Efficient and Scalable Culture of Single Dissociated Human Pluripotent Stem Cells Using Recombinant E8 Fragments of Human Laminin Isoforms

Takamichi Miyazaki1, Eihachiro Kawase1

1 Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.18
DOI:  10.1002/9780470151808.sc01c18s32
Online Posting Date:  February, 2015
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Abstract

This unit describes a protocol for efficient expansion of human pluripotent stem cells (hPSCs). A key feature of this method is subculture of hPSCs by single‐cell dissociation passaging on substrates coated with recombinant E8 fragments of human laminin isoforms (LM‐E8s). LM‐E8s, provide superior adhesion over intact laminin isoforms and Matrigel. Single hPSCs seeded on LM‐E8s show accelerated migration and rapid reconstruction of clusters, resulting in robust survival and proliferation. This protocol yields 200‐fold more hPSCs than conventional subculture methods in 1 month of culture. Furthermore, this protocol can be easily adapted to most hPSC lines in combination with the use of various xeno‐free, defined culture media, and large‐scale expansion of hPSCs is easily achievable to facilitate the practical applications of hPSCs. © 2015 by John Wiley & Sons, Inc.

Keywords: human pluripotent stem cells (hPSCs); single‐cell dissociation culture; laminin isoform; laminin fragment; culture substrate

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Coating of Substrates onto Culture Vessels
  • Basic Protocol 2: Starting the Feeder‐Free Culture
  • Basic Protocol 3: Single‐Cell Dissociation Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Coating of Substrates onto Culture Vessels

  Materials
  • Dulbecco's phosphate‐buffered saline without CaCl 2 and MgCl 2 (D‐PBS(‐); Sigma, cat. no. D5652)
  • LM‐E8 stock solution, 500 μg/ml (iMatrix‐511; see recipe)
  • Dulbecco's modified Eagle's medium (DMEM)/F12, 1:1 nutrient mixture (Sigma, cat. no. D6421)
  • Matrigel™ Basement Membrane Matrix, Growth Factor Reduced (Corning, cat. no. 354230)
  • Culture vessels: there is no specific restriction on the culture vessels used for coating LM‐E8s, but culture vessels with a hydrophobic surface, such as ultralow attachment, should be avoided

Basic Protocol 2: Starting the Feeder‐Free Culture

  Materials
  • hPSCs cultured on mouse embryonic fibroblast (MEF) feeder layer
  • hPSC detachment solution, e.g., CTK solution (ReproCELL, Inc., cat. no. RCHETP002)
  • hPSC culture medium (ReproCELL, Inc., cat. no. RCHEMD001)
  • Defined culture medium for hPSCs, e.g. N2B27 medium (see recipe): other suitable feeder‐free media include: mTeSR™1 (STEMCELL Technologies, cat. no. 05850), TeSR™2 (STEMCELL Technologies, cat. no. 05860), and STEMPRO® hESC SFM (Life Technologies, cat. no. A1000701)

Basic Protocol 3: Single‐Cell Dissociation Culture

  Materials
  • hPSC feeder‐free cultures in defined medium ( protocol 2, or daily subculture in this protocol)
  • D‐PBS(‐) (Sigma, cat. no. D5652)
  • EDTA dissociation solution (see recipe)
  • TrypLE Select (Life Technologies, cat. no. 12563‐011)
  • Defined culture medium for hPSCs, e.g. N2B27 medium (see recipe) or other suitable feeder‐free medium (see protocol 2, Materials list)
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Figures

Videos

Literature Cited

Literature Cited
   Liu, Y. , Song, Z. , Zhao, Y. , Qin, H. , Cai, J. , Zhang, H. , Yu, T. , Jiang, S. , Wang, G. , Ding, M. , and Deng, H. 2006. A novel chemical‐defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells. Biochem. Biophys. Res. Commun. 346:131‐139.
   Miner, J.H. and Yurchenco, P.D. 2004. Laminin functions in tissue morphogenesis. Annu. Rev. Cell Dev. Biol. 20:255‐284.
   Miyazaki, T. , Futaki, S. , Hasegawa, K. , Kawasaki, M. , Sanzen, N. , Hayashi, M. , Kawase, E. , Sekiguchi, K. , Nakatsuji, N. , and Suemori, H. 2008. Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem. Biophys. Res. Commun. 375:27‐32.
   Miyazaki, T. , Futaki, S. , Suemori, H. , Taniguchi, Y. , Yamada, M. , Kawasaki, M. , Hayashi, M. , Kumagai, H. , Nakatsuji, N. , Sekiguchi, K. , and Kawase, E. 2012. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat. Commun. 3:1236.
   Nishiuchi, R. , Takagi, J. , Hayashi, M. , Ido, H. , Yagi, Y. , Sanzen, N. , Tsuji, T. , Yamada, M. , and Sekiguchi, K. 2006. Ligand‐binding specificities of laminin‐binding integrins: A comprehensive survey of laminin‐integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins. Matrix Biol. 25:189‐197.
   Takagi, J. 2007. Structural basis for ligand recognition by integrins. Curr. Opin. Cell Biol. 19(5):557‐64.
   Taniguchi, Y , Ido, H , Sanzen, N , Hayashi, M , Sato‐Nishiuchi, R , Futaki, S , and Sekiguchi, K . 2009. The C‐terminal region of laminin beta chains modulates the integrin binding affinities of laminins. J. Biol. Chem. 284:7820‐7831.
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