Induced Pluripotent Stem Cell-Derived Cardiac Progenitors Differentiate to Cardiomyocytes and Form Biosynthetic Tissues

Abstract

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors’ ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

Efficient Generation of Human Embryonic Stem Cell-Derived Cardiac Progenitors Based on Tissue-Specific Enhanced Green Fluorescence Protein Expression

“…2,35 However, human PSC-derived progenitors are easy to access, can be isolated at different stages of commitment, and retain self-renewal capability and differentiation potential during long-term maintenance. 26 Steps during in vitro cardiac differentiation include the induction of mesoderm-type cells, followed by the formation of CVPCs, which are able to give rise to CMs, smooth muscle cells, and endothelial cells.…”

“…[2][3][4][5][6] However, these applications claim a large number of cardiomyocytes (CMs) that are free from any other cell types. In order to produce CMs from pluripotent stem cells, differentiation has been reported to be efficiently directed toward the cardiac lineage by various subsets of morphogenes [7][8][9][10][11] or chemical agents.…”

“…6,23,24 In contrast, cardiovascular progenitor cells (CVPCs) and cardiac progenitor cells (CPCs) may be better candidates for a functional engraftment and regeneration of a damaged myocardium. 2,3,23,25 For drug-screening applications, the isolation of progenitors and a subsequent scale-up culturing may also be more suitable than the direct purification of the quiescent CMs. 26 Therefore, methods enabling the selection of progenitors with cardiac-restricted differentiation potential, but still having a high proliferative capacity, are of utmost importance.…”

Striated muscle function, regeneration, and repair

“…With the advent of stem cell research, experiences with engineering of NRVM constructs have been applied to generate cardiac tissues from mouse [276–280] and, more recently, human embryonic and induced pluripotent stem cell-derived cardiomyocytes, ESC-CMs and iPSC-CMs [281]. In particular, numerous groups have engineered 3D cardiac tissues from hESCs and hiPSCs [163, 282–285] that generated specific forces ranging from 0.08 mN/mm 2 [286] to 11.8 mN/mm 2 [287] and conduction velocities ranging from 4.9 cm/s [288] to 25.1 cm/s [287, 289].…”

Heart-on-a-chip based on stem cell biology

“…The impact of electrical fields on cell behavior, especially embryonic cells, has been investigated for many years. In the literature, many methods of stem cells differentiation into cardiac cells performed in a macroscale were reported (Takahashi et al, 2007, Yu et al, 2010, Tchao et al, 2014, Christoforou et al, 2013. However, in the microsystems non differentiation of stem cells into the cardiac cells but the influence of electric field on the cardiac cells is often investigated.…”