Human Cardiac ECM Hydrogel Coated Biological Scaffold upon Infarction Tailored for Epicardial Regeneration upon Infarction by Colonization of Transdifferentiated Cardiac Progenitors

Objective: Upon cardiac infarction, mammals, in contrast to lower vertebrates or neonatal mice have little or no ability to regenerate the necrotic tissue. We sought to design an epicardial hybrid patch system composed of an extracellular matrix (ECM) based scaffold enhanced with induced cardiomyocyte precursors (iCMPs)—a novel tool for epicardial regeneration.

Methods: A cardiac hydrogel (gECM) was produced upon decellularization of human cardiac tissue and processing via pulverization and Pepsin digestion and analyzed with Mass spectrometry. Cell-free human amniotic membrane (DeAM) was subsequently coated by dry coating protocol with gECM (DeAM + E), visualized by electron microscopy and mechanical properties were determined. Flow cytometry and ELISA determined immunological characteristics. Enriched population of proliferative iCMPs were generated from cardiac fibroblasts (CFs) by forced expression of cardiac transcription factors Gata4, Mef2c, Tbx5, and Myocd and molecular beacon technology. Gene expression profile was assessed by RNA-Array. Upon colonization on the patch cell, compatibility was evaluated by LDH, Resazurin, and BrdU assays.

Results: Mass spectrometry (MS) identified cardiac specific proteins relevant for viability under hypoxic conditions. Electron microscopy (SEM) identified solid coating and nano-scaffold onto DeAM + E. Stability for epicardial transplantation was confirmed. Determination of immune reaction by cytokine secretion of monocytes as well as macrophage-polarization and T cell proliferation by flow cytometry displayed no pro-inflammatory activation. Immunocytology revealed expression if troponin T, α-actinin and myosin heavy chain (MHC) proteins in iCMPs. RNA sequencing of iCMPs showed upregulation of genes associated with cardiac development, differentiation and morphogenesis while they showed downregulation of genes associated to cell-proliferation in comparison to their parental cardiac fibroblasts and cardiomyocytes. Colonization of iCMPs onto DeAM + E is improved and viability increased compared to DeAM.

Conclusions: An epicardial hybrid patch system can be tailored for cardiac regeneration. The patch system specifically supports the viability of cardiac cells, and colonized iCMPs represent an excellent tool to perfectly specialize the patch for epicardial regenerative applications following infarction.