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Ghrelin promotes differentiation of human embryonic stem cells into cardiomyocytes.

ABSTRACT: AIM: Ghrelin is involved in regulating the differentiation of mesoderm-derived precursor cells. The aim of this study was to investigate whether ghrelin modulated the differentiation of human embryonic stem (hES) cells into cardiomyocytes and, if so, whether the effect was mediated by growth hormone secretagogue receptor 1α (GHS-R1α). METHODS: Cardiomyocyte differentiation from hES cells was performed according to an embryoid body (EB)-based protocol. The cumulative percentage of beating EBs was calculated. The expression of cardiac-specific markers including cardiac troponin I (cTnI) and α-myosin heavy chain (α-MHC) was detected using RT-PCR, real-time PCR and Western blot. The dispersed beating EBs were examined using immunofluorescent staining. RESULTS: The percentage of beating EBs and the expression of cTnI were significantly increased after ghrelin (0.1 and 1 nmol/L) added into the differentiation medium. From 6 to 18 d of differentiation, the increased expression of cTnI and α-MHC by ghrelin (1 nmol/L) was time-dependent, and in line with the alteration of the percentages of beating EBs. Furthermore, the dispersed beating EBs were double-positively immunostained with antibodies against cTnI and α-actinin. However, blockage of GHS-R1α with its specific antagonist D-[lys(3)]-GHRP-6 (1 μmol/L) did not alter the effects of ghrelin on cardiomyocyte differentiation. CONCLUSION: Our data show that ghrelin enhances the generation of cardiomyocytes from hES cells, which is not mediated via GHS-R1α.

SUBMITTER: Yang J

PROVIDER: S-EPMC4010076 | biostudies-other | 2011 Oct

REPOSITORIES: biostudies-other

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Project description:Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca(2+)) imaging, and intracellular recording techniques, we found that intracellular Ca(2+)-cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca(2+)-handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to influence early cardiac development. Our study is the first to demonstrate that non-cardiomyocytes influence electrophysiological maturation of early hESC-CMs in cultures. Defining the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.

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Ghrelin promotes differentiation of human embryonic stem cells into cardiomyocytes.

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