In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Summary

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

Abstract

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) — are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

Introduction

The use of pluripotent stem cells (PSCs) has revolutionized the field of cardiac regeneration and personalized medicine with patient-specific myocytes for disease modeling and drug therapies^1,2,3,4. More recently, in vitro protocols for the generation of atrial vs ventricular as well as pacemaker-like PSC-derived cardiomyocytes have been developed^5,6. However, whether cardiogenesis can be recreated in vitro to study cardiac development and subsequently generate ventricular chamber-sp....

Protocol

NOTE: In vitro generation of heart field-specific mouse cardiac progenitor cells (Figure 1).

1. Maintenance of Mouse ESCs

1. Grow mESCs (mESCs^{Tbx1-Cre; Rosa-RFP; HCN4-GFP}, mESC^Isl1-RFP)²⁵ on 0.1% (w/v) gelatin coated T25 flasks in 2i medium (870 mL of glascow minimum essential medium (GMEM), 100 mL of fetal bovine serum (FBS), 10 mL of GlutaMAX, 10 mL of non-essential amino acids, 10 mL of sodium pyruvate, 3 μL of beta-mercaptoethanol, 20 μL of Lif (200 U/mL), 0.3 μM CHIR99021 and 0.1 μM PD0325901).

Results

After approximately 132 h of differentiation, Tbx1-RFP and Hcn4-GFP CPCs can be detected using a fluorescent microscope (Figure 2). Generally, GFP and RFP cells appear approximately around the same time. The two populations of CPCs continue to expand in close proximity and commonly in a complementary pattern. Adjusting the concentrations of Activin A and BMP4 will alter the percentages of FHF vs SHF CPCs (Figure 3). CPC specification in vitro was primarily deter.......

Discussion

In our protocol, we describe a methodology to generate cardiac spheroids and isolated heart field-specific CPCs. Those can be used to study mechanisms of CPC specification and their properties, as well as for cardiac chamber-specific disease modelling. One previously published work used a mESC line with two fluorescent reporters (Mef2c/Nkx2.5) to study cardiogenesis in vitro, however, both those markers are expressed at embryonic day 9.5-10 when cardiomyocytes are already formed²⁶. To our knowledg.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
β-mercaptoethanol	Sigma	M6250
0.1% (w/v) Gelatin	EMD Millipore	ES-006-B
100 mM Sodium Pyruvate	Gibco	11360
100x Pen/Strep	Gibco	15070-063
1x PBS w/o Calcium and Magnesium	Thermo Fisher Scientific	21-040-CV
20% Paraformaldehyde	Thermo Fisher Scientific	50-980-493
5 mL Polystyrene round-bottom tube with a 40μm cell strainer	BD Falcon	35223
Activin A	R & D Systems	338-AC-010
Ascorbic Acid	Sigma	A-4544
B27 minus vitamin A (50x)	Thermo Fisher Scientific	12587010
BMP4	R & D Systems	314-BP
Bovine Serum Albumin	Sigma	A2153
Cell sorter	Sony	SH800	Sony or any other fluorescence-activated cell sorter
Cell strainer 70μm	Thermo Fisher Scientific	08-771-2
Centrifuge Sorvall Legend XT	Thermo Fisher Scientific	75004508
CHIR99021	Selleck chemicals	S2924
CO2 Incubator	Thermo Fisher Scientific	51030285
Corning Ultra Low Attachment T75 flask	Corning	07-200-875
Countless II FL automated cell counter	Thermo Fisher Scientific
Donkey anti-mouse IgG secondary antibody, Alexa Fluor 647 conjugate	Thermo Fisher Scientific	A-31571, Lot #1757130
Dulbecco's Modified Eagle's Medium high glucose (DMEM)	Gibco	11965-092
EDTA	Sigma	E6758
ESGRO (LIF)	Millipore	ESG1106
EVOS FL microscope	Thermo Fisher Scientific	AMF4300
Fetal Bovine Serum	Invitrogen	SH30071.03
Glasgow’s MEM (GMEM)	Gibco	11710035
GlutaMAX (100x)	Gibco	35050-061
Ham’s F12	Gibco	10-080-CV
HEPES	Sigma	H3375
IMDM	Gibco	12440053
Monothioglycero (MTG)	Sigma	M-6145
Mouse anti-Troponin T antibody	Thermo Fisher Scientific	MS-295-P1
N2-SUPPLEMENT	Gibco	17502-048
Non-essential amino acid solution (NEAA	Invitrogen	11140-050
PD0325901	Selleckchem	S1036
PerCP-efluor 710 conjugated anti-Cxcr4 antibody	Thermo Fisher Scientific	46-9991-82
Suspension culture dish 150 mm x 25 mm	Corning	430597
T25 flasks	Corning	353109
TrypLE (Trypsin)	Gibco	12604
Y-27632 (ROCK inhibitor)	Stem cell technologies	72304