Human pluripotent stem cells: Prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair

Abstract

Human pluripotent stem cells (PSCs) represent an attractive source of cardiomyocytes with potential applications including disease modeling, drug discovery and safety screening, and novel cell-based cardiac therapies. Insights from embryology have contributed to the development of efficient, reliable methods capable of generating large quantities of human PSC-cardiomyocytes with cardiac purities ranging up to 90%. However, for human PSCs to meet their full potential, the field must identify methods to generate cardiomyocyte populations that are uniform in subtype (e.g. homogeneous ventricular cardiomyocytes) and have more mature structural and functional properties. For in vivo applications, cardiomyocyte production must be highly scalable and clinical grade, and we will need to overcome challenges including graft cell death, immune rejection, arrhythmogenesis, and tumorigenic potential. Here we discuss the types of human PSCs, commonly used methods to guide their differentiation into cardiomyocytes, the phenotype of the resultant cardiomyocytes, and the remaining obstacles to their successful translation.

Keywords: Cardiac regeneration; Cardiac repair; Cardiovascular disease; Disease modeling; Drug discovery; Embryonic stem cells; Heart failure; Induced pluripotent stem cells.

Figure 1. Human PSCs to cardiomyocytes

Human PSCs can be isolated from the inner cell mass (ICM) of human blastocysts or generated by the reprogramming of somatic cell types via the forced expression of transcription factors (e.g. Oct4, Sox2, Klf4 and c-Myc). Using available protocols, undifferentiated PSCs can be efficiently guided into mesodermal progenitors, more restricted multipotent cardiovascular progenitor cells, and ultimately the various parenchymal cell types that populate the heart, including cardiomyocytes. Work is also ongoing to further control their differentiation into specialized cardiac subtypes including ventricular, atrial and nodal cardiomyocytes.

Figure 2. Human PSC-cardiomyocyte differentiation protocols

Diagram depicting the most commonly used methods for obtaining cardiomyocytes from human ESCs and iPSCs, including the A) embryoid body [14], B) activin A- and BMP4-based monolayer [46], C) “matrix sandwich” [50], D) Wnt inhibition [35], E) small-molecule Wnt modulation [56], F) chemically-defined, small-molecule [58], and G) "hybrid" [59] cardiac differentiation protocols. The top shaded bar of each protocol indicates the specific factors and culture conditions employed, while the bottom bar indicates the culture media and additional culture specifications used. These various interventions are depicted relative to the time-line above (with day 0 indicating the initial induction of cardiac differentiation). Abbreviations: AA, activin A; AA*, activin A with Matrigel; BMP4, bone morphogenetic protein 4; bFGF, basic fibroblast growth factor; CHIR, CHIR99021, glycogen synthase kinase 3β inhibitor; Dkk1, dickkopf homolog 1; DMEM, Dulbecco’s modified eagle’s medium; EBs, embryoid bodies; FBS, fetal bovine serum; IWP, inhibitor of Wnt protein; KO, knockout serum; MEFs, mouse embryonic fibroblasts; MEF-CM, MEF conditioned medium; RPMI, Roswell Park Memorial Institute 1640 medium; VEGF, vascular endothelial growth factor; Wnt-C59, Wnt-3a inhibitor; XAV939, tankyrase inhibitor; Y-27632, Rho kinase inhibitor.

Figure 3. Phenotype of human PSC-derived cardiomyocytes

A) Confocal photomicrograph of a representative early-stage human ESC-derived cardiomyocytes expressing α-actinin (immunostain, green), F-actin (phalloidin, red), and nuclei (Hoechst, blue). Scale bar: 100 microns. B) Ultrastructure of a late-stage human ESC-derived cardiomyocyte. Scale bar: 2 microns. Representative action potential recording from an early-stage human ESC-derived C) nodal cardiomyocyte (adapted from Zhu and colleagues [119]) and D) working cardiomyocyte. Early-stage cardiomyocytes had undergone 3–5 weeks of in vitro maturation, while late-stage cardiomyocytes had undergone 12–15 weeks of in vitro maturation.