to guide stem cell differentiation to somatic cell types, including cardiomyo- cytes47484950. "
    [Show abstract] [Hide abstract] ABSTRACT: Transdifferentiation of one cell type to another has garnered significant research efforts in recent years. As cardiomyocyte loss following myocardial infarction becomes debilitating for cardiac patients, the option of an autologous source of cardiomyocytes not derived from multi/pluripotent stem cell sources is an attractive option. Such direct programming has been clearly realized with the use of transcription factors, microRNAs and more recently small molecule delivery to enhance epigenetic modifications, all albeit with low efficiencies in vitro. In this review, we aim to present a brief overview of the current in vitro and in vivo transdifferentiation strategies in the generation of cardiomyocytes from somatic sources. The interdisciplinary fields of tissue, cell, material and regenerative engineering offer many opportunities to synergistically achieve directly programmed cardiac tissue in vitro and enhance transdifferentiation in vivo. This review aims to present a concise outlook on this topic with these fields in mind.
    Full-text · Article · Mar 2016
    • "Only the recombinant vitronectin-coated dishes supported the self-renewal and pluripotency of hESCs for at least eight passages in defined mTeSR1 medium in this study. Several other researchers have also reported that vitronectin-coated dishes support the pluripotency of hESCs (Table 1) [38,49,76,77]. Yap et al. defined a threshold surface density of vitronectin for the expansion of hESCs (H1 and HES-3) and for the maintenance of pluripotency [77]. "
    [Show abstract] [Hide abstract] ABSTRACT: This review describes recent developments regarding the use of natural and synthetic polymers to support the propagation of human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (hiPSCs) while maintaining pluripotency in feeder-free and xeno-free cultures. The development of methods for culturing these cells without using mouse embryonic fibroblasts (MEFs) as a feeder layer will enable more reproducible culture conditions and reduce the risk of xenogenic contaminants, thus increasing the potential clinical applications of differentiated hPSCs. Human or recombinant fibronectin, laminin-511, and vitronectin, which are components of the extracellular matrix (ECM), have been used instead of Matrigel for the feeder-free growth of undifferentiated hPSCs. Successful hPSC cultures have been described for the following conditions: on oligopeptide-immobilized surfaces derived from vitronectin, on microcarriers prepared from synthetic polymers, and encapsulated within three-dimensional (3D) hydrogels composed of alginate and other hydrophilic natural polymers. Recently, synthetic biomaterials that allow hPSCs to maintain pluripotency by secreting endogenous ECM components have been designed. The combination of human ECM proteins or cell adhesion molecules (e.g., oligopeptides and poly-D-lysine) and synthetic biomaterials with well-designed surfaces and/or structures (e.g., scaffolds, hydrogels, microcarriers, microcapsules, or microfibers) in the presence of a chemically defined medium containing recombinant growth factors would offer a xeno-free alternative to feeder cells for culturing hPSCs and maintaining their pluripotency.
    Article · Jul 2014
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