Co‐Differentiation of Human Pluripotent Stem Cells‐Derived Cardiomyocytes and Endothelial Cells from Cardiac Mesoderm Provides a Three‐Dimensional Model of Cardiac Microtissue

Elisa Giacomelli1, Milena Bellin1, Valeria V. Orlova1, Christine L. Mummery2

1 Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, 2 Department of Applied Stem Cell Technologies, University of Twente, Enschede
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.9
DOI:  10.1002/cphg.46
Online Posting Date:  October, 2017
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Abstract

The formation of cardiac mesodermal subtypes is highly regulated in time and space during heart development. In vitro models based on human pluripotent stem cells (hPS cells) provide opportunities to study mechanisms underlying fate choices governing lineage specification from common cardiovascular progenitors in human embryos. The generation of cardiac endothelial cells in particular allows the creation of complex models of cardiovascular disorders in which either cardiomyocytes or endothelial cells are affected. Here, a protocol for co‐differentiation of cardiomyocytes and endothelial cells from cardiac mesoderm using hPS cells is described. Precise details for the enrichment of each cell population from heterogeneous‐differentiated cultures, a description of how to maintain and dissociate enriched cardiomyocytes, and the expansion and cryopreservation of enriched endothelial cells are all provided. The generation and culture of three‐dimensional cardiac microtissues from these cell populations is described and guidelines for the characterization of microtissues by immunofluorescent staining and re‐plating for downstream applications are provided. © 2017 by John Wiley & Sons, Inc.

Keywords: human pluripotent stem cell‐derived cardiomyocytes; human pluripotent stem cell‐derived endothelial cells; cardiac mesoderm; cardiac microtissues; three‐dimensional culture model

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Co‐Differentiation of Cardiomyocytes and Endothelial Cells From Cardiac Mesoderm Using Human Pluripotent Stem Cells
  • Support Protocol 1: Isolation, Culture, and Dissociation of VCAM1+ Cardiomyocytes
  • Support Protocol 2: Isolation, Culture, and Cryopreservation of CD34+ Endothelial Cells
  • Basic Protocol 2: Generation and Culture of 3D Cardiac Microtissues From Enriched Cardiomyocytes and Endothelial Cells Differentiated From Human Pluripotent Stem Cells
  • Support Protocol 3: Characterization of 3D Cardiac Microtissues by Immunofluorescent Staining and Re‐Plating for Downstream Applications
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Co‐Differentiation of Cardiomyocytes and Endothelial Cells From Cardiac Mesoderm Using Human Pluripotent Stem Cells

  Materials
  • E8™ medium with penicillin‐streptomycin (Gibco, cat. no. A1517001; see recipe)
  • Human embryonic stem cells (NKX2.5eGFP/w hES cells; Elliott et al., ; or ) or human induced pluripotent stem cells (hiPS cells; Zhang et al., ; )
  • Liquid nitrogen (liquid N 2)
  • VTN‐N‐coated (see recipe) 6‐well plates (Greiner Bio‐One, cat. no. 657160)
  • RevitaCell™ supplement (100×; Gibco, cat. no. A2644501)
  • Dulbecco's phosphate‐buffered saline without Ca2+ or Mg2+ (1× DPBS; Gibco, cat. no. 14190‐094)
  • 0.5 mM EDTA (see recipe)
  • Matrigel‐coated (see recipe) 6‐well plates (Greiner Bio‐One, cat. no. 657160)
  • BPEL medium (see recipe)
  • BPEL medium with bone morphogenetic protein 4 (BMP4), ACTIVIN A, and CHIR99021 (see recipe)
  • BPEL medium with XAV939 and VEGF (see recipe)
  • BPEL medium with VEGF (see recipe)
  • 5‐, 10‐, and 25‐ml sterile plastic pipettes (Greiner Bio‐One, cat. no. 606180, 607180, and 760180, respectively)
  • 15‐ and 50‐ml polystyrene conical tubes (Corning Falcon, cat. no. 352097 and 352098, respectively)
  • 37ºC water bath (Julabo TW20; MCO‐18AIC, serial no. 08010017)
  • 10‐, 200‐, and 1000‐μl filter tips (Corning, cat. no. 4807, 4810, and 4809, respectively)
  • Centrifuge (Eppendorf, cat. no. 5810R)
  • Aspirator
  • 37ºC, CO 2 cell culture incubator (Sanyo)
  • Manual cell counter
  • Microscope (Nikon Eclipse T or Leitz Diavert, 987072 80 M7‐469)

