In vitro expanded skeletal myogenic progenitors from pluripotent stem cell-derived teratomas have high engraftment capacity.

One major challenge in realizing cell-based therapy for treating muscle-wasting disorders is the difficulty in obtaining therapeutically meaningful amounts of engraftable cells. We have previously described a method to generate skeletal myogenic progenitors with exceptional engraftability from pluripotent stem cells via teratoma formation. Here, we show that these cells are functionally expandable in vitro while retaining their in vivo regenerative potential. Within 37 days in culture, teratoma-derived skeletal myogenic progenitors were expandable to a billion-fold. Similar to their freshly sorted counterparts, the expanded cells expressed PAX7 and were capable of forming multinucleated myotubes in vitro. Importantly, these cells remained highly regenerative in vivo. Upon transplantation, the expanded cells formed new DYSTROPHIN+ fibers that reconstituted up to 40% of tibialis anterior muscle volume and repopulated the muscle stem cell pool. Our study thereby demonstrates the possibility of producing large quantities of engraftable skeletal myogenic cells for transplantation.

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

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 TGFß and Wnt signaling pathways using small molecules. We discovered that whereas TGFß 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 TGFß 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, TGFß inhibition and Wnt inhibition to yield a maximal cardiomyocyte output - Mesp1 was induced first, followed by dual inhibition of TGFß 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.