ABSTRACT: The influence of somatic stem cells in the stimulation of mammalian cardiac muscle regeneration is still in its early stages, and so far, it has been difficult to determine the efficacy of the procedures that have been employed. The outstanding question remains whether stem cells derived from the bone marrow or some other location within or outside of the heart can populate a region of myocardial damage and transform into tissue-specific differentiated progenies, and also exhibit functional synchronization. Consequently, this necessitates the development of an appropriate in vitro three-dimensional (3D) model of cardiomyogenesis and prompts the development of a 3D cardiac muscle construct for tissue engineering purposes, especially using the somatic stem cell, human mesenchymal stem cells (hMSCs). To this end, we have created an in vitro 3D functional prevascularized cardiac muscle construct using embryonic cardiac myocytes (eCMs) and hMSCs. First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were cocultured onto a 3D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions; hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed dense vascular networks. Next, the eCMs and hMSCs were cocultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were characterized at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated progenies revealed neo-cardiomyogenesis and neo-vasculogenesis. In this milieu, for instance, not only were hMSCs able to couple electromechanically with developing eCMs but were also able to contribute to the developing vasculature as mural cells, respectively. Hence, our unique 3D coculture system provides us a reproducible and quintessential in vitro 3D model of cardiomyogenesis and a functioning prevascularized 3D cardiac graft that can be utilized for personalized medicine.
