Tissue engineering enables the simulation of human heart physiology and pathology. It typically requires a mixture of cardiomyocytes, stromal cells, and extracellular matrix for fabrication. Here, we hypothesised that bioengineered heart muscle (BHM) can be formed directly from undifferentiated pluripotent stem cells by triggering processes of embryonic cardiogenesis.
Methods and Results:
We tested our hypothesis by applying an optimized serum-free cardiac differentiation protocol to undifferentiated pluripotent stem cells in a collagen type 1 hydrogel. During this process BHMs traversed through distinct developmental stages: early mesoderm (3 days), cardiac specification (10 days), and cardiac maturation (up to 50 days). Flow cytometry demonstrated that BHMs are comprised of primarily cardiomyocytes (α-actinin positive cells, 51 ± 5%, n = 6) and stromal cells (CD90 positive cells, 41 ± 5%, n = 6), with low yields of contaminating cells. By 22 days the BHMs exhibited measurable contractile force (207 ± 19 μN) and contained elongated cross-striated cardiomyocytes.
We next sought to optimize the force of contraction and also the maturity of the BHM, by investigating the effect of mechanical stimuli and growth factors. Mechanical stimulation was essential for BHM formation. Additionally, we found that 2 developmentally important growth factors, FGF2 and TGFβ1, induced pathological and physiological hypertrophy, respectively. This was characterized by an increase in cell size in both conditions coupled with reduced force and higher ANP expression in FGF2 treated BHM, and a higher force with reduced ANP expression and elevated β-MHC/α-MHC ratio TGFβ1 treated BHM.
Using our optimized protocol, 28 day old BHM responded to electrical pacing, preloading, and inotropic stimuli similarly as bona fide myocardium.
Conclusion:
BHM can be formed directly with undifferentiated pluripotent stem cells by recapitulating normal cardiac development. The serum-free protocol with developmentally defined stimuli provides us with a useful in vitro model to study cardiac biology and potentially provides a method of producing cardiac tissue for regenerative applications.
Migration of mouse-induced pluripotent stem cells to glioma-conditioned medium is mediated by tumor-associated specific growth factors
