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

View ORCID ProfileAlexander M. Loiben, View ORCID ProfileKun Ho Kim, View ORCID ProfileSharon Y. Soueid-Baumgarten, View ORCID ProfileVictor M. Aguilar, View ORCID ProfileJonathan Chin Cheong, View ORCID ProfileRuth F. Kopyto, View ORCID ProfilePaula Fraczek, View ORCID ProfileErn Hwei Hannah Fong, View ORCID ProfileRahul Mangal, View ORCID ProfileLynden A. Archer, View ORCID ProfileBenjamin D. Cosgrove
doi: https://doi.org/10.1101/2020.06.04.134056
Alexander M. Loiben
1Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
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Kun Ho Kim
1Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
aDepartment of Animal Sciences, Purdue University, West Lafayette, IN, USA
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Sharon Y. Soueid-Baumgarten
1Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
bDepartment of Biochemistry, Technion Israel Institute of Technology, Haifa, Israel
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Victor M. Aguilar
1Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
cDepartment of Bioengineering, University of Illinois-Chicago, Chicago, IL, USA
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Jonathan Chin Cheong
1Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
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Ruth F. Kopyto
2Biological Sciences Program, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY, USA
dProgram in Pharmacology, Weill Cornell Medicine, New York City, NY, USA
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Paula Fraczek
1Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
eDepartment of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
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Ern Hwei Hannah Fong
1Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
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Rahul Mangal
3Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
fDepartment of Chemical Engineering, Indian Institute of Technology Kanpur, Uttar Pradesh, India
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Lynden A. Archer
3Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
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Benjamin D. Cosgrove
1Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
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  • For correspondence: bdc68@cornell.edu
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Abstract

Muscle stem cells (MuSCs) are an essential stem cell population for skeletal muscle homeostasis and regeneration throughout adulthood. MuSCs are an ideal candidate for cell therapies for chronic and acute muscle injuries and diseases given their inherent ability to self-renew and generate progenitor cells capable of myogenic commitment and fusion. Given their rarity and propensity to lose stem-cell potential in prolonged culture, methods for ex vivo MuSC expansion that achieve clinical-scale stem cell yields represent a critical unmet need in muscle cell-therapeutic development. Here, we tested a microenvironment engineering approach to achieve long-term adult mouse MuSC expansion suitable for clinical demands through the combined optimization of techniques previously reported to achieve small-yield MuSC expansion in short-term cultures. We developed an optimized protocol for high-yield MuSC expansion through the combination of inflammatory cytokine and growth factor co-stimulation, temporally-staged inhibition of the p38α/β mitogen activated protein kinase signaling pathway, and modulation of substrate rigidity in long-term hydrogel cultures. We found that, on soft, muscle-mimicking (12 kPa) hydrogel substrates, a mixture of the cytokines TNF-α, IL-1α, IL-13, and IFN-γ and the growth factor FGF2 stimulated robust exponential proliferation of adult MuSCs from both wildtype and mdx dystrophic mice for up to five weeks of culture that was accompanied by a phenotype shift towards committed myocytes. After observing that the temporal variation in myogenic commitment coincided with an oscillatory activation of p38α/β signaling, we tested a late-stage p38α/β inhibition strategy and found that blocking p38α/β signaling after three weeks, but not earlier, substantially enhanced cell yield, stem-cell phenotypes, and, critically, preserved transplantation potential for up to five weeks of FGF2/cytokine mix culture on soft hydrogels. Notably, this retention of transplant engraftment potency was not observed on traditional plastic substrates. We estimate that this protocol achieves >108-fold yield in Pax7+ stem cells from each starting MuSC, which represents a substantial improvement in stem-cell yield from long-term cultures compared to established methods.

Highlights

  • TNF-α/IL-1α/IL-13/IFN-γ cytokine cocktail supports prolonged MuSC proliferation ex vivo but induces differentiation.

  • Cytokine cocktail regulates cell signaling with varied prolonged activation signatures.

  • Effects of p38α/β inhibition on cytokine-induced MuSC expansion are stage-dependent.

  • Soft hydrogels with late-stage p38α/β inhibition expand functional Pax7+ MuSCs long-term.

Short summary Cosgrove and colleagues develop a long-term muscle stem cell expansion protocol by combining a tunable stiffness hydrogel substrate, an inflammatory cytokine cocktail, and targeted inhibition of p38 MAPK signaling. They show that soft, muscle-mimicking hydrogels with delayed p38 inhibition yield robust quantities of Pax7+ functional muscle stem cells.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 05, 2020.
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Long-term high-yield skeletal muscle stem cell expansion through staged perturbation of cytokine signaling in a soft hydrogel culture platform
Alexander M. Loiben, Kun Ho Kim, Sharon Y. Soueid-Baumgarten, Victor M. Aguilar, Jonathan Chin Cheong, Ruth F. Kopyto, Paula Fraczek, Ern Hwei Hannah Fong, Rahul Mangal, Lynden A. Archer, Benjamin D. Cosgrove
bioRxiv 2020.06.04.134056; doi: https://doi.org/10.1101/2020.06.04.134056
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Long-term high-yield skeletal muscle stem cell expansion through staged perturbation of cytokine signaling in a soft hydrogel culture platform
Alexander M. Loiben, Kun Ho Kim, Sharon Y. Soueid-Baumgarten, Victor M. Aguilar, Jonathan Chin Cheong, Ruth F. Kopyto, Paula Fraczek, Ern Hwei Hannah Fong, Rahul Mangal, Lynden A. Archer, Benjamin D. Cosgrove
bioRxiv 2020.06.04.134056; doi: https://doi.org/10.1101/2020.06.04.134056

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