Dual inhibition of P38 MAPK and JNK pathways preserves stemness markers and alleviates premature activation of muscle stem cells during isolation.
p38 Mitogen-Activated Protein Kinases
/ metabolism
Animals
Humans
Mice
MAP Kinase Signaling System
/ drug effects
Muscle, Skeletal
/ metabolism
Stem Cells
/ metabolism
Cell Differentiation
/ drug effects
Mice, Inbred C57BL
Male
Anthracenes
/ pharmacology
JNK Mitogen-Activated Protein Kinases
/ metabolism
Cell therapy
Enzymatic dissociation
MAPK signaling pathways
Muscle stem cells
PAX7
Journal
Stem cell research & therapy
ISSN: 1757-6512
Titre abrégé: Stem Cell Res Ther
Pays: England
ID NLM: 101527581
Informations de publication
Date de publication:
21 Jun 2024
21 Jun 2024
Historique:
received:
03
04
2024
accepted:
08
06
2024
medline:
21
6
2024
pubmed:
21
6
2024
entrez:
20
6
2024
Statut:
epublish
Résumé
Adult skeletal muscle contains resident muscle stem cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative medicine approaches. However, purification process of MuSC remains a major hurdle to their use in the clinic. Indeed, muscle tissue enzymatic dissociation triggers a massive activation of stress signaling pathways, among which P38 and JNK MAPK, associated with a premature loss of MuSC quiescence. While the role of these pathways in the myogenic progression of MuSC is well established, the extent to which their dissociation-induced activation affects the functionality of these cells remains unexplored. We assessed the effect of P38 and JNK MAPK induction on stemness marker expression and MuSC activation state during isolation by pharmacological approaches. MuSC functionality was evaluated by in vitro assays and in vivo transplantation experiments. We performed a comparative analysis of the transcriptome of human MuSC purified with pharmacological inhibitors of P38 and JNK MAPK (SB202190 and SP600125, respectively) versus available RNAseq resources. We monitored PAX7 protein levels in murine MuSC during muscle dissociation and demonstrated a two-step decline partly dependent on P38 and JNK MAPK activities. We showed that simultaneous inhibition of these pathways throughout the MuSC isolation process preserves the expression of stemness markers and limits their premature activation, leading to improved survival and amplification in vitro as well as increased engraftment in vivo. Through a comparative RNAseq analysis of freshly isolated human MuSC, we provide evidence that our findings in murine MuSC could be relevant to human MuSC. Based on these findings, we implemented a purification strategy, significantly improving the recovery yields of human MuSC. Our study highlights the pharmacological limitation of P38 and JNK MAPK activities as a suitable strategy to qualitatively and quantitatively ameliorate human MuSC purification process, which could be of great interest for cell-based therapies.
Sections du résumé
BACKGROUND
BACKGROUND
Adult skeletal muscle contains resident muscle stem cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative medicine approaches. However, purification process of MuSC remains a major hurdle to their use in the clinic. Indeed, muscle tissue enzymatic dissociation triggers a massive activation of stress signaling pathways, among which P38 and JNK MAPK, associated with a premature loss of MuSC quiescence. While the role of these pathways in the myogenic progression of MuSC is well established, the extent to which their dissociation-induced activation affects the functionality of these cells remains unexplored.
METHODS
METHODS
We assessed the effect of P38 and JNK MAPK induction on stemness marker expression and MuSC activation state during isolation by pharmacological approaches. MuSC functionality was evaluated by in vitro assays and in vivo transplantation experiments. We performed a comparative analysis of the transcriptome of human MuSC purified with pharmacological inhibitors of P38 and JNK MAPK (SB202190 and SP600125, respectively) versus available RNAseq resources.
RESULTS
RESULTS
We monitored PAX7 protein levels in murine MuSC during muscle dissociation and demonstrated a two-step decline partly dependent on P38 and JNK MAPK activities. We showed that simultaneous inhibition of these pathways throughout the MuSC isolation process preserves the expression of stemness markers and limits their premature activation, leading to improved survival and amplification in vitro as well as increased engraftment in vivo. Through a comparative RNAseq analysis of freshly isolated human MuSC, we provide evidence that our findings in murine MuSC could be relevant to human MuSC. Based on these findings, we implemented a purification strategy, significantly improving the recovery yields of human MuSC.
CONCLUSION
CONCLUSIONS
Our study highlights the pharmacological limitation of P38 and JNK MAPK activities as a suitable strategy to qualitatively and quantitatively ameliorate human MuSC purification process, which could be of great interest for cell-based therapies.
