Advertisement

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance*

  • Despina Soteriou
    Footnotes
    Affiliations
    From the North West Embryonic Stem Cell Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9NT, United Kingdom,
    Search for articles by this author
  • Banu Iskender
    Footnotes
    Affiliations
    From the North West Embryonic Stem Cell Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9NT, United Kingdom,
    Search for articles by this author
  • Adam Byron
    Footnotes
    Affiliations
    Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
    Search for articles by this author
  • Jonathan D. Humphries
    Affiliations
    Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
    Search for articles by this author
  • Simon Borg-Bartolo
    Footnotes
    Affiliations
    From the North West Embryonic Stem Cell Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9NT, United Kingdom,
    Search for articles by this author
  • Marie-Claire Haddock
    Footnotes
    Affiliations
    From the North West Embryonic Stem Cell Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9NT, United Kingdom,
    Search for articles by this author
  • Melissa A. Baxter
    Footnotes
    Affiliations
    From the North West Embryonic Stem Cell Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9NT, United Kingdom,
    Search for articles by this author
  • David Knight
    Affiliations
    Biological Mass Spectrometry Core Facility, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
    Search for articles by this author
  • Martin J. Humphries
    Correspondence
    To whom correspondence may be addressed. Tel.: 44-161-275-5071; Fax: 44-161-275-5082;.
    Affiliations
    Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
    Search for articles by this author
  • Susan J. Kimber
    Correspondence
    To whom correspondence may be addressed. Tel.: 44-161-275-6773; Fax: 44-161-275-5600;.
    Affiliations
    From the North West Embryonic Stem Cell Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9NT, United Kingdom,
    Search for articles by this author
  • Author Footnotes
    * This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/D014638/1 (to S. J. K.), a grant from the Northwest Regional Development Agency (to S. J. K.), Wellcome Trust Grants 045225 and 074941 (to M. J. H.), a BBSRC Ph.D. studentship (to D. S.), and a Republic of Turkey Ministry of National Education Ph.D. scholarship (to B. I.). The mass spectrometer used in this study was purchased with grants from the BBSRC, Wellcome Trust, and the University of Manchester Strategic Fund.
    This article contains supplemental Tables S1–S4, Figs. S1–S7, and Files S1–S8.
    1 These authors contributed equally to this work.
    2 Present address: Stem Cell Research Group, Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 9NT, United Kingdom.
    3 Present address: Betul-Ziya Eren Genome and Stem Cell Center, Erciyes University, Kayseri 38039, Turkey.
    4 Present address: Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom.
    5 Present address: Manchester Immunology Group, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
    6 Present address: MRC Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom.
    7 Present address: Developmental Biomedicine Research Group, School of Medicine, University of Manchester, Manchester M13 9PT, United Kingdom.
Open AccessPublished:May 08, 2013DOI:https://doi.org/10.1074/jbc.M113.463372
      Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.
      Background: Interaction of stem cells with extracellular matrix (ECM) controls their fate.
      Results: MS reveals interacting ECM networks produced by human embryonic stem cells (hESCs) and their feeders; supportive and unsupportive hESC substrates comprise distinct ECM compositions.
      Conclusion: Several ECM molecules maintain hESC self-renewal.
      Significance: Better understanding of hESC self-renewal has applications in understanding development, generating cell therapies, and modeling diseases.

      Introduction

      Human embryonic stem cells (hESCs)
      The abbreviations used are:hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast; ECM, extracellular matrix; hPSF, human placental stromal fibroblast; ihPSF, immortalized human placental stromal fibroblast; Pn, passage n; LTBP, latent TGF-β-binding protein.
      are derived from the inner cell mass of the blastocyst, and they have almost unlimited self-renewal, together with the potential to differentiate into the cell types originating from all three embryonic germ layers: endoderm, mesoderm, and ectoderm. The differentiation of embryonic stem cells in vitro provides a model for studying the cellular and molecular mechanisms of early development, and hESCs can be utilized as tools for drug discovery and modeling diseases (
      • Darabi R.
      • Perlingeiro R.C.
      Lineage-specific reprogramming as a strategy for cell therapy.
      ). Although hESCs hold enormous promise for therapeutic applications, several hurdles need to be overcome before this becomes a reality (
      • Hyslop L.A.
      • Armstrong L.
      • Stojkovic M.
      • Lako M.
      Human embryonic stem cells. Biology and clinical implications.
      ). These include clearer definition of the factors that are required to maintain the self-renewal and pluripotent properties of these cells and development of approaches to direct their differentiation reproducibly into desired cell types at high efficiency. Most commonly, mouse embryonic fibroblast (MEF) feeder cells are employed to provide an environment that is suitable, although not necessarily optimal, for the maintenance of stem cell pluripotency. Routine MEF culture with medium containing animal-derived products carries the potential risk of animal pathogen or antigen transfer. To minimize such xeno-transfer, human feeder cells and autologous feeders created by differentiating hESCs have been developed (
      • Hovatta O.
      • Mikkola M.
      • Gertow K.
      • Strömberg A.M.
      • Inzunza J.
      • Hreinsson J.
      • Rozell B.
      • Blennow E.
      • Andäng M.
      • Ahrlund-Richter L.
      A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.
      ,
      • Xu C.
      • Jiang J.
      • Sottile V.
      • McWhir J.
      • Lebkowski J.
      • Carpenter M.K.
      Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth.
      ,
      • Stojkovic P.
      • Lako M.
      • Stewart R.
      • Przyborski S.
      • Armstrong L.
      • Evans J.
      • Murdoch A.
      • Strachan T.
      • Stojkovic M.
      An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells.
      ). Nonetheless, the use of any feeder cell still retains the requirement for pathogen testing and does not avoid issues of undefined culture conditions and batch-to-batch variation. As an alternative approach, feeder-free cultures using different mixtures of defined medium and human or recombinant ECM components eliminate the risk of xenogeneic transfer and at the same time increase reproducibility (
      • Amit M.
      • Shariki C.
      • Margulets V.
      • Itskovitz-Eldor J.
      Feeder layer- and serum-free culture of human embryonic stem cells.
      ,
      • Richards M.
      • Tan S.
      • Fong C.Y.
      • Biswas A.
      • Chan W.K.
      • Bongso A.
      Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.
      ,
      • Baxter M.A.
      • Camarasa M.V.
      • Bates N.
      • Small F.
      • Murray P.
      • Edgar D.
      • Kimber S.J.
      Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines.
      ). Ideally, an optimized culture system needs to be established that is xeno-free for applications such as future clinical therapies. The most successful early attempts at replacing feeders used Matrigel, an ill-defined basement membrane matrix derived from a mouse sarcoma cell line, generally together with feeder-conditioned medium (
      • Xu C.
      • Inokuma M.S.
      • Denham J.
      • Golds K.
      • Kundu P.
      • Gold J.D.
      • Carpenter M.K.
      Feeder-free growth of undifferentiated human embryonic stem cells.
      ,
      • Wang G.
      • Zhang H.
      • Zhao Y.
      • Li J.
      • Cai J.
      • Wang P.
      • Meng S.
      • Feng J.
      • Miao C.
      • Ding M.
      • Li D.
      • Deng H.
      Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers.
      ,
      • Yao S.
      • Chen S.
      • Clark J.
      • Hao E.
