Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells

Paul W. Burridge1, Alexandra Holmström1, Joseph C. Wu1

1 Department of Medicine (Division of Cardiology), Stanford University School of Medicine, Stanford, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.3
DOI:  10.1002/0471142905.hg2103s87
Online Posting Date:  October, 2015
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Abstract

Since the first discovery that human pluripotent stem cells (hPS cells) can differentiate to cardiomyocytes, efforts have been made to optimize the conditions under which this process occurs. One of the most effective methodologies to optimize this process is reductionist simplification of the medium formula, which eliminates complex animal‐derived components to help reveal the precise underlying mechanisms. Here we describe our latest, cost‐effective and efficient methodology for the culture of hPS cells in the pluripotent state using a modified variant of chemically defined E8 medium. We provide exact guidelines for cell handling under these conditions, including non‐enzymatic EDTA passaging, which have been optimized for subsequent cardiomyocyte differentiation. We describe in depth the latest version of our monolayer chemically defined small molecule differentiation protocol, including metabolic selection–based cardiomyocyte purification and the addition of triiodothyronine to enhance cardiomyocyte maturation. Finally, we describe a method for the dissociation of hPS cell–derived cardiomyocytes, cryopreservation, and thawing. © 2015 by John Wiley & Sons, Inc.

Keywords: human induced pluripotent stem cells; differentiation; cardiac; cardiomyocyte; chemically defined; monolayer

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture and Cardiomyocyte Differentiation of Human iPS Cells
  • Support Protocol 1: Characterization of Cardiomyocytes by Flow Cytometry
  • Support Protocol 2: Characterization by Immunofluorescent Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Culture and Cardiomyocyte Differentiation of Human iPS Cells

  Materials
  • Human induced pluripotent stem cells (hiPS cells; see information in step 1)
  • E8‐Y medium (see recipe)
  • E8 medium (without Y27632; see recipe)
  • 0.5 mM EDTA (see recipe)
  • CDM3‐C (with CHIR99021; see recipe)
  • CDM3‐C59 (with Wnt‐C59; see recipe)
  • CDM3‐L (without D‐glucose, with L‐lactic acid; see recipe)
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (CMF‐DPBS)
  • TrypLE Express (Life Technologies, cat. no. 12605‐036)
  • Liberase TH, 260 U/50 mg (Roche, cat. no. 05401151001), resuspend in 10 ml WFI water (Corning, cat. no. 25‐055‐CV) and make 500‐μl aliquots; store at −20°C
  • DNase I, 277 U/μl (Life Technologies, cat. no 18047‐019)
  • CDM3 (see recipe)
  • Fetal bovine serum (FBS; Life Technologies, cat. no. 10082‐147)
  • Dimethylsulfoxide (DMSO; Fisher Scientific, cat. no. BP231‐1)
  • Liquid N 2
  • CDM3‐T (with T 3; see recipe)
  • 15‐ml (Corning Falcon, cat. no. 352097) and 50‐ml (Corning Falcon, cat. no. 352098) polystyrene conical tubes
  • Matrigel‐coated (see recipe) 6, 12, 24, 96, 384‐well cell culture plates (Greiner, cat. no. 657160, 665180, 662160, 655090, 781091, respectively)
  • Centrifuge (e.g., Thermo Sorvall ST8)
  • 100‐μm cell strainer (Corning Falcon, cat. no. 352360)
  • Luna Automated Cell Counter (Logos Biosystems, cat. no. L20001)
  • Cyrovials (Greiner, cat. no. 122261)
  • Coolcell LX (Biocision, cat. no. BCS‐405)
  • Additional reagents and equipment for generating hiPS cells [unit 4.1 (Park and Daley, ) and unit 4.2 (Ohnuki et al, )]
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations are carried out in a humidified 37°C, 5% CO 2 incubator (Thermo Scientific Heracell VIOS) unless otherwise specified. We have found that 5% O 2 (hypoxic) incubators are not essential for the success of this protocol.NOTE: We do not place any media in a 37°C water bath before use due to concerns regarding the temperature stability of the FGF2 in the media (Chen et al., ). Bringing the media to room temperature is sufficient, and we have found no noticeable effects on cell growth by using 4°C media.

