Introduction: Advancements in generating and differentiating human induced pluripotent stem cells (hiPSCs) have made hiPSC-derived cardiomyocytes (hiPSC-CMs) a viable cell source for engineered cardiac tissues. However, these hiPSC-CMs display underdeveloped structure and functionality when compared to adult CMs. Although engineered hiPSC-derived cardiac tissues have demonstrated varying degrees of functionality, we still lack a viable approach for coupling engineered tissues electrically and mechanically to the heart. Here we showed that treating hiPSC-CMs with synthetic cells that are rich in Cx43 improves electrochemical coupling of hiPSC-CMs, evident by their synchronous beating.
Materials and
Methods: Three different hiPSCs cell lines: non-repoter hiPSCs (Thermo Fisher), reporter hiPSCs expressing mEGFP-tagged connexin-43 (hiPSC-Cx43GFP, Allen Institute for Cell Science), and hiPSCs transfected with CMV-GCaMP6f (hiPSC-GCaMP; a gift from Dr. Bruce Conklin) were differentiated into CMs using the Wnt pathway modulation [1]. Donor cells for extraction of Connectosomes, including reporter hiPSC-Cx43GFPs, HeLa-Cx43YFPs, and wild-type HeLa cells, were induced to form Connectosomes using an established protocol [2]. Day 20 hiPSC-GCaMP-CMs were treated with Connectosomes and synchronicity of beating was analyzed. Changes in Cx43 expression in treated cells were monitored on both gene and protein levels.
Results, Conclusions, and Discussions:
Results: We successfully harvested Connectosomes from the plasma membrane of donor hiPSCs. The advantage of using hiPSCs as donor cells is that they are inherently rich in Cx43. We demonstrate that harvested hiPSC-Connectosomes retain the high Cx43 expression on their surfaces and that Cx43 hemichannels on hiPSC-Connectosomes are functional. We found that Connectosomes preferentially locate at the interface between adjacent hiPSC-CMs, causing significant increases in Cx43 expression and protein synthesis of target hiPSC-CMs. Most significantly, we demonstrated that hiPSC-Connectosomes significantly improve the synchronization of differentiated CMs compared to cells that were not treated with hiPSC-Connectosomes. The observed synchronization effects persisted for more than 8 days and are Cx43 dependent (Figure 1).
Conclusions: Here, we generated Connexin-rich biomaterials that promote coupling between hiPSC-CMs by reinforce sing the gap junctions and interconnectivity between these cells. A key advantage of this strategy is that it uses the cells’ machinery to accomplish the functional insertion of connexin transmembrane proteins into membranes. This approach has the potential to overcome the key hurdles currently preventing the successful implantation of hiPSC-derived tissue replacements in the heart: the lack of electrochemically coupled beating cells.
