Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming

Abstract

Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes, both in vitro and in vivo. Beyond uses in cell replacement therapy, patient-specific cardiomyocytes may find applications in drug testing, drug discovery, and disease modeling. Recently, approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs), adult heart-derived cardiac progenitor cells (CPCs), and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts, highlighting potential applications and future challenges.

Figure 1. The mouse as a model for human cardiogenesis

A, Gastrulation begins with the formation of the primitive streak. Anterior primitive ectoderm cells perform an epithelial to mesenchymal transition (EMT), pass through the primitive streak, and move laterally between the primitive ectoderm and visceral endoderm. B, Cells in the most proximal portion of the primitive streak will form the extraembryonic mesoderm, mid-primitive streak cells will form the embryonic tissues such as heart and blood, and cells of the distal portion of the primitive streak will form the endoderm. C, In the case of cardiac progenitors, this mid-streak mesoderm progresses laterally, then ventrally, around both sides of the embryo, becoming the lateral plate mesoderm from which the first heart field (FHF) is derived. The lateral plate mesoderm then delaminates to form the splanchnic mesoderm (on the ventral side), which forms the second heart field (SHF) and somatic mesoderm (on the dorsal side). D, The FHF progenitors form the cardiac crescent, whereas SHF progenitors are found medial to the crescent. The cells of both heart fields then move to the midline, where the FHF progenitors form a linear heart tube that later contributes to the left ventricle. Cells of the SHF proliferate, migrate, and join with the cardiomyocytes of the first heart field, resulting in the rightward looping of the cardiac tube, a process that culminates in a segmented structure and the formation of cardiac chambers.

Figure 2. Methods for the differentiation of human pluripotent stem cells

Three methods for differentiating hPSCs, highlighting commonalities at each of the six steps: pluripotent culture, pre-differentiation culture, differentiation format, and treatment with mesoderm induction factors, cardiac specification factors, and cardiac differentiation factors. A, Yang et al. suspension EB in StemPro34; B, Forced aggregation; C, Laflamme et al. monolayer differentiation. Abbreviations: KSR, Knockout Serum Replacement; FGF2, fibroblast growth factor 2; BMP4, bone morphogenic protein 4; VEGFA, vascular endothelial growth factor A; DKK1, dickkopf homolog 1; SB431542, TGFB/activin/NODAL signaling inhibitor (ALK4,5,7); dorsomorphin, BMP signaling inhibitor (ALK2,3,6); IWR-1, WNT signaling inhibitor; MEF CM, mouse embryonic fibroblast conditioned hESC medium; RPMI, Roswell Park Memorial Insitute 1640 basal medium; B27, media supplement; NOGGIN, BMP signaling inhibitor; RAi, retinoic acid signaling inhibitor; LIAPEL, low insulin, Albucult, polyvinyl alcohol, essential lipids media; SCF, stem cell factor (KITLG); LI-BEL, low insulin, bovine serum albumin, essential lipids media; Y27632, Rho kinase inhibitor; CHIR99021, GSK3 inhibitor; IWP-2, WNT signaling inhibitor; FBS, fetal bovine serum; DMEM, basal media; RPMI+PVA, RPMI based media supplemented with polyvinyl alcohol; RPMI-INS, RPMI-based media without insulin.

Figure 3. Schematic of current knowledge of factors involved in hPSC cardiac differentiation

Factors that influence the progression through each of the six major steps of hPSC cardiomyogenesis: epithelial to mesenchymal transition, mesoderm differentiation, mesoderm speciation, cardiac specification, cardiomyocyte differentiation, and electrical maturation. Data shown are derived from developmental biology models that have been directly assessed and proved functional in hPSC cardiac differentiation, along with knowledge gained directly from hPSC differentiation. Below are the markers associated with each of the seven cell types during differentiation; surface markers are marked with an asterisk.

Figure 4. Schematic of de novo cardiomyocyte generation and applications

Demonstrating peripheral blood and skin fibroblasts as a cell source, hiPSC derivation and differentiation, direct reprogramming using GMT, and partial reprogramming using OSKM and a full reprogramming inhibitor. Applications in disease modeling, engraftment into the heart, drug discovery, and cardiotoxicity analysis. Adult heart sources of cardiomyocytes from cardiac progenitor cells and application for in vivo expansion and differentiation. Direct reprogramming of adult cardiac fibroblasts in vivo. Abbreviations: OSKM, Oct4, Sox2, Klf4, c-Myc; JAKi, JAK/STAT inhibitor; GMT. Gata4, Mef2c, Tbx5; CPC, cardiac progenitor cell.