Single‐Cell Functional Analysis of Stem‐Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates

Jan David Kijlstra1, Dongjian Hu2, Peter van der Meer1, Ibrahim J. Domian3

1 Department of Experimental Cardiology, University Medical Center Groningen, University of Groningen, Groningen, 2 Department of Biomedical Engineering, Boston University, Boston, Massachusetts, 3 Harvard Stem Cell Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.20
DOI:  10.1002/cpsc.40
Online Posting Date:  November, 2017
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Abstract

Human pluripotent stem–cell derived cardiomyocytes (hPSC‐CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC‐CM function, such as contractile and electrical activity, calcium cycling, and force generation, is therefore of paramount importance. hPSC‐CMs cultured on stiff substrates like glass or polystyrene do not have the ability to shorten during contraction, making them less suitable for the study of hPSC‐CM contractile function. Other approaches require highly specialized hardware and are difficult to reproduce. Here we describe a protocol for the preparation of hPSC‐CMs on soft substrates that enable shortening, and subsequently the simultaneous quantitative analysis of their contractile and electrical activity, calcium cycling, and force generation at single‐cell resolution. This protocol requires only affordable and readily available materials and works with standard imaging hardware. © 2017 by John Wiley & Sons, Inc.

Keywords: stem‐cell derived cardiomyocytes; functional analysis; contractility; force generation; calcium cycling; electrophysiology; microcontact printing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of PDMS Substrates
  • Basic Protocol 2: Microcontact Printing on Soft PDMS Substrates
  • Basic Protocol 3: Dissociation of HPSC‐CMS Into Single‐Cell Suspension and Plating Onto Protein Micropattern
  • Basic Protocol 4: Live Cell Imaging Including Calcium and Action Potential Imaging and Data Analysis
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of PDMS Substrates

  Materials
  • PDMS (Dow Corning, Sylgard 527 A&B Silicone Dielectric Gel)
  • 50‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • 10‐ml serological pipet
  • Vortex mixer
  • Vacuum chamber that can hold at least one 50‐ml tube
  • Fluorodish (World Precision Instruments, cat. no. FD35‐100) or other 35‐mm glass‐bottom dishes with a glass‐bottom well at least 23 mm in diameter
  • 150‐mm Petri dish
  • 60°C oven

Basic Protocol 2: Microcontact Printing on Soft PDMS Substrates

  Materials
  • Polyvinyl alcohol (PVA; Sigma, cat. no. P8136)
  • 70% ethanol
  • Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning, cat. no. 354230)
  • 1:1 DMEM/F‐12 Mixture (Lonza, cat. no. BE12‐719F)
  • Phosphate‐buffered saline (PBS) without Mg2+ or Ca2+ (Thermo Fisher Scientific, cat. no. 10010015)
  • Aluminum foil
  • Erlenmeyer flask
  • Magnetic hotplate stirrer
  • Magnetic stir bar
  • 100‐µm nylon cell strainer (Corning Falcon, cat. no. 352360) or similar filter
  • Polystyrene 150‐mm Petri dish
  • Scissors
  • Scalpel
  • Tweezer
  • PDMS‐coated dishes ( protocol 1)
  • Micropatterned PDMS stamps of 1 × 1 cm (protocol for stamp fabrication not provided here; see Théry & Piel, )
  • 50 g rod weight (stack of coins wrapped in Parafilm)
  • Gas duster can (Sigma, cat. no. Z379522), or similar product

Basic Protocol 3: Dissociation of HPSC‐CMS Into Single‐Cell Suspension and Plating Onto Protein Micropattern

