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.

Amniotic fluid-derived stem cells demonstrate limited cardiac differentiation following small molecule-based modulation of Wnt signaling pathway

“…Additionally, previous studies found that more than 90% of pluripotent cells will express Nkx-2.5 and Islet-1 at day 8 of the differentiation Following the attempted differentiation protocol, connexin 43 preferentially located to the cell membranes between cells. This occurrence has been observed following other cardiac differentiation attempts in AFSC [9][10][11]. Connexin 43, also known as gap junction alpha-1 protein, is a dominant connexin expressed in cardiovascular tissue and is a key component to gap junctions [28].…”

“…Upon direct co-culture of AFSC with neonatal rat ventricular myocytes (NRVM), human cells positive for cardiac markers such as sarcomeric proteins have been observed [8][9][10]. However, when AFSC are co-cultured with NRVM in systems that do not allow for cell fusion, including on opposite sides of a porous membrane, in shared media dishes, and AFSC in conditioned medium from NRVM, it is observed that AFSC form functional gap junctions but do not express sarcomere proteins [11]. It has also been observed that treatment of AFSC with the demethylating agent 5-aza-2'-deoxycytidine for 24 hours at the beginning of a 10 day culture period results in phenotypic changes of the cells consistent with muscle cells and protein expression of cardiac troponin I (cTnI) and cardiac troponin T (cTNT), but no mature sarcomeres or spontaneous contraction [9].…”

In situ vascularization of injectable fibrin/poly(ethylene glycol) hydrogels by human amniotic fluid‐derived stem cells

“…[22,39] The experimental protocol and informed consent forms were approved by the Institutional Review Boards of Baylor College of Medicine and Rice University.…”

Mesenchymal Stem Cells: An Innovative Approach in Pharmacokinetics

“…Human MSCs can make cardiac connexins (Cx43, Cx 40 and Cx 45) and form gap junctional complex. It is recently reported that amniotic fluid-derived stem cells differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes [50]. Similar to other cells, toxic agents also affect viability, morphology, and function of MSCs.…”