Efficient Generation of Cardiac Purkinje‐like Cells from Embryonic Stem Cells by Activating cAMP Signaling

Su‐Yi Tsai1, Shuibing Chen2, Todd Evans2

1 Department of Life Science, National Taiwan University, 2 Department of Surgery, Weill Cornell Medical College, New York
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.16
DOI:  10.1002/cpsc.20
Online Posting Date:  February, 2017
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Strategies to derive cardiac conduction system (CCS) cells including Purkinje cells (PC) would facilitate models for mechanistic studies and drug discovery, and also provide new cellular materials for regenerative therapies. However, using current cardiac differentiation protocols, the differentiation efficiency of CCS cells is extremely low, typically below 1% of the culture. High‐throughput chemical screening is a powerful strategy for identifying small molecules that can activate signaling pathways to enhance embryonic stem cell (ESC) differentiation. We describe how to carry out a high‐ throughput screen to identify small molecules that can efficiently promote CCS generation from mouse ESCs. We also describe several assays, including immunofluorescence staining, electrophysiology, FACS analysis and quantitative real‐ time PCR, to characterize the phenotype of ESC‐derived PC. In summary, we describe an efficient way to identify small molecules that enhance cardiac PC generation. These protocols can be adapted to identify other rare cell lineages by directed differentiation from ESCs. © 2017 by John Wiley & Sons, Inc.

Keywords: high‐throughput chemical screen; directed differentiation; protocol; heart

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Table of Contents

  • Significance Statement
  • Introduction
  • Basic Protocol 1: High‐Throughput Screening
  • Basic Protocol 2: Characterization of ESC‐Derived Cardiac Purkinje Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: High‐Throughput Screening

  • mESC cells
  • mESC medium (2i + LIF)
  • 0.1% gelatin (Stem Cell technologies, cat. no. 07903)
  • Accutase (Ebioscience, cat. no. 00‐4555‐56)
  • Serum‐free (SF) wash medium (see recipe)
  • Serum‐free differentiation (SFD) medium (see recipe)
  • Trypan blue
  • Phosphate‐buffered saline (PBS)1×
  • Recombinant human Activin‐A (R&D system, cat. no. 338‐AC): Reconstitute at 10 µg/ml in sterile PBS containing 0.1% bovine serum albumin
  • BMP4 (R&D system, cat. no. 314‐BP): Reconstitute at 10 µg/ml in sterile 4 mM HCl containing 0.1% bovine serum albumin
  • VEGF (R&D system, cat 293‐VE): Reconstitute at 10 µg/ml in sterile PBS containing 0.1% bovine serum albumin
  • FACS buffer (see recipe)
  • Blocking buffer (see recipe)
  • Ice
  • FLK1‐PE rat anti‐mouse antibody (eBioscience, cat. no. 12‐5820)
  • PDGFR‐α‐APC rat anti‐mouse antibody (eBioscience, cat. no. 17‐1401)
  • Cardiomyocyte (CM) medium (see recipe)
  • Chemical libraries (see Reagents and Solutions)
  • Dimethyl sulfoxide (DMSO)
  • 100% ethanol
  • Beta‐Glo assay substrate kit (Promega, cat. no. E4740)
  • 6‐well and 384‐well tissue culture plates
  • 37°C incubator
  • Centrifuge
  • 10‐cm2 petri dish
  • 37°C water bath
  • 1.5‐ml microcentrifuge tubes
  • 35‐μm cell strainer
  • 12‐channel pipettor (30 to 300 µl; ThermoFisher, cat. no. 4661070)
  • 384 solid Pin multi‐blot replicator (V&P Scientific, cat. no. VP386)
  • 24‐channel wand (V&P Scientific, cat. no. VP186L‐1)
  • UV light
  • 12‐channel pipettor (5 to 50 µl; ThermoFisher, cat. no. 4661050)
  • EnVision automated micro‐plate reader system (Perkin Elmer)
NOTE: Additional reagents and equipment for culturing and differentiating mESCs (see Reagents and Solutions)NOTE: Coat the tissue culture plate with 0.1% gelatin for 20 min before use.