  Materials
  • Human pluripotent stem–cell derived cardiomyocytes (hPSC‐CMs; Lian et al., ) growing in 6‐well culture plate
  • Collagenase A and B dissolved in cell medium (5 mg/ml each), for a total collagenase concentration of 10 mg/ml
  • Phosphate‐buffered saline (PBS) without Mg2+ or Ca2+ (Thermo Fisher Scientific, cat. no. 10010015)
  • 0.05% trypsin‐EDTA (Thermo Fisher, cat. no. 25300054)
  • G21 NeuroPlex Serum‐Free Supplement (Gemini Bioproducts, cat. no. 400‐160) dissolved in DMEM/F12 (Lonza, cat. no. BE12‐719F) to 1×
  • Cell medium: G21 Neuroplex Serum‐Free Supplement (Gemini Bioproducts, cat. no. 400‐160) dissolved in RPMI 1640 to 1× (Thermo Fisher Scientific, cat. no. 11875093)
  • 15‐ml conical tube (e.g., Corning Falcon)
  • 10‐ml serological pipet
  • Centrifuge
  • Dishes with protein micropattern ( protocol 2)

Basic Protocol 4: Live Cell Imaging Including Calcium and Action Potential Imaging and Data Analysis

  Materials
  • hPSC‐CMs plated on micropatterned PDMS substrate ( protocol 3)
  • Cell medium: G21 Neuroplex Serum‐Free Supplement (Gemini Bioproducts, cat. no. 400‐160) dissolved in RPMI 1640 to 1× (Thermo Fisher Scientific, cat. no. 11875093)
  • Fluo‐4 AM (Thermo Fisher, cat. no. F14201)
  • Fluovolt Membrane Potential Kit (Thermo Fisher, cat. no. F10488)
  • Nikon A1R confocal laser scanning microscope
  • Visible video analysis software (from Reify Corporation; available on request)
  • Fiji image analysis software (https://imagej.net/Fiji/Downloads)
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Figures

Videos

Literature Cited

Literature Cited
  Bray, M. A., Sheehy, S. P., & Parker, K. K. (2008). Sarcomere alignment is regulated by myocyte shape. Cell Motility and the Cytoskeleton, 65, 641–651. doi: 10.1002/cm.20290.
  Kijlstra, J. D., Hu, D., Mittal, N., Kausel, E., van der Meer, P., Garakani, A., & Domian, I. J. (2015). Integrated analysis of contractile kinetics, force generation, and electrical activity in single human stem cell‐derived cardiomyocytes. Stem Cell Reports, 5, 1226–1238. doi: 10.1016/j.stemcr.2015.10.017.
  Lian, X., Hsiao, C., Wilson, G., Zhu, K., Hazeltine, L. B., Azarin, S. M., … Palecek, S. P. (2012). Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proceedings of the National Academy of Sciences of the United States of America, 109, E1848–1857. doi: 10.1073/pnas.1200250109.
  Lundy, S. D., Zhu, W. Z., Regnier, M., & Laflamme, M. A. (2013). Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cells and Development, 22, 1991–2002. doi: 10.1089/scd.2012.0490.
  Musunuru, K., Domian, I. J., & Chien, K. R. (2010). Stem cell models of cardiac development and disease. Annual Review of Cell and Developmental Biology, 26, 667–687. doi: 10.1146/annurev‐cellbio‐100109‐103948.
  Palchesko, R. N., Zhang, L., Sun, Y., & Feinberg, A. W. (2012). Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve. PloS One, 7, e51499. doi: 10.1371/journal.pone.0051499.
  Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., & Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–872. doi: 10.1016/j.cell.2007.11.019.
  Thery, M., & Piel, M. (2009). Adhesive micropatterns for cells: A microcontact printing protocol. Cold Spring Harbor Protocols, 2009, pdb prot5255. doi: 10.1101/pdb.prot5255.
  Thomson, J. A., Itskovitz‐Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., & Jones, J. M. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147. doi: 10.1126/science.282.5391.1145.
  Yu, H., Xiong, S., Tay, C. Y., Leong, W. S., & Tan, L. P. (2012). A novel and simple microcontact printing technique for tacky, soft substrates and/or complex surfaces in soft tissue engineering. Acta Biomaterialia, 8, 1267–1272. doi: 10.1016/j.actbio.2011.09.006.
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