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      Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells

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          Abstract

          Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to provide an infinite source of tissues for regenerative medicine. Although defined xeno-free media have been developed, culture conditions for reliable propagation of hESCs still require considerable improvement. Here we show that recombinant E8 fragments of laminin isoforms (LM-E8s), which are the minimum fragments conferring integrin-binding activity, promote greater adhesion of hESCs and hiPSCs than do Matrigel and intact laminin isoforms. Furthermore, LM-E8s sustain long-term self-renewal of hESCs and hiPSCs in defined xeno-free media with dissociated cell passaging. We successfully maintained three hESC and two hiPSC lines on LM-E8s in three defined media for 10 passages. hESCs maintained high level expression of pluripotency markers, had a normal karyotype after 30 passages and could differentiate into all three germ layers. This culture system allows robust proliferation of hESCs and hiPSCs for therapeutic applications.

          Abstract

          The use of animal products as culture substrates for human embryonic stem cell and induced pluripotent stem cell culture raises numerous safety concerns in a therapeutic setting. Miyazaki et al.. show that minimal fragments of human laminins provide a more effective support for the culture of these cell types.

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          Most cited references35

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          Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.

          Embryonic stem (ES) cell lines derived from human blastocysts have the developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture. Here we describe the clonal derivation of two human ES cell lines, H9.1 and H9.2. At the time of the clonal derivation of the H9.1 and H9.2 ES cell lines, the parental ES cell line, H9, had already been continuously cultured for 6 months. After an additional 8 months of culture, H9.1 and H9.2 ES cell lines continued to: (1) actively proliferate, (2) express high levels of telomerase, and (3) retain normal karyotypes. Telomere lengths, while somewhat variable, were maintained between 8 and 12 kb in high-passage H9.1 and H9.2 cells. High-passage H9.1 and H9.2 cells both formed teratomas in SCID-beige mice that included differentiated derivatives of all three embryonic germ layers. These results demonstrate the pluripotency of single human ES cells, the maintenance of pluripotency during an extended period of culture, and the long-term self-renewing properties of cultured human ES cells. The remarkable developmental potential, proliferative capacity, and karyotypic stability of human ES cells distinguish them from adult cells. Copyright 2000 Academic Press.
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            Cellular functions of FAK kinases: insight into molecular mechanisms and novel functions.

            Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are related tyrosine kinases that have important cellular functions, primarily through regulation of the cytoskeleton. Recent studies have identified multiple molecular mechanisms that regulate cytoskeletal responses, and have provided important and exciting insights into how FAK and Pyk2 control cellular processes such as cell migration. Equally exciting are reports of novel and originally unanticipated functions of these kinases, providing the groundwork for future avenues of investigation. This Commentary summarizes some of these recent discoveries that are relevant to the control of biological responses of the cell.
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              Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.

              We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511, a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component, human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages), after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps, hES cells spread out in a monolayer, maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on alpha6beta1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Pub. Group
                2041-1723
                04 December 2012
                : 3
                : 1236
                Affiliations
                [1 ]Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku , Kyoto 606-8507, Japan
                [2 ]Laboratory of Extracellular Matrix Biochemistry, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita , Osaka 565-0871, Japan
                [3 ]Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku , Kyoto 606-8507, Japan
                [4 ]Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Ushinomiya-cho, Yoshida, Sakyo-ku , Kyoto 606-8501, Japan
                Author notes
                Article
                ncomms2231
                10.1038/ncomms2231
                3535336
                23212365
                03bcaaa4-867f-474a-9424-d320b70b3e21
                Copyright © 2012, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/

                History
                : 30 July 2012
                : 29 October 2012
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