Support Protocol 1: Characterization of Cardiomyocytes by Flow Cytometry

  Materials
  • Cardiomyocytes ( protocol 1Basic Protocol)
  • 1% (w/v) PFA in CMF‐DPBS (prepare from 20% PFA; Electron Microscopy Sciences, cat. no. 15713‐S)
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (CMF‐DPBS)
  • 90% (v/v) methanol (Fisher, cat. no. A412‐1)
  • 0.5% (w/v) BSA (Sigma‐Aldrich, cat. no. A3311) in CMF‐DPBS
  • 0.5% (w/v) BSA (Sigma‐Aldrich, cat. no. A3311) in CMF‐DPBS containing 0.1% (v/v) Triton X‐100 (Sigma Aldrich, cat. no. X100)
  • TNNT2 mouse monoclonal (13‐11) primary antibody (Thermo Scientific, cat. no. MS‐295‐P)
  • AlexaFluor 488–conjugated goat anti–mouse IgG 1 (Life Technologies, cat. no. A21121)
  • Flow cytometry tubes (Corning Falcon, cat. no. 352235)
  • Centrifuge accommodating TX‐750 rotor
  • 5/7 ml tube buckets with decanting aid for TX‐750 rotor (Thermo, cat. no. 75003732)
  • Flow cytometer capable for analyzing FITC and Texas Red such as Beckman Coulter CytoFLEX

Support Protocol 2: Characterization by Immunofluorescent Staining

  Material
  • Cardiomyocytes ( protocol 1Basic Protocol)
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (CMF‐DPBS)
  • 4% (w/v) PFA in CMF‐DPBS (prepare from 20% PFA; Electron Microscope Sciences, cat. no. 15713‐S)
  • 0.5% (v/v) Triton X‐100 (Sigma‐Aldrich, cat. no. X100) in CMF‐DPBS
  • 3% (w/v) BSA (Sigma‐Aldrich, cat. no. A3311) in CMF‐DPBS
  • TNNT2 (Troponin T) primary antibody, rabbit polyclonal IgG (Abcam, cat. no. ab45932)
  • ACTN2 (α‐actinin) primary antibody, mouse monoclonal IgG 1, clone EA‐53 (Sigma‐Aldrich, cat. no. A7811)
  • AlexaFluor 488–conjugated goat anti‐rabbit IgG (Life Technologies, cat. no. A11008)
  • AlexaFluor 594–conjugated goat anti‐mouse IgG1 (Life Technologies, cat. no. A21125)
  • Prolong Diamond with DAPI (Life Technologies, cat. no. P36962)
  • 8‐well Lab‐Tek II chamber slides (Thermo Nunc, cat .no. 154534)
  • 12‐well Matek glass No. 1.5 plates (Matek, cat no. P12G‐1.5‐14‐F)
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Figures

