Dual TGFβ and Wnt inhibition promotes Mesp1-mediated mouse pluripotent stem cell differentiation into functional cardiomyocytes

Abstract

Efficient derivation of cardiomyocytes from mouse pluripotent stem cells has proven challenging, and existing approaches rely on expensive supplementation or extensive manipulation. Mesp1 is a transcription factor that regulates cardiovascular specification during embryo development, and its overexpression has been shown to promote cardiogenesis. Here, we utilize a doxycycline-inducible Mesp1-expressing mouse embryonic stem cell system to develop an efficient differentiation protocol to generate functional cardiomyocytes. Our cardiac differentiation method involves transient Mesp1 induction following by subsequent dual inhibition of TGFβ and Wnt signaling pathways using small molecules. We discovered that whereas TGFβ inhibition promoted Mesp1-induced cardiac differentiation, Wnt inhibition was ineffective. Nevertheless, a combined inhibition of both pathways was superior to either inhibition alone in generating cardiomyocytes. These observations suggested a potential interaction between TGFβ and Wnt signaling pathways in the context of Mesp1-induced cardiac differentiation. Using a step-by-step approach, we have further optimized the windows of Mesp1 induction, TGFβ inhibition and Wnt inhibition to yield a maximal cardiomyocyte output – Mesp1 was induced first, followed by dual inhibition of TGFβ and Wnt signaling. Our protocol is capable of producing approximately 50% of cardiomyocytes in 12 days, which is comparable to existing methods, and have the advantages of being technically simple and inexpensive. Moreover, cardiomyocytes thus derived are functional, displaying intrinsic contractile capacity and contraction in response to electric stimulus. Derivation of mouse cardiomyocytes without the use of growth factors or other costly supplementation provides an accessible cell source for future applications.