      • Beattie G.M.
      • Hayek A.
      • Ding S.
      Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions.
      ). This system still retains the possibility of xenopathogen transfer and batch variation. However, newer defined serum-free media have now been developed that avoid the need for conditioning.
      Our understanding of how hESCs are regulated in vivo is limited because of their transient nature and their tendency to differentiate easily (
      • Liu N.
      • Lu M.
      • Tian X.
      • Han Z.
      Molecular mechanisms involved in self-renewal and pluripotency of embryonic stem cells.
      ). However, observations in vitro indicate that stem cell fate is controlled by many factors, both intrinsic genetic and epigenetic signals and extrinsic regulators, such as growth factors and extracellular matrix (ECM) components. Although much attention has been paid to the influence of growth factors on stem cell fate (
      • Amit M.
      • Shariki C.
      • Margulets V.
      • Itskovitz-Eldor J.
      Feeder layer- and serum-free culture of human embryonic stem cells.
      ,
      • Liu N.
      • Lu M.
      • Tian X.
      • Han Z.
      Molecular mechanisms involved in self-renewal and pluripotency of embryonic stem cells.
      ), the role of the ECM has been relatively neglected. ECM components, which form dynamic adhesive structures that affect cell proliferation, survival, shape, migration, and differentiation, are important candidates for establishing an optimized feeder-free hESC culture system (
      • Li L.
      • Xie T.
      Stem cell niche. Structure and function.
      ,
      • Ohlstein B.
      • Kai T.
      • Decotto E.
      • Spradling A.
      The stem cell niche. Theme and variations.
      ,
      • Scadden D.T.
      The stem-cell niche as an entity of action.
      ,
      • Unwin R.D.
      • Gaskell S.J.
      • Evans C.A.
      • Whetton A.D.
      The potential for proteomic definition of stem cell populations.
      ). In our laboratory, we developed a defined culture medium, which allows maintenance of several hESC lines for at least 15 passages (
      • Baxter M.A.
      • Camarasa M.V.
      • Bates N.
      • Small F.
      • Murray P.
      • Edgar D.
      • Kimber S.J.
      Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines.
      ). Using this system, we showed that hESCs grow well on human plasma fibronectin (
      • Baxter M.A.
      • Camarasa M.V.
      • Bates N.
      • Small F.
      • Murray P.
      • Edgar D.
      • Kimber S.J.
      Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines.
      ). Other studies have also reported the maintenance of stem cells using fibronectin or laminin substrates (
      • Amit M.
      • Shariki C.
      • Margulets V.
      • Itskovitz-Eldor J.
      Feeder layer- and serum-free culture of human embryonic stem cells.
      ,
      • Beattie G.M.
      • Lopez A.D.
      • Bucay N.
      • Hinton A.
      • Firpo M.T.
      • King C.C.
      • Hayek A.
      Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers.
      ), and more recently, these molecules have been used together for suspension culture of stem cells (
      • Steiner D.
      • Khaner H.
      • Cohen M.
      • Even-Ram S.
      • Gil Y.
      • Itsykson P.
      • Turetsky T.
      • Idelson M.
      • Aizenman E.
      • Ram R.
      • Berman-Zaken Y.
      • Reubinoff B.
      Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension.
      ). In addition, other ECM molecules, such as vitronectin, have been shown to support stem cell self-renewal (
      • Baxter M.A.
      • Camarasa M.V.
      • Bates N.
      • Small F.
      • Murray P.
      • Edgar D.
      • Kimber S.J.
      Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines.
      ,
      • Braam S.R.
      • Zeinstra L.
      • Litjens S.
      • Ward-van Oostwaard D.
      • van den Brink S.
      • van Laake L.
      • Lebrin F.
      • Kats P.
      • Hochstenbach R.
      • Passier R.
      • Sonnenberg A.
      • Mummery C.L.
      Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via αvβ5 integrin.
      ,
      • Prowse A.B.
      • Doran M.R.
      • Cooper-White J.J.
      • Chong F.
      • Munro T.P.
      • Fitzpatrick J.
      • Chung T.L.
      • Haylock D.N.
      • Gray P.P.
      • Wolvetang E.J.
      Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media.
      ), and hESC culture on ECM derived from MEF feeders has been reported (
      • Klimanskaya I.
      • Chung Y.
      • Meisner L.
      • Johnson J.
      • West M.D.
      • Lanza R.
      Human embryonic stem cells derived without feeder cells.
      ). Therefore, we set out to analyze comprehensively the ECM of hESC-supportive feeder cells using a proteomic approach.
      Several previous studies have used proteomic approaches to identify proteins that regulate stem cell pluripotency. Some studies analyzed stem cell-conditioned Matrigel (
      • Hughes C.
      • Radan L.
      • Chang W.Y.
      • Stanford W.L.
      • Betts D.H.
      • Postovit L.M.
      • Lajoie G.A.
      Mass spectrometry-based proteomic analysis of the matrix microenvironment in pluripotent stem cell culture.
      ) or medium conditioned by feeder cells capable of maintaining hESCs (
      • Chin A.C.
      • Fong W.J.
      • Goh L.T.
      • Philp R.
      • Oh S.K.
      • Choo A.B.
      Identification of proteins from feeder conditioned medium that support human embryonic stem cells.
      ,
      • Lim J.W.
      • Bodnar A.
      Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells.
      ), whereas others analyzed membrane proteins of hESCs (
      • Harkness L.
      • Christiansen H.
      • Nehlin J.
      • Barington T.
      • Andersen J.S.
      • Kassem M.
      Identification of a membrane proteomic signature for human embryonic stem cells independent of culture conditions.
      ,
      • Intoh A.
      • Kurisaki A.
      • Yamanaka Y.
      • Hirano H.
      • Fukuda H.
      • Sugino H.
      • Asashima M.
      Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells.
      ,
      • McQuade L.R.
      • Schmidt U.
      • Pascovici D.
      • Stojanov T.
      • Baker M.S.
      Improved membrane proteomics coverage of human embryonic stem cells by peptide IPG-IEF.
      ) or the hESC phosphoproteome (
      • Brill L.M.
      • Xiong W.
      • Lee K.B.
      • Ficarro S.B.
      • Crain A.
      • Xu Y.
      • Terskikh A.
      • Snyder E.Y.
      • Ding S.
      Phosphoproteomic analysis of human embryonic stem cells.
      ,
      • Van Hoof D.
      • Heck A.J.
      • Krijgsveld J.
      • Mummery C.L.
      Proteomics and human embryonic stem cells.
      ). Here, we used an MS-based proteomic approach to identify ECM proteins released by mouse and human feeders in order to characterize the range of ECM components that support the growth of self-renewing hESCs. We aimed to determine both similarities and differences between supportive and unsupportive feeder cells and so to dissect important and novel components of the ECM that maintain the pluripotent self-renewing state. We compared ECM derived from conventional MEFs, primary human placental stromal fibroblasts (hPSFs), and immortalized human placental stromal fibroblasts (ihPSFs) produced in our laboratory, which have been shown to support pluripotent hESC growth for over 25 passages (
      • McKay T.R.
      • Camarasa M.V.
      • Iskender B.
      • Ye J.
      • Bates N.
      • Miller D.
      • Fitzsimmons J.C.
