Differentiation and Application of Human Pluripotent Stem Cells Derived Cardiovascular Cells for Treatment of Heart Diseases: Promises and Challenges

Abstract

Human pluripotent stem cells (hPSCs) are derived from human embryos (human embryonic stem cells) or reprogrammed from human somatic cells (human induced pluripotent stem cells). They can differentiate into cardiovascular cells, which have great potential as exogenous cell resources for restoring cardiac structure and function in patients with heart disease or heart failure. A variety of protocols have been developed to generate and expand cardiovascular cells derived from hPSCs in vitro. Precisely and spatiotemporally activating or inhibiting various pathways in hPSCs is required to obtain cardiovascular lineages with high differentiation efficiency. In this concise review, we summarize the protocols of differentiating hPSCs into cardiovascular cells, highlight their therapeutic application for treatment of cardiac diseases in large animal models, and discuss the challenges and limitations in the use of cardiac cells generated from hPSCs for a better clinical application of hPSC-based cardiac cell therapy.

Keywords: cardiovascular cells; differentiation; human pluripotent stem cells (hPSCs); large animal; therapeutic application.

FIGURE 1

Schematic diagram of the development of heart cells in vivo. (A) Mutual regulation between epiblast and distal visceral endoderm (DVE) through Nodal, Cerberus, Lefty1, and DRP1 signals leads to a gradient distribution of the concentrations of Nodal and WNT, which results in the formation of primitive streak. During primitive streak migration, a small number of cells express mesoderm posterior protein 1 (MESP1), marking the beginning of heart development. MESP1⁺ cells finally differentiate into various cells that form the heart, such as endothelium, smooth muscle, and myocardium. (B) The migration of the primitive streak from posterior to anterior also marks the beginning of gastrulation, a crucial event in embryonic development. During this period, the embryo becomes a trilaminar embryonic disk, and the heart develops from the mesoderm.

FIGURE 2

Schematic diagram of differentiation of hPSCs into cardiomyocytes. (A) Schematic diagram of matrix sandwich protocol. Extracellular matrix application promoted epithelial–mesenchymal transition of human PSCs. (B) Schematic diagram of GiWi small molecule differentiation protocol, which proved that timing regulation of Wnt signal was critical. (C) Schematic diagram of activin-A/BMP-4/VEGF protocol, which efficiently differentiated cardiomyocytes from both integrated and non-integrated hiPSCs. (D) Schematic diagram of flexible Wnt signal suppression protocol, which indicated that the differentiation of various cell types can be flexibly changed.

FIGURE 3

Schematic diagram of differentiation of hPSCs into endothelial cells. (A) Schematic diagram of chemically defined protocol. Cells were differentiated to mesoderm by GSK3 inhibition or BMP4 treatment and treated with VEGF to induce ECs. (B,C) Schematic diagram of two-stage treated EB protocols. (D,E) Schematic diagram of single-cell and EB protocols, respectively, and both methods applied a two-stage cytokine treatment procedure.

FIGURE 4

Schematic diagram of differentiation of hPSCs into smooth muscle cells. (A,B) Schematic diagram of chemically defined protocols. GSK3 inhibition or BMP4 treatment followed by activin-A and PDGF-BB treatment induced VSMCs from hPSCs, and subsequent applications of PDGF-BB or heparin with activin-A obtained synthetic or contractile VSMCs, respectively. (C) Schematic diagram of EB protocol for differentiation of VSMCs from hPSCs. (D,E) Schematic diagram of chemically defined protocols that efficiently induced hPSCs to differentiate into VSMCs with different phenotypes. Both methods first used GSK3 inhibition and BMP4 to stimulate differentiation into mesoderm cells and then treated with VEGF-A and FGFβ. Synthetic VSMCs (D) were produced by culturing the cells with VEGF-A and FGFβ and with PDGF-β and TGF-β in order. Contractile VSMCs (E) were induced by culturing the cells with PDGF-β and TGF-β directly.

FIGURE 5

Schematic diagram of differentiation of hPSCs into cardiac progenitor cells. (A) MESP1⁺ cells were obtained after treatment of GSK3 inhibition and then were cultured in four different conditions: (a) suspension culture of spheroids, (b) adherent culture of spheroids on gelatin, (c) adherent culture of single cells on gelatin, and (d) adherent culture of single cells on Matrigel. (B) Isoxazole (ISX-9), a cardiogenic small molecule, induced CPCs from hPSCs, which further differentiated into three cardiac lineages in vitro.