Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro

Abstract

Several types of terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of Klf4, Oct3/4, Sox2, and c-Myc. Such induced pluripotent stem (iPS) cells have great potential to serve as an autologous source of cells for tissue repair. In the process of developing iPS-cell-based therapies, the major goal is to determine whether differentiated cells derived from iPS cells, such as cardiomyocytes (CMs), have the same functional properties as their physiological in vivo counterparts. Therefore, we differentiated murine iPS cells to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. As key markers of cardiac lineages, transcripts for Nkx2.5, alphaMHC, Mlc2v, and cTnT could be identified. Immunocytochemical stainings revealed the presence of organized sarcomeric actinin but the absence of mature atrial natriuretic factor. We examined characteristics and developmental changes of action potentials, as well as functional hormonal regulation and sensitivity to channel blockers. In addition, we determined expression patterns and functionality of cardiac-specific voltage-gated Na+, Ca2+, and K+ channels at early and late differentiation stages and compared them with CMs derived from murine embryonic stem cells (ESCs) as well as with fetal CMs. We conclude that iPS cells give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed cardiac ion channels; CMs generated from iPS cells have a ventricular phenotype; and cardiac development of iPS cells is delayed compared with maturation of native fetal CMs and of ESC-derived CMs. This difference may reflect the incomplete reprogramming of iPS cells and should be critically considered in further studies to clarify the suitability of the iPS model for regenerative medicine of heart disorders.

Cardiomyocytes Derived From Pluripotent Stem Cells Recapitulate Electrophysiological Characteristics of an Overlap Syndrome of Cardiac Sodium Channel Disease

“…As others have reported, [21][22][23]32 we observed an increase in I Na in mPSC-CMs with prolonged culture, reaching a plateau at around day 17 of differentiation ( Figure IVA and IVB in the online-only Data Supplement). Therefore, only cardiomyocytes after 17 or 18 days of differentiation were analyzed further.…”

“…17 In human iPSC-derived cardiomyocytes (hiPSC-CMs), similar low upstroke velocities have also been reported. 18 -20 Although mouse (m)PSC-CMs show an increase in I Na and upstroke velocity during prolonged in vitro differentiation, [21][22][23] the latter is still smaller than that observed in isolated primary adult mouse cardiomyocytes. 8 Therefore, it remains uncertain whether PSC-CMs can recapitulate the electrophysiological features of Na ϩ channel loss-of-function mutations.…”

Myocardial Reprogramming Medicine: The Development, Application, and Challenge of Induced Pluripotent Stem Cells

“…These cells are reflective of very early human cardiac development, limiting their utility in the generation of in vitro models of mature myocardium. Immature differentiated cardiomyocytes lack the consistent properties of adult mature ventricular cardiomyocytes such as gene expression profile, morphology, and electrophysiological function [158,259,260]. For instance, the majority of hiPSC-derived cardiomyocytes lack the action potential notch characteristic which is central to the disease phenotype of inherited cardiac arrhythmia syndromes [261].…”

Directed Differentiation of Mesendoderm Derivatives from Embryonic Stem Cells

“…However, hES differentiate at a slower pace and with a lower efficiency. The miPS are similarly slow to differentiate, form smaller CMs, and may have a predilection for a ventricular phenotype (Kuzmenkin et al, 2009;Mauritz et al, 2008).…”