      • Foxler D.
      • Mee M.
      • Sharp T.V.
      • Aplin J.
      • Brison D.R.
      • Kimber S.J.
      Human feeder cell line for derivation and culture of hESc/hiPSc.
      ). All tested mouse and human feeder cells supported hESC self-renewal, but only ECM derived from CD1×CD1 (referred to herein as CD1) MEFs or ihPSFs supported hESC self-renewal, whereas ECM derived from MF1×CD1 MEFs or hPSFs was unsupportive. We found that many ECM proteins are expressed by both mouse and human feeders and are also produced by hESCs. Intriguingly, quantitative differences were identified between supportive and unsupportive matrices, and some proteins were only detected in supportive ECMs; these proteins might play a role in the maintenance of pluripotency. We tested candidate ECM molecules, including perlecan, fibrillin-1, fibulin-2, collagen VI, and tenascin C, as substrates for feeder-free growth of hESCs. Our results show that some of these molecules can support attachment and self-renewal of hESCs alone or in combination with a low, unsupportive concentration of fibronectin, in the absence of feeders. Thus, this study further illuminates the role that ECM interactions play in the hESC phenotype, which has until recently been a neglected area of hESC biology.

      DISCUSSION

      The stem cell niche has been defined as a microenvironment that regulates stem cell self-renewal, proliferation, and differentiation via external signals, and its importance for proper stem cell function and fate determination is well established (
      • Rodin S.
      • Domogatskaya A.
      • Ström S.
      • Hansson E.M.
      • Chien K.R.
      • Inzunza J.
      • Hovatta O.
      • Tryggvason K.
      Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.
      ,
      • Montes R.
      • Ligero G.
      • Sanchez L.
      • Catalina P.
      • de la Cueva T.
      • Nieto A.
      • Melen G.J.
      • Rubio R.
      • García-Castro J.
      • Bueno C.
      • Menendez P.
      Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media. Distinct requirements for TGF-β and IGF-II.
      ,
      • Xiao L.
      • Yuan X.
      • Sharkis S.J.
      Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells.
      ). hESCs are known to require precise conditions for culture and are routinely cultured in the presence of feeder cells, which provide a complex conditioning environment (
      • Lu J.
      • Hou R.
      • Booth C.J.
      • Yang S.H.
      • Snyder M.
      Defined culture conditions of human embryonic stem cells.
      ). However, there has been an increasing effort to refine hESC culture systems using defined conditions (with well established growth factors), including the use of single ECM substrates (
      • Amit M.
      • Shariki C.
      • Margulets V.
      • Itskovitz-Eldor J.
      Feeder layer- and serum-free culture of human embryonic stem cells.
      ,
      • Baxter M.A.
      • Camarasa M.V.
      • Bates N.
      • Small F.
      • Murray P.
      • Edgar D.
      • Kimber S.J.
      Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines.
      ,
      • Braam S.R.
      • Zeinstra L.
      • Litjens S.
      • Ward-van Oostwaard D.
      • van den Brink S.
      • van Laake L.
      • Lebrin F.
      • Kats P.
      • Hochstenbach R.
      • Passier R.
      • Sonnenberg A.
      • Mummery C.L.
      Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via αvβ5 integrin.
      ,
      • Rodin S.
      • Domogatskaya A.
      • Ström S.
      • Hansson E.M.
      • Chien K.R.
      • Inzunza J.
      • Hovatta O.
      • Tryggvason K.
      Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.
      ). There is, therefore, a pressing need to analyze the ECM components produced by feeder cells that contribute to a favorable niche in vitro and to assess the contribution of individual ECM proteins to the support of stem cell maintenance. Here, we have employed a proteomic approach to identify the ECM components produced by feeder cells that maintain hESC self-renewal, by feeder cells that do not maintain self-renewal, and by hESCs cultured on a single, favorable substrate, fibronectin. We show that many ECM components are produced by supportive and unsupportive MEF and human PSF feeder cells, whereas some proteins are only expressed in supportive ECM, suggesting a role in the maintenance of hESC self-renewal. We demonstrate that, in the absence of feeders, fibrillin-1 alone and either perlecan or fibulin-2 in combination with fibronectin can support attachment and maintenance of hESCs. Together with interaction network analysis, these data highlight the importance of the balance between ECM network properties and molecular composition in the regulation of hESC phenotype and provide a resource for further studies of hESC self-renewal.
      MS analysis of extracted ECMs revealed that both feeder cells and hESCs produce a complex network of ECM proteins. We showed that CD1 MEFs at P4 and P9 were supportive as feeders for hESC maintenance, as were the ECMs derived from these cells. MF1×CD1 MEFs at P4 and P9 were supportive as feeders, but their ECMs were unable to support hESCs in culture. Interaction network analysis revealed different network architectures between the ECMs of the two mouse feeder crosses. The MF1×CD1 ECM interaction network displayed a highly clustered module of collagens and thrombospondin-2, which was not present in the CD1 ECM network, which suggested that these molecules might play an inhibitory role in the support of hESC maintenance. This finding was supported by the loss of attachment of hESCs plated on collagen VI with fibronectin as compared with fibronectin alone. We speculate that this inhibition may arise by altering the interaction partners of a hub protein, such as fibronectin, which is known to play a key role in stem cell self-renewal (
      • Baxter M.A.
      • Camarasa M.V.
      • Bates N.
      • Small F.
      • Murray P.
      • Edgar D.
      • Kimber S.J.
      Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines.
      ,
      • Hunt G.C.
      • Singh P.
      • Schwarzbauer J.E.
      Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.
      ). Collagens and thrombospondins have been implicated in the maturation of cartilage by proteomic analysis of mouse neocartilage ECM (
      • Wilson R.
      • Diseberg A.F.
      • Gordon L.
      • Zivkovic S.
      • Tatarczuch L.
      • Mackie E.J.
      • Gorman J.J.
      • Bateman J.F.
      Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics.
      ). Furthermore, transforming growth factor β (TGF-β), a known regulator of hESC pluripotency (
      • James D.
      • Levine A.J.
      • Besser D.
      • Hemmati-Brivanlou A.
      TGFβ/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells.
      ), was enriched in CD1 ECM but absent from MF1×CD1 ECM, suggesting that insufficient levels of growth factors might also affect the supportive capacity of these ECMs.
      ihPSFs have been previously shown to support hESC proliferation and self-renewal for up to 25 passages, whereas hPSFs could not (
      • McKay T.R.
      • Camarasa M.V.
      • Iskender B.
      • Ye J.
      • Bates N.
      • Miller D.
      • Fitzsimmons J.C.
      • Foxler D.
      • Mee M.
      • Sharp T.V.
      • Aplin J.
      • Brison D.R.
      • Kimber S.J.
      Human feeder cell line for derivation and culture of hESc/hiPSc.
      ). Here, we showed that ECM derived from ihPSFs was able to support hESC maintenance, whereas ECM from hPSFs was not. Our proteomic data revealed several differences between the composition of the ECMs from ihPSFs and hPSFs. The interaction network of ihPSF ECM was notably more interconnected and denser than that of hPSF ECM. In addition to the presence of additional collagens in ihPSF ECM compared with hPSF ECM, which were also present in the unsupportive MF1×CD1 ECM, ihPSF ECM contained several laminin chains that were not detected in the hPSF ECM. Laminin has been previously shown to support stem cell maintenance (
      • Rodin S.