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Literature Cited

Literature Cited
  Beers, J., Gulbranson, D.R., George, N., Siniscalchi, L.I., Jones, J., Thomson, J.A., and Chen, G. 2012. Passaging and colony expansion of human pluripotent stem cells by enzyme‐free dissociation in chemically defined culture conditions. Nat. Protoc. 7:2029‐2040. doi: 10.1038/nprot.2012.130.
  Burridge, P.W., Anderson, D., Priddle, H., Barbadillo Munoz, M.D., Chamberlain, S., Allegrucci, C., Young, L.E., and Denning, C. 2007. Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V‐96 plate aggregation system highlights interline variability. Stem Cells 25:929‐938. doi: 10.1634/stemcells.2006‐0598.
  Burridge, P.W., Thompson, S., Millrod, M.A., Weinberg, S., Yuan, X., Peters, A., Mahairaki, V., Koliatsos, V.E., Tung, L., and Zambidis, E.T. 2011. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One 6:e18293. doi: 10.1371/journal.pone.0018293.
  Burridge, P.W., Matsa, E., Shukla, P., Lin, Z.C., Churko, J.M., Ebert, A.D., Lan, F., Diecke, S., Huber, B., Mordwinkin, N.M., Plews, J.R., Abilez, O.J., Cui, B., Gold, J.D., and Wu, J.C. 2014. Chemically defined generation of human cardiomyocytes. Nat. Methods 11:855‐860. doi: 10.1038/nmeth.2999.
  Chen, K.G., Hamilton, R.S., Robey, P.G., and Mallon, B.S. 2014. Alternative cultures for human pluripotent stem cell production, maintenance, and genetic analysis. J. Vis. Exp. Jul 24;(89). doi: 10.3791/51519.
  Chen, G., Gulbranson, D.R., Yu, P., Hou, Z., and Thomson, J.A. 2012. Thermal stability of fibroblast growth factor protein is a determinant factor in regulating self‐renewal, differentiation, and reprogramming in human pluripotent stem cells. Stem Cells 30:623‐630. doi: 10.1002/stem.1021.
  Chen, G., Gulbranson, D.R., Hou, Z., Bolin, J.M., Ruotti, V., Probasco, M.D., Smuga‐Otto, K., Howden, S.E., Diol, N.R., Propson, N.E., Wagner, R., Lee, G.O., Antosiewicz‐Bourget, J., Teng, J.M., and Thomson, J.A. 2011. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8:424‐429. doi: 10.1038/nmeth.1593.
  Denning, C., Allegrucci, C., Priddle, H., Barbadillo‐Munoz, M.D., Anderson, D., Self, T., Smith, N.M., Parkin, C.T., and Young, L.E. 2006. Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES‐7. Int. J. Dev. Biol. 50:27‐37. doi: 10.1387/ijdb.052107cd.
  Ezashi, T., Das, P., and Roberts, R.M. 2005. Low O2 tensions and the prevention of differentiation of hES cells. Proc. Natl. Acad. Sci. U. S. A. 102:4783‐4788. doi: 10.1073/pnas.0501283102.
  Forristal, C.E., Wright, K.L., Hanley, N.A., Oreffo, R.O., and Houghton, F.D. 2010. Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions. Reproduction 139:85‐97. doi: 10.1530/REP‐09‐0300.
  Gonzalez, R., Lee, J.W., and Schultz, P.G. 2011. Stepwise chemically induced cardiomyocyte specification of human embryonic stem cells. Angew. Chem. Int. Ed. Engl. 50:11181‐11185. doi: 10.1002/anie.201103909
  Lian, X., Hsiao, C., Wilson, G., Zhu, K., Hazeltine, L.B., Azarin, S.M., Raval, K.K., Zhang, J., Kamp, T.J., and Palecek, S.P. 2012. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc. Natl. Acad. Sci. U. S. A. 109:E1848‐1857. doi: 10.1073/pnas.1200250109.
  Ludwig, T.E., Levenstein, M.E., Jones, J.M., Berggren, W.T., Mitchen, E.R., Frane, J.L., Crandall, L.J., Daigh, C.A., Conard, K.R., Piekarczyk, M.S., Llanas, R.A., and Thomson, J.A. 2006. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24:185‐187. doi: 10.1038/nbt1177.
  Marx, V. 2015. Stem cells: Disease models that show and tell. Nat. Methods 12:111‐114. doi: 10.1038/nmeth.3263.
  McKernan, R. and Watt, F.M. 2013. What is the point of large‐scale collections of human induced pluripotent stem cells? Nat. Biotechnol. 31:875‐877. doi: 10.1038/nbt.2710.
  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. doi: 10.1038/ncomms2231.
  Narva, E., Pursiheimo, J.P., Laiho, A., Rahkonen, N., Emani, M.R., Viitala, M., Laurila, K., Sahla, R., Lund, R., Lahdesmaki, H., Jaakkola, P., and Lahesmaa, R. 2013. Continuous hypoxic culturing of human embryonic stem cells enhances SSEA‐3 and MYC levels. PLoS One 8:e78847. doi: 10.1371/journal.pone.0078847.
  Ohnuki, M., Takahashi, K., and Yamanaka, S. 2009. Generation and characterization of human induced pluripotent stem cells. Curr. Protoc. Stem Cell Biol. 9:4A.2.1‐4A.2.25.
  Park, I.‐H. and Daley, G.Q. 2009. Human iPS cell derivation/reprogramming. Curr. Protoc. Stem Cell Biol. 8:4A.1.1‐4A.1.8.
  Tohyama, S., Hattori, F., Sano, M., Hishiki, T., Nagahata, Y., Matsuura, T., Hashimoto, H., Suzuki, T., Yamashita, H., Satoh, Y., Egashira, T., Seki, T., Muraoka, N., Yamakawa, H., Ohgino, Y., Tanaka, T., Yoichi, M., Yuasa, S., Murata, M., Suematsu, M., and Fukuda, K. 2013. Distinct metabolic flow enables large‐scale purification of mouse and human pluripotent stem cell‐derived cardiomyocytes. Cell Stem Cell 12:127‐137. doi: 10.1016/j.stem.2012.09.013.
  Xu, C., He, J.Q., Kamp, T.J., Police, S., Hao, X., O'Sullivan, C., Carpenter, M.K., Lebkowski, J., and Gold, J.D. 2006. Human embryonic stem cell‐derived cardiomyocytes can be maintained in defined medium without serum. Stem Cells Dev. 15:931‐941. doi: 10.1089/scd.2006.15.931.
  Yang, X., Rodriguez, M., Pabon, L., Fischer, K.A., Reinecke, H., Regnier, M., Sniadecki, N.J., Ruohola‐Baker, H., and Murry, C.E. 2014. Tri‐iodo‐l‐thyronine promotes the maturation of human cardiomyocytes‐derived from induced pluripotent stem cells. J. Mol. Cell. Cardiol. 72:296‐304. doi: 10.1016/j.yjmcc.2014.04.005.
  Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., and Yamanaka, S. 2009. Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell 5:237‐241. doi: 10.1016/j.stem.2009.08.001.
  Yu, J., Chau, K.F., Vodyanik, M.A., Jiang, J., and Jiang, Y. 2011. Efficient feeder‐free episomal reprogramming with small molecules. PLoS One 6:e17557. doi: 10.1371/journal.pone.0017557.
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