Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Summary

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Introduction

Primary human cardiomyocytes are difficult to obtain because of the requirement for invasive cardiac biopsies, difficulty in dissociating to single cells, and because of poor long-term cell survival in culture. Given this lack of primary human cardiomyocytes, patient-specific human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology has been regarded as a powerful alternative cardiomyocyte source for basic research as well as clinical and translational applications such as disease modeling and drug discovery¹. Early efforts in differentiating pluripotent stem cells into cardiomyocytes employed differentiation protocols using embryoid b....

Protocol

NOTE: Vendor information for all reagents used in this protocol has been listed in Table 1 and Materials List. All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly. Perform all culture incubations in a humidified 37 °C, 5% CO₂ incubator unless otherwise specified. In this protocol, all differentiations are performed in 6-well plates, in which the hiPSCs are seeded. Following differentiation and purification, cells can be dissociated and replated for downstream use.

1. Medium Preparation

1. For the E8 medium, mak....

Results

The morphological changes during hiPSC differentiation.

The hiPSC cultured in feeder-free plates grew as flat, two-dimensional colonies. Upon reaching around 85% confluency, hiPSCs were treated with 6 µM CHIR for differentiation (Figure 1A). Substantial amounts of cell death, a normal and common phenomenon, were observed after 24 hr of CHIR treatment. After two days of CHIR treatment, the hiPSCs continued to differentiate towards a mesodermal fate. In comparis.......

Discussion

Obtaining a large amount of highly purified hiPSC-derived cardiomyocytes is critical for basic cardiac research as well as clinical and translational applications. Cardiac differentiation protocols have undergone tremendous improvements in recent years, transitioning from embryoid body-based methods utilizing cardiogenic growth factors², to matrix sandwich methods¹², and finally to small molecule-modulated and monolayer-based methods⁵. Of the aforementioned protocols, the protocol describ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Matrigel (9-12 mg/ml)	BD Biosciences	354277
RPMI media	Invitrogen	11835055
Glucose free RPMI media	Invitrogen	11879-020
B27 Minus Insulin	Invitrogen	A1895601
B27 Supplement (w/ insulin)	Invitrogen	17504-044
Pen-strep antibiotic	Invitrogen	15140122
Fetal bovine serum	BenchMark	100-106
DMSO	Sigma	D-2650
ROCK inhibitor Y-27632	EMD Millipore	688000
CHIR99021	Thermo Fisher	508306
IWR1	Sigma	I0161
EDTA	Invitrogen	15575-020
Accutase	Millipore	SCR005
Cell lifter	Fisher	08-100-240
Cryovial	Fisher (NUNC tubes)	375418
TrypLE Select Enzyme	Invitrogen	12563-011