      • Domogatskaya A.
      • Ström S.
      • Hansson E.M.
      • Chien K.R.
      • Inzunza J.
      • Hovatta O.
      • Tryggvason K.
      Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.
      ,
      • Miyazaki T.
      • Futaki S.
      • Suemori H.
      • Taniguchi Y.
      • Yamada M.
      • Kawasaki M.
      • Hayashi M.
      • Kumagai H.
      • Nakatsuji N.
      • Sekiguchi K.
      • Kawase E.
      Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells.
      ) and to be expressed by supportive feeder cells (
      • Hongisto H.
      • Vuoristo S.
      • Mikhailova A.
      • Suuronen R.
      • Virtanen I.
      • Otonkoski T.
      • Skottman H.
      Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture.
      ), so we speculate that ECM interactions that might be inhibitory to hESC growth, such as those potentially provided by collagens, may be overcome by the presence of key supportive components, such as laminin. Thus, the balance between ECM network properties and molecular composition appears critical for the support of stem cell self-renewal.
      hESC-supportive ECM from mouse and human feeders shared many common components not detected in unsupportive hPSF ECM, including collagen XII, collagen I, nidogen-1, fibulin-2, fibulin-5, and collagen III. The ECM molecules laminin 511, which was shown to support hESC growth in a xeno-free medium (
      • Rodin S.
      • Domogatskaya A.
      • Ström S.
      • Hansson E.M.
      • Chien K.R.
      • Inzunza J.
      • Hovatta O.
      • Tryggvason K.
      Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.
      ), and collagen IV, which maintained hESC self-renewal only with MEF-conditioned medium (
      • Braam S.R.
      • Zeinstra L.
      • Litjens S.
      • Ward-van Oostwaard D.
      • van den Brink S.
      • van Laake L.
      • Lebrin F.
      • Kats P.
      • Hochstenbach R.
      • Passier R.
      • Sonnenberg A.
      • Mummery C.L.
      Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via αvβ5 integrin.
      ), were detected in ihPSF and HUES1 ECM but not in CD1 MEF ECM, suggesting that the hESCs can tolerate certain molecules that are not essential for maintenance. Because these molecules were present in supportive ihPSF ECM, this would suggest that, in the absence of conditioned medium, they would not be detrimental as part of a mixed ECM in a chemically defined culture system. Furthermore, ihPSF ECM shared many compositional and network similarities with HUES1 ECM, which suggests that hESCs may secrete all of the ECM components necessary for maintenance of pluripotency if exposed to the “trigger” of a supportive substrate.
      Fibronectin, EMILIN-1, tenascin C, fibulin-1, and collagen VI α3 chain were expressed in all types of feeders. ihPSFs have been previously shown to produce a larger amount of fibronectin than hPSFs (
      • McKay T.R.
      • Camarasa M.V.
      • Iskender B.
      • Ye J.
      • Bates N.
      • Miller D.
      • Fitzsimmons J.C.
      • Foxler D.
      • Mee M.
      • Sharp T.V.
      • Aplin J.
      • Brison D.R.
      • Kimber S.J.
      Human feeder cell line for derivation and culture of hESc/hiPSc.
      ). Indeed, normalized spectrum count data showed that fibronectin was enriched in ihPSF ECM 24-fold over hPSF ECM (Table 1). Because fibronectin is known to support hESC growth in the absence of feeders (
      • Amit M.
      • Shariki C.
      • Margulets V.
      • Itskovitz-Eldor J.
      Feeder layer- and serum-free culture of human embryonic stem cells.
      ,
      • Baxter M.A.
      • Camarasa M.V.
      • Bates N.
      • Small F.
      • Murray P.
      • Edgar D.
      • Kimber S.J.
      Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines.
      ,
      • Xu C.
      • Inokuma M.S.
      • Denham J.
      • Golds K.
      • Kundu P.
      • Gold J.D.
      • Carpenter M.K.
      Feeder-free growth of undifferentiated human embryonic stem cells.
      ,
      • Rodin S.
      • Domogatskaya A.
      • Ström S.
      • Hansson E.M.
      • Chien K.R.
      • Inzunza J.
      • Hovatta O.
      • Tryggvason K.
      Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.
      ), the lower fibronectin content of hPSF ECM may contribute to its failure to support hESC maintenance. Indeed, an increase or decrease in fibronectin concentration away from an optimal, intermediate concentration has been shown to induce a switch in focal adhesion kinase signaling and promote differentiation of mouse embryonic stem cells (
      • Hunt G.C.
      • Singh P.
      • Schwarzbauer J.E.
      Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.
      ). Our feeder-free system for culturing hESCs on fibronectin-coated plates means that it is difficult to assess the levels of endogenous fibronectin produced by hESCs. However, we can deduce from immunostaining that once hESCs start undergoing the differentiation process, cells begin to organize fibronectin into fibrillar-like structures. In previous proteomic studies of conditioned medium, a high percentage of proteins identified comprised ECM components (
      • Chin A.C.
      • Fong W.J.
      • Goh L.T.
      • Philp R.
      • Oh S.K.
      • Choo A.B.
      Identification of proteins from feeder conditioned medium that support human embryonic stem cells.
      ,
      • Prowse A.B.
      • McQuade L.R.
      • Bryant K.J.
      • Van Dyk D.D.
      • Tuch B.E.
      • Gray P.P.
      A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells.
      ,
      • Prowse A.B.
      • McQuade L.R.
      • Bryant K.J.
      • Marcal H.
      • Gray P.P.
      Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media.
      ), such as perlecan, fibronectin, and fibrillin-1, which were also identified in our MS analysis of hESC-derived ECM as well as mouse and human feeder-derived ECMs. One of the aims of this study was to define an ECM substrate that sustains undifferentiated self-renewing hESCs. Exploiting our MS data, we identified and tested a number of ECM proteins as potential culture substrates. Some of these molecules were found to maintain hESCs for three passages, including fibrillin-1 as a single substrate, and perlecan and fibulin-2 in combination with a low, otherwise unsupportive, concentration of fibronectin. Other substrates tested, including tenascin C, collagen VI, biglycan, and versican, did not support hESC self-renewal. Single ECM molecules, such as fibronectin (
      • Amit M.
      • Shariki C.
      • Margulets V.
      • Itskovitz-Eldor J.
      Feeder layer- and serum-free culture of human embryonic stem cells.
      ,
      • Baxter M.A.
      • Camarasa M.V.
      • Bates N.
      • Small F.
      • Murray P.
      • Edgar D.
      • Kimber S.J.
      Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines.
      ), laminin (
      • Rodin S.
      • Domogatskaya A.
      • Ström S.
      • Hansson E.M.
      • Chien K.R.
      • Inzunza J.
      • Hovatta O.
      • Tryggvason K.
      Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.
      ), and vitronectin (
      • Braam S.R.
      • Zeinstra L.
      • Litjens S.
      • Ward-van Oostwaard D.
      • van den Brink S.
      • van Laake L.
      • Lebrin F.
      • Kats P.
      • Hochstenbach R.
      • Passier R.
      • Sonnenberg A.
      • Mummery C.L.
      Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via αvβ5 integrin.
