Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

Summary

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

Abstract

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70–90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Introduction

Large scale production of human pluripotent stem cells (hPSCs)-derived cardiac progenitors and cardiomyocytes is a pre-requisite for stem cell-based therapies¹, disease modeling^2,3 and the rapid characterization of novel pathways regulating cardiac differentiation^4,5,6 and physiology^7,8. Although a number of studies^9,10,11,

Protocol

1. Id1 Virus Preparation and Infection

1. Generate Id1 overexpressing lentivirus by co-transfecting pCMVDR8.74, pMD2.G, and pCDH-EF1-Id1-PGK-PuroR (Addgene: plasmid #107735) into HEK293T cells. Collect viral particles, filtrate, purify from the supernatant, and store at -80 °C as in Kitamura et al. (2003)¹⁹. Alternatively, produce Id1 lentivirus commercially by sending pCDH-EF1-Id1-PGK-PuroR to a lentivirus-producing vendor.
   Caution: Please follow lentiviral safety regulations and standard operation protocols.
   Note: IMPORTANT: Optimal virus titer should be at least 10⁸ to 10⁹ Transducin....

Results

Generation of hPSCs^Id1lines
hPSCs are infected with a lentivirus mediating Id1 overexpression (Figure 1A). Once hPSC^Id1 are generated, transgene expression is quantified by qRT-PCR (Figure 1B). Only hPSC^Id1 lines expressing Id1 mRNA at levels greater than 0.005 fold of that of GAPDH should be.......

Discussion

For successful differentiations, make sure to closely follow instructions listed above. In addition, here we highlight key parameters that strongly influence differentiation outcomes. Before starting a differentiation, the following three morphological parameters should be observed: a stem morphology of hPSCs^Id1, a high cellular compaction and a high confluence (>90%) of the culture at day 0. In that regard, optimal differentiation conditions are best created by plating dissociated hPSCs^Id1 as s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ACTC1 antibody	Sigma	A7811
Activin A	Stem Cell Technologies	Hu Recom Activin A
Antibiotic Antimycotic (Anti-Anti)	Thermo Fisher Scientific	15240062
B27 supplement	Thermo Fisher Scientific	17504044
B27 supplement w/o - insulin	Thermo Fisher Scientific	A1895601
B27 supplement w/o - vitamin A	Thermo Fisher Scientific	12587001
CDH5 antibody	R&D Systems	AF938
CryoStor CS10	Stem Cell Technologies	7930	Cryopreservation reagent
DMEM high Glucose	Mediatech	10-013-CV
DPBS w/ Ca & Mg	Corning	21-030-CV
EDTA	Thermo Fisher Scientific	15575-038
FBS	VWR	89510-186
FluoVolt membrane potential kit	Thermo Fisher Scientific	F10488	For optical action potential acquisition, please refer to McKeithan et al. 2017
KnockOut Serum Replacement	Gibco	10828010
Matrigel, Growth Factor Reduced	Corning	356231	Coating reagent
mTeSR1 media kit	Stem Cell Technologies	5850
PBS w/o Ca & Mg	Corning	21-040-CV
Penicillin-Streptomycin	Gibco
Puromycin	Acros	227422500
ReLeSR	Stem Cell Technologies	5872	Enzyme-free dissociation reagent
RPMI 1640	Thermo Fisher Scientific	11875-093
TAGLN antibody	Abcam	ab14106
Thiazovivin	Stem Cell Technologies	72254	RHO/ROCK pathway inhibitor
TrypLE Express	Thermo Fisher Scientific	12605 -010	1X enzyme-containing dissociation reagent
Tyrodes solution mix packets	Sigma	T2145-10X1L	(For optical action potential acquisition, please refer to McKeithan et al. 2017)