Cardiac tissue engineering (CTE) aims to generate potential scaffolds to mimic extracellular matrix (ECM) for recreating the injured myocardium. Highly porous scaffolds with properties that aid cell adhesion, migration and proliferation are critical in CTE. In this study, electrospun porous poly (l-lactic acid) (PLLA) porous scaffolds were fabricated and modified with different ECM derived proteins such as collagen, gelatin, fibronectin and poly-L-lysine. Subsequently, adult human cardiac fibroblasts (AHCF) were cultured on the protein modified and unmodified fibers to study the cell behavior and guidance. Further, the cytotoxicity and reactive oxygen species (ROS) assessments of the respective fibers were performed to determine their biocompatibility. Excellent cell adhesion and proliferation of the cardiac fibroblasts was observed on the PLLA porous fibers regardless of the surface modifications. The metabolic rate of cells was on par with the conventional cell culture ware while the proliferation rate surpassed the latter by nearly two-folds. Proteome profiling revealed that apart from being an anchorage platform for cells, the surface topography has modulated significant expression of the cellular proteome with many crucial proteins responsible for cardiac fibroblast growth and proliferation.

Cardiovascular disease remains a major cause of deaths globally. Post heart infarction, the most abundant cell type of the heart, fibroblasts, undergo a series of culminating events that lead to fibrotic scar tissue. In many organisms, injury to the heart can be restored, but the adult human heart is unable to efficiently regenerate after ischemic injury. So, the inefficiency of the heart at regenerating on its own after ischemic injury accounts for its reprogramming. Herein, we demonstrate the effect of microRNAs encapsulating poly(lactic-co-glycolic acid) (PLGA)-polyethylenimine (PEI) nanocarriers for direct reprogramming of cardiac fibroblast to cardiomyocyte-like cells. Dual, cardiac-muscle-specific miRNA (miR-1 and miR-133a) polyplexes were encapsulated in biodegradable PLGA nanospheres. Cytocompatibility of the nanocomplexes were evaluated by various in vitro assays, confirming their safety profile. The change in cardiac fibroblast phenotype to cardiomyocyte was identified by the expression of late-stage signature markers. The PLGA-PEI-miRNA nanocomplex improved the intracellular internalization of cargo, exhibited pH-dependent release of the genetic material, and efficiently reprogrammed cardiac fibroblasts to cardiomyocyte-like cells. This is a first report of development of a nanovector targeting cardiac fibroblast for direct genetic reprogramming. Thus, this nanoscale approach serves as an ideal system for gene delivery and a promising therapeutic strategy for direct reprogramming of the heart.

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Human pluripotent stem cells can recapitulate significant features of mammalian organ development in vitro, including key aspects of heart development. We hypothesized that the organoids thus created can be made substantially more relevant by mimicking aspects of in utero gestation, leading to higher physiological and anatomical resemblance to their in vivo counterparts. Here, we report steps towards generating developmentally inspired maturation methodologies to differentiate early human heart organoids into patterned heart-tube-like structures in a reproducible and high-throughput fashion by complete self-organization. The maturation strategy consists of the controlled and stepwise exposure to metabolic (glucose, fatty acids) and hormonal signals (T3, IGF-1) as present during early heart development. These conditions elicit important transcriptomic, cellular, morphological, metabolomic, and functional changes over a 10-day period consistent with continuously increasing heart complexity, maturation, and patterning. Our data reveals the emergence of atrial and ventricular cardiomyocyte populations, valvular cells, epicardial cells, proepicardial-derived cells, endothelial cells, stromal cells, conductance cells, and cardiac progenitors, all of them cell types present in the primitive heart tube. Anatomically, the organoids elongate and develop well-differentiated atrial and ventricular chambers with compacted myocardial muscle walls and a proepicardial organ. For the first time in a completely self-organizing heart organoid, we show anterior-posterior patterning due to an endogenous retinoic acid gradient originating at the atrial pole, where proepicardial and atrial populations reside, mimicking the developmental process present within the primitive heart tube. Collectively, these findings highlight the ability of self-organization and developmental maturation strategies to recapitulate human heart development. Our patterned human heart tube model constitutes a powerful in vitro tool for dissecting the role of different cell types and genes in human heart development, as well as disease modeling congenital heart defects, and represents a step forward in creating fully synthetic human hearts.