Introduction
Bioengineered human myocardium (BHM) has a vast range of current and potential applications including human developmental biology, pharmacological studies, disease modelling and regenerative medicine. Ordinarily, human tissue engineered myocardium is created by firstly differentiating pluripotent stem cells into cardiomyocytes and also deriving/isolating other required cell types (eg fibroblasts), and subsequently forming tissue. In this investigation we hypothesized that we could form the BHMs directly with undifferentiated pluripotent stem cells and instructing the formation of myocardial muscle including all the required cell types.
Methods and Results
We then tested our hypothesis by applying an optimized cardiac differentiation protocol (including mechanical stimulation) to undifferentiated pluripotent stem cells in a collagen type 1 hydrogel. qPCR analysis revealed that the development of BHMs followed the temporal progression of known developmental pathways, with very low levels of other contaminating cell types. This analysis also revealed the early and transient expression of various indicators of different heart progenitor populations (TBX5, ISL1, GATA5 and NKX2-5). Flow cytometric analysis demonstrated that these constructs were comprised of mainly cardiomyocytes (α-actinin positive cells) and stromal cells (CD90 positive cells), with low yields of remaining pluripotent stem cells (TRA-1-60/OCT4 positive cells). The BHMs also had a measurable contractile force and contained elongated cross-striated cardiomyocytes together with stromal cells as demonstrated in whole mount immuno-staining analysis.
We also tested the effect of different external stimuli on BHM properties including mechanical stimulation, growth factors and small molecules and demonstrate that these different stimuli modulate different BHM properties leading to changes in active force generation.
Conclusion
BHMs can be formed directly with undifferentiated pluripotent stem cells by recapitulating cardiogenesis. The BHM provides a useful system to study the biology of human heart muscle under serum-free conditions.