Basic Protocol 2: Characterization of ESC‐Derived Cardiac Purkinje Cells

  • Phosphate‐buffered saline (PBS), 1×
  • 4% paraformaldehyde (PFA; see recipe)
  • Blocking buffer (see recipe)
  • Primary antibodies (HCN4, ab85023; GFP, AF4240; TNNT2, MA5‐12960; αMHC ab15; Connexin43, ab11370)
  • 1× PBST (see recipe)
  • Secondary antibodies (Alexa‐488, Alexa‐555 and Alexa647 conjugated donkey secondary antibodies against mouse, goat or Rabbit, Invitrogen)
  • DAPI
  • Gelatin‐coated coverslips
  • Tyrode's solution
  • KCl
  • K‐aspartic acid
  • MgCl 2
  • EGTA
  • Na 2‐ATP
  • KOH
  • Digestion buffer (see recipe)
  • FACS buffer (see recipe)
  • Absolutely RNA Nanoprep kit (Agilent technologies, cat. no. 400753)
  • High capacity cDNA reverse transcription kit (Applied Biosystems, cat. no. 4374966)
  • Molecular biology grade water
  • LightCycler DNA master SYBR Green I reagents
  • Digestion buffer (see recipe)
  • Electrodes
  • Amplifier (Axon Instruments, MultiClamp 700B)
  • Digitizer (Axon Instruments, Model DIGIDATA 1440A)
  • Personal computer
  • CLAMPEX 10.2 and CLAMFIT 10.2 software (Axon Instruments)
  • 37°C incubator
  • Centrifuge
  • 35‐μm cell strainer
  • BD FACSAria cell sorter
  • C16 flow cytometer (Accuri)
  • FCS express (De Novo Software)
  • NanoDrop spectrophotometer (Thermo Scientific)
  • LightCycler 480 (Roche)
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Literature Cited

Literature Cited
  Kattman, S.J., Witty, A.D., Gagliardi, M., Dubois, N.C., Niapour, M., Hotta, A., Ellis, J., and Keller, G. 2011. Stage‐specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem. Cell 8:228‐240. doi: 10.1016/j.stem.2010.12.008.
  Kleger, A., Seufferlein, T., Malan, D., Tischendorf, M., Storch, A., Wolheim, A., Latz, S., Protze, S., Porzner, M., Proepper, C., Brunner, C., Katz, S.F., Varma Pusapati, G., Bullinger, L., Franz, W.M., Koehntop, R., Giehl, K., Spyrantis, A., Wittekindt, O., Lin, Q., Zenke, M., Fleischmann, B.K., Wartenberg, M., Wobus, A.M., Boeckers, T.M., and Liebau, S. 2010. Modulation of calcium‐activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker‐like cells. Circulation 122:1823‐1836. doi: 10.1161/CIRCULATIONAHA.110.971721.
  Laflamme, M.A. and Murry, C.E. 2011. Heart regeneration. Nature 473:326‐335. doi: 10.1038/nature10147.
  Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real‐time quantitative PCR and the 2(‐Delta Delta C(T)) Method. Methods 25:402‐408. doi: 10.1006/meth.2001.1262.
  Maass, K., Shekhar, A., Lu, J., Kang, G., See, F., Kim, E., Delgado, C., Shen, S., Cohen, L., and Fishman, G.I. 2015. Isolation and Characterization of ESC‐Derived Cardiac Purkinje Cells. Stem Cells 33:1102‐1112. doi: 10.1002/stem.1921.
  Pallante, B.A., Giovannone, S., Fang‐Yu, L., Zhang, J., Liu, N., Kang, G., Dun, W., Boyden, P.A., and Fishman, G.I. 2010. Contactin‐2 expression in the cardiac Purkinje fiber network. Circ. Arrhythm. Electrophysiol. 3:186‐194. doi: 10.1161/CIRCEP.109.928820.
  Rentschler, S., Vaidya, D.M., Tamaddon, H., Degenhardt, K., Sassoon, D., Morley, G.E., Jalife, J., and Fishman, G.I. 2001. Visualization and functional characterization of the developing murine cardiac conduction system. Development 128:1785‐1792.
  Saito, M., Sasaki, T., and Matsuoka, H. 2009. Vitamin B(12) promotes Cx40 and HCN4 gene expression at an early stage of cardiomyocyte differentiation. Exp. Anim. 58:57‐60. doi: 10.1538/expanim.58.57.
  Spooner, P.M., Albert, C., Benjamin, E.J., Boineau, R., Elston, R.C., George, A.L., Jr., Jouven, X., Kuller, L.H., MacCluer, J.W., Marban, E., Muller, J.E., Schwartz, P.J., Siscovick, D.S., Tracy, R.P., Zareba, W., and Zipes, D.P. 2001. Sudden cardiac death, genes, and arrhythmogenesis: Consideration of new population and mechanistic approaches from a national heart, lung, and blood institute workshop, part I. Circulation 103:2361‐2364. doi: 10.1161/01.CIR.103.19.2361.
  Tsai, S.Y., Maass, K., Lu, J., Fishman, G.I., Chen, S., and Evans, T. 2015. Efficient Generation of Cardiac Purkinje Cells from ESCs by Activating cAMP Signaling. Stem Cell Reports 4:1089‐1102. doi: 10.1016/j.stemcr.2015.04.015.
  Wiese, C., Nikolova, T., Zahanich, I., Sulzbacher, S., Fuchs, J., Yamanaka, S., Graf, E., Ravens, U., Boheler, K.R., and Wobus, A.M. 2011. Differentiation induction of mouse embryonic stem cells into sinus node‐like cells by suramin. Int. J. Cardiol. 147:95‐111. doi: 10.1016/j.ijcard.2009.08.021.
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