Structural and Functional Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells

Abstract

Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cellderived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)-and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20-40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80-120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calciumhandling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including b-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue.

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

“…When clinical applications are required, it should be possible to optimize the protocol to work without the addition of FBS and on an alternative matrix that is xeno-free and fully defined, though these have not been evaluated here. Cells can be successfully maintained for more than 90 days in culture when using serum-free RPMI-B27 plus L-glutamine on 0.1% polyethyleneimine coated plates as described 34 .…”

Human pluripotent stem cell-derived cardiomyocyte based models for cardiotoxicity and drug discovery

“…Generating 3D micro ECTs of variable cell compositions, using human-derived cardiac cells, has been demonstrated as discussed. Moreover, varying degrees of hPSC-CM maturation have been demonstrated by means of prolonged culture [26], topographical cues [14], electrical stimulation [14], mechanical stretching [19], and/or chemical manipulation [27], as well as coculture with cardiac fibroblasts [28] and endothelial cells [25]. Yet, there are currently no robust methods of promoting the functional maturation of hPSC-CMs within a short time frame.…”

Concise Review: Mechanotransduction via p190RhoGAP Regulates a Switch Between Cardiomyogenic and Endothelial Lineages in Adult Cardiac Progenitors

“…Although the CDCs have entered in phase II clinical trial, there are many other competing cell sources, including cardiomyocytes from pluripotent stem cells or other adult stem cell types [75][76][77][78][79][80]. Considering that the biomimetic mechanical cues used in our experiments result in different fate decisions of CDCs, there is a potential to regulate the fate of the grafted cells according to the needs of a particular patient.…”