Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

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

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.......

Materials

Name	Company	Catalog Number	Comments
Reagent
Accutase (stem cell detachment solution)	STEMCELL Technologies	#07920
B27 minus insulin	Fisher Scientific	A1895601
B27 Supplement	Fisher Scientific	17-504-044
CHIR99021	Stem Cell Technologies	72054
DMEM (1x), high glucose, HEPES, no phenol red	Thermofisher	20163029
Fetal bovine serum	Atlanta Biologicals	S11150
Fluo-4 AM (calcium indicator)	Invitrogen/Thermofisher	F14201
Glucose-free RPMI 1640	Fisher Scientific	11879020
IWR1	Stem Cell Technologies	72562
Matrigel (extracellular matrix )	Fisher Scientific	CB-40230C
mTeSR (human pluripotent stem cells medium)	STEMCELL Technologies	85850
Pen-strep antibiotic	Fisher Scientific	15-140-122
Pluronic F-127 (surfactant polyol)	Sigma-Aldrich	P2443
Rho activator II	Cytoskeleton	CN03
RPMI1640	Fisher Scientific	11875119
Sodium DL-lactate	Sigma-Aldrich	L4263
TrypLE (cell-dissociation enzymes)	Fisher Scientific	12-605-010
Verapamil	Sigma-Aldrich	V4629
Y-27632	STEMCELL Technologies	72304
Name	Company	Catalog Number	Comments
Equipment and Supplies
IVIS Lumina III Bioluminescence Instruments	PerkinElmer	CLS136334
15 mm Coverslips	Warner	CS-15R15
Centrifuge	Eppendorf	5415R
Confocal Microscope	Olympus	IX81
Cryostat	Thermo Scientific	NX50
Dual Automatic Temperature Controller	Warner Instruments	TC-344B
Electrophoresis Power Supply	BIO-RAD	1645050
Fluoresence Microscope	Olympus	IX83
High Speed Camera	pco	1200 s
Laser Scan Head	Olympus	FV-1000
Low Profile Open Bath Chamber (mounts into above microincubation system)	Warner Instruments	RC-42LP
Microincubation System	Warner Instruments	DH-40iL
Minivent Mouse Ventilator	Harvard Apparatus	845
NOD/SCID mice	Jackson Laboratory	001303
Precast Protein Gels	BIO-RAD	4561033
PVDF Transfer Packs	BIO-RAD	1704156
Trans-Blot System	BIO-RAD	Trans-Blot Turbo
Hot bead sterilizer	Fine Science Tools	18000-45
Name	Company	Catalog Number	Comments
Antibody
Anti-human Nucleolin (Alexa Fluor 647)	Abcam	ab198580
Cardiac Troponin T	R&D Systems	MAB1874
Cardiac Troponin C	Abcam	ab137130
Cardiac Troponin I	Abcam	ab47003
Cy5-donkey anti-mouse	Jackson ImmunoResearch Laboratory	715-175-150
Cy3-donkey anti-rabbit	Jackson ImmunoResearch Laboratory	711-165-152
Fitc-donkey anti-mouse	Jackson ImmunoResearch Laboratory	715-095-150
GAPDH	Abcam	ab22555
Human Cardiac Troponin T	Abcam	ab91605
Integrin β1	Abcam	ab24693
Ki67	EMD Millipore	ab9260
N-cadherin	Abcam	ab18203
Phospho-Myosin Light Chain 2	Cell Signaling Technology	3671s
Name	Company	Catalog Number	Comments
Software
Matlab	MathWorks	R2016A
Image J	NIH	1.52g