      ), have been used previously as substrates for hESC culture. Our MS analysis of the ECM produced by hESCs cultured on fibronectin identified a number of other ECM components, including fibrillin-2, perlecan, thrombospondin, metalloproteinases, and growth factors. This lends weight to the idea that, even when hESCs are cultured on a single substrate, they produce their own specialized niche that may be involved in regulating pluripotency. Our data suggest that when hESCs are grown on a single substrate, they deposit a complex ECM, and it is likely that the interactions between the ECM components are crucial in providing the supportive niche conducive to continued stem cell self-renewal.
      Many proteins comprising or associated with fibrillin microfibrils were identified in our proteomic data sets, including fibrillin-1 and -2, fibulin-2, EMILIN-1, and latent TGF-β-binding protein (LTBP)-1 and -2. Fibrillin-1 was identified in both feeder ECMs and HUES1 ECM and was tested successfully as a substrate for at least short term culture of hESCs. Fibrillin-1 has been shown to mediate cell adhesion via integrin α5β1 (
      • Bax D.V.
      • Bernard S.E.
      • Lomas A.
      • Morgan A.
      • Humphries J.
      • Shuttleworth C.A.
      • Humphries M.J.
      • Kielty C.M.
      Cell adhesion to fibrillin-1 molecules and microfibrils is mediated by α5β1 and αvβ3 integrins.
      ), which we showed here was expressed by hESCs. Furthermore, fibrillin-1 has been reported to regulate the bioavailability of TGF-β (
      • Chaudhry S.S.
      • Cain S.A.
      • Morgan A.
      • Dallas S.L.
      • Shuttleworth C.A.
      • Kielty C.M.
      Fibrillin-1 regulates the bioavailability of TGFβ1.
      ), whose role in maintaining pluripotency through Smad pathway activation is well established (
      • James D.
      • Levine A.J.
      • Besser D.
      • Hemmati-Brivanlou A.
      TGFβ/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells.
      ). Indeed, our MS data revealed that ECMs produced by feeder cells and HUES1 cells contained TGF-β as well as LTBP-1 and thrombospondin-1, which are known to activate latent TGF-β (
      • Chaudhry S.S.
      • Cain S.A.
      • Morgan A.
      • Dallas S.L.
      • Shuttleworth C.A.
      • Kielty C.M.
      Fibrillin-1 regulates the bioavailability of TGFβ1.
      ,
      • Annes J.P.
      • Munger J.S.
      • Rifkin D.B.
      Making sense of latent TGFβ activation.
      ). Activation of TGF-β is normally tightly regulated, and the effects of TGF-β family signaling on stem cell pluripotency are diverse (
      • Sakaki-Yumoto M.
      • Katsuno Y.
      • Derynck R.
      TGF-β family signaling in stem cells.
      ). Because both high and very low concentrations of TGF-β family members can induce hESC differentiation, fibrillin-1, along with appropriate networks of ECM molecules, may function to modulate levels of TGF-β signaling appropriate to control stem cell maintenance or differentiation. Thus, the function of ECM molecules in regulating the availability of growth factors is likely to play a critical role in the maintenance of hESC pluripotency.
      Perlecan was identified in both the feeder ECMs and the HUES1 ECM, which is consistent with previous reports analyzing conditioned media and feeder cells (
      • Prowse A.B.
      • McQuade L.R.
      • Bryant K.J.
      • Marcal H.
      • Gray P.P.
      Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media.
      ,
      • Abraham S.
      • Riggs M.J.
      • Nelson K.
      • Lee V.
      • Rao R.R.
      Characterization of human fibroblast-derived extracellular matrix components for human pluripotent stem cell propagation.
      ). Perlecan interacts with fibronectin, fibulin-2, nidogen, and collagen IV, all of which were identified in our proteomic data sets. hESCs cultured on 20 μg/ml perlecan in combination with 5 μg/ml fibronectin retained their Oct4 expression after three passages, which is in agreement with a recently published paper by Abraham et al. (
      • Abraham S.
      • Riggs M.J.
      • Nelson K.
      • Lee V.
      • Rao R.R.
      Characterization of human fibroblast-derived extracellular matrix components for human pluripotent stem cell propagation.
      ) that also showed that perlecan in combination with fibronectin can support hESC pluripotency. Perlecan binds FGF through its heparan sulfate side chains (
      • Vigny M.
      • Ollier-Hartmann M.P.
      • Lavigne M.
      • Fayein N.
      • Jeanny J.C.
      • Laurent M.
      • Courtois Y.
      Specific binding of basic fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor.
      ) and promotes FGF receptor binding to modulate angiogenesis (
      • Aviezer D.
      • Hecht D.
      • Safran M.
      • Eisinger M.
      • David G.
      • Yayon A.
      Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis.
      ). Indeed, in endothelial cells, heparan sulfate chains interact with integrins to regulate binding of endostatin, an inhibitor of angiogenesis (
      • Faye C.
      • Moreau C.
      • Chautard E.
      • Jetne R.
      • Fukai N.
      • Ruggiero F.
      • Humphries M.J.
      • Olsen B.R.
      • Ricard-Blum S.
      Molecular interplay between endostatin, integrins, and heparan sulfate.
      ). FGF is an important self-renewal component in routine hESC culture, and it has been shown that heparin in solution can increase hESC survival under certain conditions (
      • Furue M.K.
      • Na J.
      • Jackson J.P.
      • Okamoto T.
      • Jones M.
      • Baker D.
      • Hata R.
      • Moore H.D.
      • Sato J.D.
      • Andrews P.W.
      Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium.
      ) and that heparin-binding surfaces are supportive of pluripotent hESCs in the long term (
      • Klim J.R.
      • Li L.
      • Wrighton P.J.
      • Piekarczyk M.S.
      • Kiessling L.L.
      A defined glycosaminoglycan-binding substratum for human pluripotent stem cells.
      ).
      Fibulin-2 is an ECM glycoprotein that binds other ECM molecules and, in fibroblasts, is deposited into a meshwork that contains fibronectin (
      • Sasaki T.
      • Göhring W.
      • Pan T.C.
      • Chu M.L.
      • Timpl R.
      Binding of mouse and human fibulin-2 to extracellular matrix ligands.
      ). In this study, fibulin-2 was identified in ECM produced by mouse feeder cells and HUES1 cells, in accordance with published data (
      • Sasaki T.
      • Göhring W.
      • Pan T.C.
      • Chu M.L.
      • Timpl R.
      Binding of mouse and human fibulin-2 to extracellular matrix ligands.
      ). Fibulin-2 was able to support maintenance of Oct4-positive hESCs when used in combination with 5 μg/ml fibronectin but not when used as a single substrate. Because fibulin-2 has been shown to colocalize with fibronectin in fibrils deposited by human fibroblasts, it is possible that the interaction of these two molecules may facilitate the correct organization of the ECM needed for hESC attachment and growth. Little is known about the influence of fibulin-2 on stem cell self-renewal, so further work is necessary to determine the role of fibulin-2 in conjunction with fibronectin in supporting pluripotency.
