Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Summary

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

Abstract

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Introduction

Pluripotent stem cells are a powerful tool for generating and studying cells from a host of difficult to access tissues for basic research and pre-clinical studies, especially in humans^1-5. Proper modulation of developmental signaling pathways can direct the differentiation of pluripotent stem cells to the desired phenotypic fate. Many protocols have been developed to generate cardiomyocytes (CMs) from pluripotent stem cells^6-14. These protocols generally involve modulation of TFGβ superfamily (Activin, BMP, and TGFβ) and Wnt pathways through timed addition of exogenous growth factors and/or small molecules^10,12-15. These pro....

Protocol

1. Preparation of Cell Culture Media, Solutions, and Reagents.

1. Prepare 500 ml of Mouse embryonic stem cell (mESC) media by mixing and sterile filtering (0.2 μm pore size) 445 ml Glasgow Minimum Essential Medium (GMEM), 50 ml heat inactivated fetal bovine serum (FBS), 5 ml 100X concentrated L-Glutamine replacement, 5 µl recombinant mouse Leukemia Inhibitory Factor (LIF) at 1 x 10⁷ units per ml, and 1.43 µl ß-Mercaptoethanol (final concentration is 50 µM). Store at 4 ˚C until ready to use.
2. Prepare 500 ml of Embryoid Body (EB) differentiation media by mixing and sterile filtering 391 ml Iscove's Modified ....

Results

Prior to differentiation, pluripotent stem cells should be compact and free of spontaneous differentiation. The cells shown in Figure 1A are ready to be singularized and used for hanging drops as described in 5.1 of the protocol section. The cells shown in Figure 1B are spontaneously differentiating and should not be used for preparation of hanging drops.

The panels included in Figure 2<.......

Discussion

This protocol routinely produces cultures with a high percentage of CMs that are characteristic of the atrial lineage. As with any differentiation protocol, the quality of the mESCs prior to differentiation should be given particular attention. mESCs should be routinely monitored for proper morphology (Figure 1A). Any spontaneous differentiation that occurs prior to formation of EBs will severely limit the efficiency of cardiogenesis and should be removed before passaging (Figure 1B). EB.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
GMEM	Life Technologies	11710
FBS	Life Technologies	10082
Glutamax	Life Technologies	35050
LIF	EMD Millipore	ESG1107
ß-Mercaptoethanol	Sigma	M3148
IMDM	Sigma	I3390
Non-Essential Amino Acids	Sigma	M7145
10 cm Tissue Culture Plates	Sarstedt	83.3902
10 cm Bacterial Petri Dishes	VWR	25384-342
6 cm Bacterial Petri Dishes	VWR	25384-092
6-well tissue culture plates	Sarstedt	83.3920
Gremlin 2 recombinant protein	R&D Systems	2069-PR-050
Sterile filter units	Thermo Fisher	09-741-02
Gelatin (from porcine skin)	Sigma	G1890
10X PBS, Sterile	Sigma	P5493
BSA	Sigma	5470
0.05% Trypsin-EDTA solution	Life Technologies	25300054
DPBS, no Calcium, no Magnesium	Life Technologies	14200