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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

Abstract

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

Introduction

Stem cell-based therapies have shown considerable potential as a treatment for cardiac damage caused by MI1. The use of differentiated hiPSCs provides an inexhaustible source of hiPSC-CMs2 and opens the door for the rapid development of breakthrough treatments. However, many limitations to therapeutic translation remain, including the challenge of the severely low engraftment rate of implanted cells.

Dissociating cells with trypsin initiates anoikis3, which is only accelerated once these cells are injected into harsh environments like the ischemic myocardium, where the ....

Protocol

All animal procedures in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham and were based on the National Institutes of Health Laboratory Animal Care and Use Guidelines (NIH Publication No 85-23).

1. Preparation of Culture Media and Culture Plates

  1. Medium preparation
    1. For hiPSC medium, mix 400 mL of human pluripotent stem cell (hPSC) basal medium (Table of Materials 1

Representative Results

The hiPSC-CMs used in this study were derived from human origin with luciferase reporter gene; therefore, the survival rate of the transplanted cells in vivo was detected by bioluminescence imaging (BLI)17 (Figure 1A,B). For histological heart sections, human-specific cardiac troponin T (hcTnT) and human nuclear antigen (HNA) double-positive cells were classified as engrafted hiPSC-CMs (Figure 1C

Discussion

The key steps of this study include obtaining pure hiPSC-CMs, improving the activity of hiPSC-CMs through Y-27632 pretreatment, and finally, transplanting a precise amount of hiPSC-CMs into a mouse MI model.

The key issues addressed here were that, first, we optimized the glucose-free purification methods19 and established a novel efficient purification system. The system procedure included applying cell-dissociation enzymes, replanting cells in gelatin-coated plates, c.......

Acknowledgements

The authors thank Dr. Joseph C. Wu (Stanford University) for kindly providing the Fluc-GFP construct and Dr. Yanwen Liu for excellent technical assistance. This study is supported by the National Institutes of Health RO1 grants HL95077, HL114120, HL131017, HL138023, UO1 HL134764 (to J.Z.), and HL121206A1 (to L.Z.), and a R56 grant HL142627 (to W.Z.), an American Heart Association Scientist Development Grant 16SDG30410018, and the University of Alabama at Birmingham Faculty Development Grant (to W.Z.).

....

Materials

NameCompanyCatalog NumberComments
Reagent
Accutase (stem cell detachment solution)STEMCELL Technologies#07920
B27 minus insulinFisher ScientificA1895601
B27 SupplementFisher Scientific17-504-044
CHIR99021Stem Cell Technologies72054
DMEM (1x), high glucose, HEPES, no phenol redThermofisher20163029
Fetal bovine serumAtlanta BiologicalsS11150
Fluo-4 AM (calcium indicator)Invitrogen/ThermofisherF14201
Glucose-free RPMI 1640Fisher Scientific11879020
IWR1Stem Cell Technologies72562
Matrigel (extracellular matrix )Fisher ScientificCB-40230C
mTeSR (human pluripotent stem cells medium)STEMCELL Technologies85850
Pen-strep antibioticFisher Scientific15-140-122
Pluronic F-127 (surfactant polyol)Sigma-AldrichP2443
Rho activator IICytoskeletonCN03
RPMI1640Fisher Scientific11875119
Sodium DL-lactateSigma-AldrichL4263
TrypLE (cell-dissociation enzymes)Fisher Scientific12-605-010
VerapamilSigma-AldrichV4629
Y-27632STEMCELL Technologies72304
NameCompanyCatalog NumberComments
Equipment and Supplies
IVIS Lumina III Bioluminescence InstrumentsPerkinElmerCLS136334
15 mm CoverslipsWarnerCS-15R15
CentrifugeEppendorf5415R
Confocal MicroscopeOlympusIX81
CryostatThermo ScientificNX50
Dual Automatic Temperature ControllerWarner InstrumentsTC-344B
Electrophoresis Power SupplyBIO-RAD1645050
Fluoresence MicroscopeOlympusIX83
High Speed Camerapco1200 s
Laser Scan HeadOlympusFV-1000
Low Profile Open Bath Chamber (mounts into above microincubation system)Warner InstrumentsRC-42LP
Microincubation SystemWarner InstrumentsDH-40iL
Minivent Mouse VentilatorHarvard Apparatus845
NOD/SCID miceJackson Laboratory001303
Precast Protein GelsBIO-RAD4561033
PVDF Transfer PacksBIO-RAD1704156
Trans-Blot SystemBIO-RADTrans-Blot Turbo
Hot bead sterilizerFine Science Tools18000-45
NameCompanyCatalog NumberComments
Antibody
Anti-human Nucleolin (Alexa Fluor 647)Abcamab198580
Cardiac Troponin TR&D SystemsMAB1874
Cardiac Troponin CAbcamab137130
Cardiac Troponin IAbcamab47003
Cy5-donkey anti-mouseJackson ImmunoResearch Laboratory715-175-150
Cy3-donkey anti-rabbitJackson ImmunoResearch Laboratory711-165-152
Fitc-donkey anti-mouseJackson ImmunoResearch Laboratory715-095-150
GAPDHAbcamab22555
Human Cardiac Troponin TAbcamab91605
Integrin β1Abcamab24693
Ki67EMD Milliporeab9260
N-cadherinAbcamab18203
Phospho-Myosin Light Chain 2Cell Signaling Technology3671s
NameCompanyCatalog NumberComments
Software
MatlabMathWorksR2016A
Image JNIH1.52g

References

  1. Menasche, P., et al. Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience. European Heart Journal. 36 (12), 743-750 (2015).
  2. Burridge, P. W., Keller, G., Gold, J. D., Wu, J. C.

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HiPSC derived CardiomyocytesRho Kinase ActivityCell EngraftmentCell AdhesionCell SurvivalCardiac InfarctionHeart FailureY 27632VerapamilCell TransplantationCell DissociationCell CultureCardiac FunctionVascularizationApoptosis

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