      In summary, our proteomic analysis allowed the cataloguing and comparison of ECMs that are supportive and unsupportive for hESC self-renewal. Some ECM proteins were enriched or expressed only in supportive ECM, and we demonstrated that several of these candidates alone or in combination with fibronectin could act as substrates to support at least short term self-renewal of hESCs. Furthermore, the presence of key supportive proteins in native ECMs may be sufficient to permit successful hESC growth, even in the presence of unsupportive components. Indeed, mouse and human feeder cells produced complex networks of ECM proteins with distinct compositions and network topologies, which suggests that the balance between ECM network properties, molecular composition, and specific protein-protein interactions plays a role in the maintenance of pluripotency. Given the outstanding need for a better understanding of stem cell maintenance, our data provide a useful resource for the further study of stem cell growth in vitro and microenvironmental control of stem cell function and fate in vivo.

      Acknowledgments

      We thank S. Warwood for mass spectrometric data acquisition; M. C. Jackson for flow cytometric data acquisition; J. N. Selley for bioinformatic support; N. Bates, S. E. Craig, and E.-J. Keevill for technical assistance; and K. A. Jones and M. D. Bass for discussions. We are grateful to T. Sasaki, S. A. Cain, C. M. Kielty, J. M. Whitelock, M. Koch, and D. R. Garrod for generous gifts of reagents.

      References

        • Darabi R.
        • Perlingeiro R.C.
        Lineage-specific reprogramming as a strategy for cell therapy.
        Cell Cycle. 2008; 7: 1732-1737
        • Hyslop L.A.
        • Armstrong L.
        • Stojkovic M.
        • Lako M.
        Human embryonic stem cells. Biology and clinical implications.
        Expert. Rev. Mol. Med. 2005; 7: 1-21
        • Hovatta O.
        • Mikkola M.
        • Gertow K.
        • Strömberg A.M.
        • Inzunza J.
        • Hreinsson J.
        • Rozell B.
        • Blennow E.
        • Andäng M.
        • Ahrlund-Richter L.
        A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.
        Hum. Reprod. 2003; 18: 1404-1409
        • Xu C.
        • Jiang J.
        • Sottile V.
        • McWhir J.
        • Lebkowski J.
        • Carpenter M.K.
        Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth.
        Stem Cells. 2004; 22: 972-980
        • Stojkovic P.
        • Lako M.
        • Stewart R.
        • Przyborski S.
        • Armstrong L.
        • Evans J.
        • Murdoch A.
        • Strachan T.
        • Stojkovic M.
        An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells.
        Stem Cells. 2005; 23: 306-314
        • Amit M.
        • Shariki C.
        • Margulets V.
        • Itskovitz-Eldor J.
        Feeder layer- and serum-free culture of human embryonic stem cells.
        Biol. Reprod. 2004; 70: 837-845
        • Richards M.
        • Tan S.
        • Fong C.Y.
        • Biswas A.
        • Chan W.K.
        • Bongso A.
        Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.
        Stem Cells. 2003; 21: 546-556
        • Baxter M.A.
        • Camarasa M.V.
        • Bates N.
        • Small F.
        • Murray P.
        • Edgar D.
        • Kimber S.J.
        Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines.
        Stem Cell Res. 2009; 3: 28-38
        • Xu C.
        • Inokuma M.S.
        • Denham J.
        • Golds K.
        • Kundu P.
        • Gold J.D.
        • Carpenter M.K.
        Feeder-free growth of undifferentiated human embryonic stem cells.
        Nat. Biotechnol. 2001; 19: 971-974
        • Wang G.
        • Zhang H.
        • Zhao Y.
        • Li J.
        • Cai J.
        • Wang P.
        • Meng S.
        • Feng J.
        • Miao C.
        • Ding M.
        • Li D.
        • Deng H.
        Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers.
        Biochem. Biophys. Res. Commun. 2005; 330: 934-942
        • Yao S.
        • Chen S.
        • Clark J.
        • Hao E.
        • Beattie G.M.
        • Hayek A.
        • Ding S.
        Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6907-6912
        • Liu N.
        • Lu M.
        • Tian X.
        • Han Z.
        Molecular mechanisms involved in self-renewal and pluripotency of embryonic stem cells.
        J. Cell. Physiol. 2007; 211: 279-286
        • Li L.
        • Xie T.
        Stem cell niche. Structure and function.
        Annu. Rev. Cell Dev. Biol. 2005; 21: 605-631
        • Ohlstein B.
        • Kai T.
        • Decotto E.
        • Spradling A.
        The stem cell niche. Theme and variations.
        Curr. Opin. Cell Biol. 2004; 16: 693-699
        • Scadden D.T.
        The stem-cell niche as an entity of action.
        Nature. 2006; 441: 1075-1079
        • Unwin R.D.
        • Gaskell S.J.
        • Evans C.A.
        • Whetton A.D.
        The potential for proteomic definition of stem cell populations.
        Exp. Hematol. 2003; 31: 1147-1159
        • Beattie G.M.
        • Lopez A.D.
        • Bucay N.
        • Hinton A.
        • Firpo M.T.
        • King C.C.
        • Hayek A.
        Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers.
        Stem Cells. 2005; 23: 489-495
        • Steiner D.
        • Khaner H.
        • Cohen M.
        • Even-Ram S.
        • Gil Y.
        • Itsykson P.
        • Turetsky T.
        • Idelson M.
        • Aizenman E.
        • Ram R.
        • Berman-Zaken Y.
        • Reubinoff B.
        Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension.
        Nat. Biotechnol. 2010; 28: 361-364
        • Braam S.R.
        • Zeinstra L.
        • Litjens S.
        • Ward-van Oostwaard D.
        • van den Brink S.
        • van Laake L.
        • Lebrin F.
        • Kats P.
        • Hochstenbach R.
        • Passier R.
        • Sonnenberg A.
        • Mummery C.L.
        Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via αvβ5 integrin.
        Stem Cells. 2008; 26: 2257-2265
        • Prowse A.B.
        • Doran M.R.
        • Cooper-White J.J.
        • Chong F.
        • Munro T.P.
        • Fitzpatrick J.
        • Chung T.L.
        • Haylock D.N.
        • Gray P.P.
        • Wolvetang E.J.
        Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media.
        Biomaterials. 2010; 31: 8281-8288
        • Klimanskaya I.
        • Chung Y.
        • Meisner L.
        • Johnson J.
        • West M.D.
        • Lanza R.
        Human embryonic stem cells derived without feeder cells.
        Lancet. 2005; 365: 1636-1641
        • Hughes C.
        • Radan L.
        • Chang W.Y.
        • Stanford W.L.
        • Betts D.H.
        • Postovit L.M.
        • Lajoie G.A.
        Mass spectrometry-based proteomic analysis of the matrix microenvironment in pluripotent stem cell culture.
        Mol. Cell. Proteomics. 2012; 11: 1924-1936
        • Chin A.C.
        • Fong W.J.
        • Goh L.T.
        • Philp R.
        • Oh S.K.
        • Choo A.B.
        Identification of proteins from feeder conditioned medium that support human embryonic stem cells.
        J. Biotechnol. 2007; 130: 320-328
        • Lim J.W.
        • Bodnar A.
        Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells.
        Proteomics. 2002; 2: 1187-1203
        • Harkness L.
        • Christiansen H.
        • Nehlin J.
        • Barington T.
        • Andersen J.S.
        • Kassem M.
        Identification of a membrane proteomic signature for human embryonic stem cells independent of culture conditions.
