Central to this method is the activation
Central to this method is the activation of canonical WNT signaling by the GSK3β inhibitor CHIR. Expression profiling of hPSCs over the course of guided differentiation showed progression through defined developmental milestones leading to myogenesis. This transition was initiated by a strong induction of TBX6, MESP1, and MSGN1 in CHIR-treated hPSCs, followed by high levels of PARAXIS expression, indicating progression into somitic mesoderm. In addition to the induction of paraxial mesoderm, activation of WNT signaling by CHIR was responsible for generating dorsal tissues, such as dorsal neural tube tachykinin receptor marked by LMX1A expression, along with SOX10+ neural crest cells and AP2α+ nonneural ectoderm (Figure S1). It has been established that myogenic patterning of the dermomyotome requires WNT signaling from the dorsal neural tube and overlying ectoderm (Tajbakhsh and Buckingham, 2000), together with transient, neural-crest-mediated notch activation of myogenic precursors (Rios et al., 2011). Early GSK3β inhibition during hPSC differentiation allowed us to reproduce the conditions necessary for the specification of skeletal muscle cells, closely replicating the events that occur during normal development in vivo. We speculate that the generation of dorsal tissues played an essential role in delivering the appropriate signals required for the patterning of the presomitic mesoderm within our culture system. Conversely, prolonged exposure to CHIR for up to 10 days was shown to have a negative effect on muscle derivation, and no muscle cells were identified in the treated dishes (data not shown). Although we show that CHIR alone is sufficient for myogenic induction, prolonged FGF2 exposure proved to play a proliferative role by significantly increasing the number of myogenic precursors.
Progress in considering hPSC-derived muscle as a valid source of cells for basic and translational research applications has been hindered by the lack of an efficient method to isolate muscle precursors. To overcome this limitation, we developed a FACS strategy to purify muscle precursors generated in our differentiation system. Since we detected LBX1 transcripts during directed myogenic commitment of hPSCs, we considered the use of two markers that are known to be highly expressed in hypaxial migratory muscle precursors during development: C-MET and CXCR4. FACS selection of two populations, CXCR4−/C-MET+ and CXCR4+/C-MET+, allowed the isolation of PAX3+/PAX7+ precursors at high purity. Notably, the negative cell population (HNK−/AChR−/CXCR4−/C-MET−) was devoid of any muscle markers, indicating not only that our sorting strategy is sufficient to isolate all skeletal muscle cells generated in our culture system but also that all PAX3+/PAX7+ precursors are of hypaxial origin. The specificity of this strategy is also confirmed by the complete absence of CXCR4+/C-MET+ cells and by a nonmuscle identity of CXCR4-/C-MET+ cells in the absence of early GSK3β inhibition during hPSC differentiation.