LAUSR

Endogenous adult cardiac regenerative machinery is not capable of replacing the lost cells following myocardial infarction, often leading to permanent alterations in structure‐function‐mechanical properties. Regenerative therapies based on delivering autologous stem cells within an appropriate 3D milieu could meet such demand, by enabling homing and directed differentiation of the transplanted cells into lost specialized cell populations. Since type I collagen is the predominant cardiac tissue matrix protein, we here optimized the 3D niche which could promote time‐dependent evolution of cardiomyogenesis from human bone marrow‐derived mesenchymal stem cells (BM‐MSC). 3D collagen gel physical and mechanical characteristics were assessed using SEM and AFM, respectively, while the standalone and combined effects of collagen concentration, culture duration, and 5‐azacytidine (aza) dose on the phenotype and genotype of MSC spheroids were quantified using immunofluorescence labeling and RT‐PCR analysis. Increasing collagen concentration led to a significant increase in Young's modulus (p < 0.01) but simultaneous decrease in the mean pore size, resulting in stiffer gels. Spheroid formation significantly modulated MSC differentiation and genotype, mostly due to better cell–cell interactions. Among the aza dosages tested, 10 μM appears to be optimal, while 3 mg/ml gels resulted in significantly lower cell viability compared to 1 or 2 mg/ml gels. Stiffer gels (2 and 3 mg/ml) and exposure to 10 μM aza upregulated early and late cardiac marker expressions in a time‐dependent fashion. On the other hand, cell–cell signaling within the MSC spheroids seem to have a strong role in influencing mature cardiac markers expression, since neither aza nor gel stiffness seem to significantly improve their expression. Western blot analysis suggested that canonical Wnt/β‐catenin signaling pathway might be primarily mediating the observed benefits of aza on cardiac differentiation of MSC spheroids. In conclusion, 2 mg/ml collagen and 10 μM aza appears to offer optimal 3D microenvironment in terms of cell viability and time‐dependent evolution of cardiomyogenesis from human BM‐MSCs, with significant applications in cardiac tissue engineering and stem cell transplantation for regenerating lost cardiac tissue.