        Stem Cell Res. 2008; 1: 219-227
        • Intoh A.
        • Kurisaki A.
        • Yamanaka Y.
        • Hirano H.
        • Fukuda H.
        • Sugino H.
        • Asashima M.
        Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells.
        Proteomics. 2009; 9: 126-137
        • McQuade L.R.
        • Schmidt U.
        • Pascovici D.
        • Stojanov T.
        • Baker M.S.
        Improved membrane proteomics coverage of human embryonic stem cells by peptide IPG-IEF.
        J. Proteome Res. 2009; 8: 5642-5649
        • Brill L.M.
        • Xiong W.
        • Lee K.B.
        • Ficarro S.B.
        • Crain A.
        • Xu Y.
        • Terskikh A.
        • Snyder E.Y.
        • Ding S.
        Phosphoproteomic analysis of human embryonic stem cells.
        Cell Stem Cell. 2009; 5: 204-213
        • Van Hoof D.
        • Heck A.J.
        • Krijgsveld J.
        • Mummery C.L.
        Proteomics and human embryonic stem cells.
        Stem Cell Res. 2008; 1: 169-182
        • McKay T.R.
        • Camarasa M.V.
        • Iskender B.
        • Ye J.
        • Bates N.
        • Miller D.
        • Fitzsimmons J.C.
        • Foxler D.
        • Mee M.
        • Sharp T.V.
        • Aplin J.
        • Brison D.R.
        • Kimber S.J.
        Human feeder cell line for derivation and culture of hESc/hiPSc.
        Stem Cell Res. 2011; 7: 154-162
        • Thomson J.A.
        • Itskovitz-Eldor J.
        • Shapiro S.S.
        • Waknitz M.A.
        • Swiergiel J.J.
        • Marshall V.S.
        • Jones J.M.
        Embryonic stem cell lines derived from human blastocysts.
        Science. 1998; 282: 1145-1147
        • Camarasa M.V.
        • Kerr R.W.
        • Sneddon S.F.
        • Bates N.
        • Shaw L.
        • Oldershaw R.A.
        • Small F.
        • Baxter M.A.
        • Mckay T.R.
        • Brison D.R.
        • Kimber S.J.
        Derivation of Man-1 and Man-2 research grade human embryonic stem cell lines.
        In Vitro Cell. Dev. Biol. Anim. 2010; 46: 386-394
        • Cowan C.A.
        • Klimanskaya I.
        • McMahon J.
        • Atienza J.
        • Witmyer J.
        • Zucker J.P.
        • Wang S.
        • Morton C.C.
        • McMahon A.P.
        • Powers D.
        • Melton D.A.
        Derivation of embryonic stem-cell lines from human blastocysts.
        N. Engl. J. Med. 2004; 350: 1353-1356
        • Lu J.
        • Hou R.
        • Booth C.J.
        • Yang S.H.
        • Snyder M.
        Defined culture conditions of human embryonic stem cells.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 5688-5693
        • Cain S.A.
        • McGovern A.
        • Baldwin A.K.
        • Baldock C.
        • Kielty C.M.
        Fibrillin-1 mutations causing Weill-Marchesani syndrome and acromicric and geleophysic dysplasias disrupt heparan sulfate interactions.
        PLoS One. 2012; 7: e48634
        • Shevchenko A.
        • Wilm M.
        • Vorm O.
        • Mann M.
        Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.
        Anal. Chem. 1996; 68: 850-858
        • Humphries J.D.
        • Byron A.
        • Bass M.D.
        • Craig S.E.
        • Pinney J.W.
        • Knight D.
        • Humphries M.J.
        Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6.
        Sci. Signal. 2009; 2: ra51
        • Perkins D.N.
        • Pappin D.J.
        • Creasy D.M.
        • Cottrell J.S.
        Probability-based protein identification by searching sequence databases using mass spectrometry data.
        Electrophoresis. 1999; 20: 3551-3567
        • Keller A.
        • Nesvizhskii A.I.
        • Kolker E.
        • Aebersold R.
        Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.
        Anal. Chem. 2002; 74: 5383-5392
        • Nesvizhskii A.I.
        • Vitek O.
        • Aebersold R.
        Analysis and validation of proteomic data generated by tandem mass spectrometry.
        Nat. Methods. 2007; 4: 787-797
        • Barsnes H.
        • Vizcaíno J.A.
        • Eidhammer I.
        • Martens L.
        PRIDE Converter. Making proteomics data-sharing easy.
        Nat. Biotechnol. 2009; 27: 598-599
        • Vizcaíno J.A.
        • Côté R.
        • Reisinger F.
        • Barsnes H.
        • Foster J.M.
        • Rameseder J.
        • Hermjakob H.
        • Martens L.
        The Proteomics Identifications database. 2010 update.
        Nucleic Acids Res. 2010; 38: D736-D742
        • Liu H.
        • Sadygov R.G.
        • Yates 3rd, J.R.
        A model for random sampling and estimation of relative protein abundance in shotgun proteomics.
        Anal. Chem. 2004; 76: 4193-4201
        • Old W.M.
        • Meyer-Arendt K.
        • Aveline-Wolf L.
        • Pierce K.G.
        • Mendoza A.
        • Sevinsky J.R.
        • Resing K.A.
        • Ahn N.G.
        Comparison of label-free methods for quantifying human proteins by shotgun proteomics.
        Mol. Cell. Proteomics. 2005; 4: 1487-1502
        • Zybailov B.
        • Coleman M.K.
        • Florens L.
        • Washburn M.P.
        Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling.
        Anal. Chem. 2005; 77: 6218-6224
        • Byron A.
        • Humphries J.D.
        • Bass M.D.
        • Knight D.
        • Humphries M.J.
        Proteomic analysis of integrin adhesion complexes.
        Sci. Signal. 2011; 4: pt2
        • Huang da W.
        • Sherman B.T.
        • Lempicki R.A.
        Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
        Nat. Protoc. 2009; 4: 44-57
        • Barrell D.
        • Dimmer E.
        • Huntley R.P.
        • Binns D.
        • O'Donovan C.
        • Apweiler R.
        The GOA database in 2009. An integrated Gene Ontology Annotation resource.
        Nucleic Acids Res. 2009; 37: D396-D403
        • Binns D.
        • Dimmer E.
        • Huntley R.
        • Barrell D.
        • O'Donovan C.
        • Apweiler R.
        QuickGO. A web-based tool for Gene Ontology searching.
        Bioinformatics. 2009; 25: 3045-3046
        • Shannon P.
        • Markiel A.
        • Ozier O.
        • Baliga N.S.
        • Wang J.T.
        • Ramage D.
        • Amin N.
        • Schwikowski B.
        • Ideker T.
        Cytoscape. A software environment for integrated models of biomolecular interaction networks.
        Genome Res. 2003; 13: 2498-2504
        • Wu J.
        • Vallenius T.
        • Ovaska K.
        • Westermarck J.
        • Mäkelä T.P.
        • Hautaniemi S.
        Integrated network analysis platform for protein-protein interactions.
        Nat. Methods. 2009; 6: 75-77
        • Chautard E.
        • Ballut L.
        • Thierry-Mieg N.
        • Ricard-Blum S.
