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Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes.

ABSTRACT: Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2-week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co-culture group. Protein expression of cardiac calsequestrin 2 was up-regulated in direct transmembrane co-cultures and media control cultures compared to the other experimental groups, but all groups were up-regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape-loading dye transfer assay, was significantly increased in direct transmembrane co-cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co-culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co-culture does enhance connexin-43-mediated gap junction communication between AFSC.

SUBMITTER: Connell JP

PROVIDER: S-EPMC3823181 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes.

Connell Jennifer Petsche JP Augustini Emily E Moise Kenneth J KJ Johnson Anthony A Jacot Jeffrey G JG

Journal of cellular and molecular medicine 20130502 6

Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compa ...[more]

PMID: 23634988

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Project description:: The establishment of an abundant source of autologous cardiac progenitor cells would represent a major advance toward eventual clinical translation of regenerative medicine strategies in children with prenatally diagnosed congenital heart disease. In support of this concept, we sought to examine whether functional, transgene-free human cardiomyocytes (CMs) with potential for patient-specific and autologous applications could be reliably generated following routine amniocentesis. Under institutional review board approval, amniotic fluid specimens (8-10 ml) at 20 weeks gestation were expanded and reprogrammed toward pluripotency using nonintegrating Sendai virus (SeV) expressing OCT4, SOX2, cMYC, and KLF4. Following exposure of these induced pluripotent stem cells to cardiogenic differentiation conditions, spontaneously beating amniotic fluid-derived cardiomyocytes (AF-CMs) were successfully generated with high efficiency. After 6 weeks, quantitative gene expression revealed a mixed population of differentiated atrial, ventricular, and nodal AF-CMs, as demonstrated by upregulation of multiple cardiac markers, including MYH6, MYL7, TNNT2, TTN, and HCN4, which were comparable to levels expressed by neonatal dermal fibroblast-derived CM controls. AF-CMs had a normal karyotype and demonstrated loss of NANOG, OCT4, and the SeV transgene. Functional characterization of SIRPA+ AF-CMs showed a higher spontaneous beat frequency in comparison with dermal fibroblast controls but revealed normal calcium transients and appropriate chronotropic responses after ?-adrenergic agonist stimulation. Taken together, these data suggest that somatic cells present within human amniotic fluid can be used to generate a highly scalable source of functional, transgene-free, autologous CMs before a child is born. This approach may be ideally suited for patients with prenatally diagnosed cardiac anomalies.This study presents transgene-free human amniotic fluid-derived cardiomyocytes (AF-CMs) for potential therapy in tissue engineering and regenerative medicine applications. Using 8-10 ml of amniotic fluid harvested at 20 weeks gestation from normal pregnancies, a mixed population of atrial, ventricular, and nodal AF-CMs were reliably generated after Sendai virus reprogramming toward pluripotency. Functional characterization of purified populations of beating AF-CMs revealed normal calcium transients and appropriate chronotropic responses after ?-adrenergic agonist stimulation in comparison with dermal fibroblast controls. Because AF-CMs can be generated in fewer than 16 weeks, this approach may be ideally suited for eventual clinical translation at birth in children with prenatally diagnosed cardiac anomalies.

Dataset Information

Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes.

Publications

Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes.

Similar Datasets

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