Stem cell therapy has emerged as a promising treatment for myocardial infarction. A population of stem cells that resides in the human heart (cardiomyocyte progenitor cells, CMPCs) can be isolated and differentiated into beating cardiomyocytes using biochemical factors in vitro. Upon injection in the heart, these cells will experience a three dimensional (3D) rather than a 2D environment and are exposed to mechanical forces. To investigate further the cardiac regeneration potential of CMPCs, we studied the behavior of these cells in a 3D constrained environment.Methods
Human fetal CMPCs were encapsulated in either longitudinally constrained or free floating hydrogels composed of collagen-Matrigel (n = 3 each). Resulting constructs were cultured for 9 days in control or differentiation medium, and analyzed for compaction, cell survival, proliferation, alignment and cardiac differentiation using immunofluorescent stainings.Results
Cell survival was higher in constrained constructs compared to free floating constructs. Furthermore, proliferation in constrained constructs was decreased, but 14% of the cells was still dividing. In contrast, in free floating gels cell proliferation was abolished, indicating that static strain stimulates proliferation of CMPCs in 3D. Moreover, cells cultured in constrained constructs aligned in the direction of the constraint, probably as a result of internal stress formation as demonstrated by the compaction of the constructs (20% after 1 day of culture, 27% after 9 days) and the formation of intracellular stress fibers. Construct compaction was greatly enhanced after culture in differentiation medium (51%). Lastly, cells cultured in constrained constructs showed expression of the cardiac markers Nkx2.5 and α-actinin after 9 days of culture and independent of culture medium. Expression of those cardiac markers was lost in free floating constructs, suggesting that static strain is necessary to preserve the cardiac differentiation potential of CMPCs in 3D. The early cardiac marker GATA4 was only expressed in constructs cultured in differentiated medium.Conclusion
Constraining appeared to be a crucial factor for the survival, proliferation and cardiac differentiation of CMPCs cultured in 3D hydrogel-based constructs. Moreover, constraining resulted in cell alignment, which is important for proper tissue integration. Optimization of the culture system may be of great relevance for cardiac regeneration.