Long-term high-yield skeletal muscle stem cell expansion through staged perturbation of cytokine signaling in a soft hydrogel culture platform

Abstract

30Muscle stem cells (MuSCs) are an essential stem cell population for skeletal muscle homeostasis and 31 regeneration throughout adulthood. MuSCs are an ideal candidate for cell therapies for chronic and acute 32 muscle injuries and diseases given their inherent ability to self-renew and generate progenitor cells 33 capable of myogenic commitment and fusion. Given their rarity and propensity to lose stem-cell potential 34 in prolonged culture, methods for ex vivo MuSC expansion that achieve clinical-scale stem cell yields 35 represent a critical unmet need in muscle cell-therapeutic development. Here, we tested a 36 microenvironment engineering approach to achieve long-term adult mouse MuSC expansion suitable for 37 clinical demands through the combined optimization of techniques previously reported to achieve small-38 yield MuSC expansion in short-term cultures. We developed an optimized protocol for high-yield MuSC 39 expansion through the combination of inflammatory cytokine and growth factor co-stimulation, temporally-40 staged inhibition of the p38α/β mitogen activated protein kinase signaling pathway, and modulation of 41 substrate rigidity in long-term hydrogel cultures. We found that, on soft, muscle-mimicking (12 kPa) 42 hydrogel substrates, a mixture of the cytokines TNF-α, IL-1α, IL-13, and IFN-γ and the growth factor 43 FGF2 stimulated robust exponential proliferation of adult MuSCs from both wildtype and mdx dystrophic 44 mice for up to five weeks of culture that was accompanied by a phenotype shift towards committed 45 myocytes. After observing that the temporal variation in myogenic commitment coincided with an 46 oscillatory activation of p38α/β signaling, we tested a late-stage p38α/β inhibition strategy and found that 47 blocking p38α/β signaling after three weeks, but not earlier, substantially enhanced cell yield, stem-cell 48 phenotypes, and, critically, preserved transplantation potential for up to five weeks of FGF2/cytokine mix 49 culture on soft hydrogels. Notably, this retention of transplant engraftment potency was not observed on 50 traditional plastic substrates. We estimate that this protocol achieves >10 8 -fold yield in Pax7 + stem cells 51 from each starting MuSC, which represents a substantial improvement in stem-cell yield from long-term 52 cultures compared to established methods. 53 Highlights 54 • TNF-α/IL-1α/IL-13/IFN-γ cytokine cocktail supports prolonged MuSC proliferation ex vivo but 55 induces differentiation. 56 • Cytokine cocktail regulates cell signaling with varied prolonged activation signatures. 57 • Effects of p38α/β inhibition on cytokine-induced MuSC expansion are stage-dependent. 58 • Soft hydrogels with late-stage p38α/β inhibition expand functional Pax7 + MuSCs long-term. 59 60 Short summary 61 Cosgrove and colleagues develop a long-term muscle stem cell expansion protocol by combining a 62 tunable stiffness hydrogel substrate, an inflammatory cytokine cocktail, and targeted inhibition of p38 63 MAPK signaling. They show that soft, muscle-mimicking hydr...