Support Protocol 1: Isolation, Culture, and Dissociation of VCAM1+ Cardiomyocytes

  Additional Materials (see also protocol 1)
  • hPS cell‐derived cardiomyocytes (see protocol 1)
  • 1 × and 2× TrypLE Select (Gibco, cat. no. 12563029; see recipe)
  • FACS buffer (see recipe)
  • VCAM1‐PE antibody (R&D Systems, cat. no. FAB5649P)
  • EasySep™ buffer (see recipe)
  • EasySep™ PE Positive Selection Kit (StemCell Technologies, cat. no. 18557)
  • 5‐ml FACS round‐bottom tubes (BD Biosciences, cat. no. 352058)
  • EasySep™ Purple Easy magnet (StemCell Technologies, cat. no. 18000)
  • MACSQuant® VYB (Miltenyi Biotech) equipped with a violet (405‐nm), blue (488‐nm), and yellow (561‐nm) laser

Support Protocol 2: Isolation, Culture, and Cryopreservation of CD34+ Endothelial Cells

  Additional Materials (also see protocol 1 and protocol 2)
  • hPS cell‐derived endothelial cells (see protocol 1)
  • EasySep™ CD34 Human Cord Blood Isolation Kit II (StemCell Technologies, cat. no. 18309)
  • Anti‐CD34‐APC antibody (Miltenyi Biotech, cat. no.130‐090‐954)
  • Fibronectin‐coated (see recipe) 6‐well plates (Greiner Bio‐One, cat. no. 657160)
  • CryoStor® CS10 medium (StemCell Technologies)
  • Cryovials (Greiner Bio‐One, cat. no. 123263)
  • Nalgene Cryo 1ºC freezing container (Thermo Scientific, cat. no. 6100‐0001)

Basic Protocol 2: Generation and Culture of 3D Cardiac Microtissues From Enriched Cardiomyocytes and Endothelial Cells Differentiated From Human Pluripotent Stem Cells

  Additional Materials (also see protocol 1)
  • Enriched VCAM1+ cardiomyocytes (see protocol 2)
  • Enriched CD34+ endothelial cells (see protocol 3)
  • 96‐well V‐bottom microplates (Greiner Bio‐one; cat. no. 651161)
  • Multi‐channel 5‐ to 50‐μl pipette (FinnPipette, Thermo Scientific; cat. no. 4610130)

Support Protocol 3: Characterization of 3D Cardiac Microtissues by Immunofluorescent Staining and Re‐Plating for Downstream Applications

  Additional Materials (also see protocol 1)
  • MT‐CM and MT‐CMEC microtissues (see protocol 4)
  • Fixative solution (see recipe)
  • Permeabilization solution (see recipe)
  • Blocking solution (see recipe)
  • Rabbit anti‐human TNNI primary antibody, polyclonal (Santa Cruz, cat. no. Sc‐15368)
  • Mouse anti‐human CD31 primary antibody, monoclonal (Dako, cat. no. M0823)
  • Donkey anti‐mouse Cy®3 secondary antibody, polyclonal (Dianova, cat. no. 715‐165‐150)
  • Donkey anti‐rabbit AF488 secondary antibody, polyclonal (Invitrogen, cat. no. A21206)
  • DAPI nucleic acid stain, dilactate (Molecular Probes, cat. no. D3571)
  • ProLong® Gold antifade Mountant with DAPI (Life Technologies, cat. no. P36931)
  • Matrigel‐coated (see recipe) 24‐well plates (Greiner Bio‐One; cat. no. 662160), optional
  • 1.5‐ml microcentrifuge tubes (Eppendorf, cat. no. 0030 120.086)
  • Microscope slides (VWR Collection, cat. no. ECN 631‐1553)
  • 12‐mm, round, glass coverslips (Menzel‐Glaser, cat. no. CB00140RA1)
  • SP8WLL fluorescent confocal laser‐scanning microscope (Leica) equipped with a violet (405‐nm), blue (488‐nm), and orange (532‐nm) laser
  • Plastic tissue‐culture coverslips, 13‐mm (Sarstedt, cat. no. 83.1840.002), optional
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Figures

Videos

Literature Cited

Literature Cited
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