        MatrixDB, a database focused on extracellular protein-protein and protein-carbohydrate interactions.
        Bioinformatics. 2009; 25: 690-691
        • Zaidel-Bar R.
        • Itzkovitz S.
        • Ma'ayan A.
        • Iyengar R.
        • Geiger B.
        Functional atlas of the integrin adhesome.
        Nat. Cell Biol. 2007; 9: 858-867
        • Ostlund G.
        • Schmitt T.
        • Forslund K.
        • Köstler T.
        • Messina D.N.
        • Roopra S.
        • Frings O.
        • Sonnhammer E.L.
        InParanoid 7. New algorithms and tools for eukaryotic orthology analysis.
        Nucleic Acids Res. 2010; 38: D196-D203
        • Assenov Y.
        • Ramírez F.
        • Schelhorn S.E.
        • Lengauer T.
        • Albrecht M.
        Computing topological parameters of biological networks.
        Bioinformatics. 2008; 24: 282-284
        • Park J.H.
        • Kim S.J.
        • Oh E.J.
        • Moon S.Y.
        • Roh S.I.
        • Kim C.G.
        • Yoon H.S.
        Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line.
        Biol. Reprod. 2003; 69: 2007-2014
        • Humphries J.D.
        • Byron A.
        • Humphries M.J.
        Integrin ligands at a glance.
        J. Cell Sci. 2006; 119: 3901-3903
        • Byron A.
        • Humphries J.D.
        • Humphries M.J.
        Defining the extracellular matrix using proteomics.
        Int. J. Exp. Pathol. 2013; 94: 75-92
        • Yook S.H.
        • Oltvai Z.N.
        • Barabási A.L.
        Functional and topological characterization of protein interaction networks.
        Proteomics. 2004; 4: 928-942
        • Seebacher J.
        • Gavin A.C.
        SnapShot. Protein-protein interaction networks.
        Cell. 2011; 144: 1000-1000.e1
        • Rodin S.
        • Domogatskaya A.
        • Ström S.
        • Hansson E.M.
        • Chien K.R.
        • Inzunza J.
        • Hovatta O.
        • Tryggvason K.
        Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.
        Nat. Biotechnol. 2010; 28: 611-615
        • Miyazaki T.
        • Futaki S.
        • Suemori H.
        • Taniguchi Y.
        • Yamada M.
        • Kawasaki M.
        • Hayashi M.
        • Kumagai H.
        • Nakatsuji N.
        • Sekiguchi K.
        • Kawase E.
        Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells.
        Nat. Commun. 2012; 3: 1236
        • Montes R.
        • Ligero G.
        • Sanchez L.
        • Catalina P.
        • de la Cueva T.
        • Nieto A.
        • Melen G.J.
        • Rubio R.
        • García-Castro J.
        • Bueno C.
        • Menendez P.
        Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media. Distinct requirements for TGF-β and IGF-II.
        Cell Res. 2009; 19: 698-709
        • Xiao L.
        • Yuan X.
        • Sharkis S.J.
        Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells.
        Stem Cells. 2006; 24: 1476-1486
        • Hunt G.C.
        • Singh P.
        • Schwarzbauer J.E.
        Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.
        Exp. Cell Res. 2012; 318: 1820-1831
        • Wilson R.
        • Diseberg A.F.
        • Gordon L.
        • Zivkovic S.
        • Tatarczuch L.
        • Mackie E.J.
        • Gorman J.J.
        • Bateman J.F.
        Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics.
        Mol. Cell. Proteomics. 2010; 9: 1296-1313
        • James D.
        • Levine A.J.
        • Besser D.
        • Hemmati-Brivanlou A.
        TGFβ/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells.
        Development. 2005; 132: 1273-1282
        • Hongisto H.
        • Vuoristo S.
        • Mikhailova A.
        • Suuronen R.
        • Virtanen I.
        • Otonkoski T.
        • Skottman H.
        Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture.
        Stem Cell Res. 2012; 8: 97-108
        • Prowse A.B.
        • McQuade L.R.
        • Bryant K.J.
        • Van Dyk D.D.
        • Tuch B.E.
        • Gray P.P.
        A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells.
        Proteomics. 2005; 5: 978-989
        • Prowse A.B.
        • McQuade L.R.
        • Bryant K.J.
        • Marcal H.
        • Gray P.P.
        Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media.
        J. Proteome Res. 2007; 6: 3796-3807
        • Bax D.V.
        • Bernard S.E.
        • Lomas A.
        • Morgan A.
        • Humphries J.
        • Shuttleworth C.A.
        • Humphries M.J.
        • Kielty C.M.
        Cell adhesion to fibrillin-1 molecules and microfibrils is mediated by α5β1 and αvβ3 integrins.
        J. Biol. Chem. 2003; 278: 34605-34616
        • Chaudhry S.S.
        • Cain S.A.
        • Morgan A.
        • Dallas S.L.
        • Shuttleworth C.A.
        • Kielty C.M.
        Fibrillin-1 regulates the bioavailability of TGFβ1.
        J. Cell Biol. 2007; 176: 355-367
        • Annes J.P.
        • Munger J.S.
        • Rifkin D.B.
        Making sense of latent TGFβ activation.
        J. Cell Sci. 2003; 116: 217-224
        • Sakaki-Yumoto M.
        • Katsuno Y.
        • Derynck R.
        TGF-β family signaling in stem cells.
        Biochim. Biophys. Acta. 2013; 1830: 2280-2296
        • Abraham S.
        • Riggs M.J.
        • Nelson K.
        • Lee V.
        • Rao R.R.
        Characterization of human fibroblast-derived extracellular matrix components for human pluripotent stem cell propagation.
        Acta Biomater. 2010; 6: 4622-4633
        • Vigny M.
        • Ollier-Hartmann M.P.
        • Lavigne M.
        • Fayein N.
        • Jeanny J.C.
        • Laurent M.
        • Courtois Y.
        Specific binding of basic fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor.
        J. Cell. Physiol. 1988; 137: 321-328
        • Aviezer D.
        • Hecht D.
        • Safran M.
        • Eisinger M.
        • David G.
        • Yayon A.
        Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis.
        Cell. 1994; 79: 1005-1013
        • Faye C.
        • Moreau C.
        • Chautard E.
        • Jetne R.
        • Fukai N.
        • Ruggiero F.
        • Humphries M.J.
        • Olsen B.R.
        • Ricard-Blum S.
        Molecular interplay between endostatin, integrins, and heparan sulfate.
        J. Biol. Chem. 2009; 284: 22029-22040
        • Furue M.K.
        • Na J.
        • Jackson J.P.
        • Okamoto T.
        • Jones M.
        • Baker D.
        • Hata R.
        • Moore H.D.
        • Sato J.D.
        • Andrews P.W.
        Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13409-13414
        • Klim J.R.
        • Li L.
        • Wrighton P.J.
        • Piekarczyk M.S.
        • Kiessling L.L.
        A defined glycosaminoglycan-binding substratum for human pluripotent stem cells.
        Nat. Methods. 2010; 7: 989-994
        • Sasaki T.
        • Göhring W.
        • Pan T.C.
        • Chu M.L.
        • Timpl R.
        Binding of mouse and human fibulin-2 to extracellular matrix ligands.
        J. Mol. Biol. 1995; 254: 892-899