Identifiants
pubmed: 38902774
doi: 10.1186/s13287-024-03795-0
pii: 10.1186/s13287-024-03795-0
doi:
Substances chimiques
p38 Mitogen-Activated Protein Kinases
EC 2.7.11.24
Anthracenes
0
JNK Mitogen-Activated Protein Kinases
EC 2.7.11.24
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
179Subventions
Organisme : AFM-Téléthon
ID : Translamuscle PROJECT 19507
Organisme : Agence Nationale de la Recherche
ID : ANR-22-ASTR-0034 HydrACell
Organisme : Fondation des Gueules Cassées
ID : Dossier n°59
Informations de copyright
© 2024. The Author(s).
Références
Hung M, Lo HF, Jones GEL, Krauss RS. The muscle stem cell niche at a glance. J Cell Sci. 2023;136(24):jcs261200.
pubmed: 38149870
doi: 10.1242/jcs.261200
Relaix F, Bencze M, Borok MJ, Der Vartanian A, Gattazzo F, Mademtzoglou D, et al. Perspectives on skeletal muscle stem cells. Nat Commun. 2021;12(1):692.
pubmed: 33514709
pmcid: 7846784
doi: 10.1038/s41467-020-20760-6
Chargé SBP, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev. 2004;84(1):209–38.
pubmed: 14715915
doi: 10.1152/physrev.00019.2003
Zammit PS, Golding JP, Nagata Y, Hudon V, Partridge TA, Beauchamp JR. Muscle satellite cells adopt divergent fates: a mechanism for self-renewal. J Cell Biol. 2004;166(3):347–57.
pubmed: 15277541
pmcid: 2172269
doi: 10.1083/jcb.200312007
Rocheteau P, Gayraud-Morel B, Siegl-Cachedenier I, Blasco MA, Tajbakhsh S. A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division. Cell. 2012;148(1):112–25.
pubmed: 22265406
doi: 10.1016/j.cell.2011.11.049
Von Maltzahn J, Jones AE, Parks RJ, Rudnicki MA. Pax7 is critical for the normal function of satellite cells in adult skeletal muscle. Proc Natl Acad Sci. 2013;110(41):16474–9.
doi: 10.1073/pnas.1307680110
Judson RN, Rossi FMV. Towards stem cell therapies for skeletal muscle repair. Npj Regen Med. 2020;5(1):1–6.
doi: 10.1038/s41536-020-0094-3
Montarras D, Morgan J, Collins C, Relaix F, Zaffran S, Cumano A, et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science. 2005;309(5743):2064–7.
pubmed: 16141372
doi: 10.1126/science.1114758
Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM. Self-renewal and expansion of single transplanted muscle stem cells. Nature. 2008;456(7221):502–6.
pubmed: 18806774
pmcid: 2919355
doi: 10.1038/nature07384
Renault V, Rolland E, Thornell LE, Mouly V, Butler-Browne G. Distribution of satellite cells in the human vastus lateralis muscle during aging. Exp Gerontol. 2002;37(12):1513–4.
pubmed: 12559420
doi: 10.1016/S0531-5565(02)00095-5
Bigot A, Jacquemin V, Debacq-Chainiaux F, Butler-Browne GS, Toussaint O, Furling D, et al. Replicative aging down-regulates the myogenic regulatory factors in human myoblasts. Biol Cell. 2008;100(3):189–99.
pubmed: 17988214
doi: 10.1042/BC20070085
Cooper RN, Thiesson D, Furling D, Di Santo JP, Butler-Browne GS, Mouly V. Extended amplification in vitro and replicative senescence: key factors implicated in the success of human myoblast transplantation. Hum Gene Ther. 2003;14(12):1169–79.
pubmed: 12908968
doi: 10.1089/104303403322168000
Castiglioni A, Hettmer S, Lynes MD, Rao TN, Tchessalova D, Sinha I, et al. Isolation of progenitors that exhibit myogenic/osteogenic bipotency in vitro by fluorescence-activated cell sorting from human fetal muscle. Stem Cell Rep. 2014;2(1):92–106.
doi: 10.1016/j.stemcr.2013.12.006
Gheller BJ, Blum J, Souigeid-Baumgarten S, Bender E, Cosgrove BD, Thalacker-Mercer A. Isolation, culture, characterization, and differentiation of human muscle progenitor cells from the skeletal muscle biopsy procedure. J Vis Exp. 2019;150:59580. https://doi.org/10.3791/59580 .
doi: 10.3791/59580
Liu L, Cheung TH, Charville GW, Rando TA. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat Protoc. 2015;10(10):1612–24.
pubmed: 26401916
pmcid: 4793971
doi: 10.1038/nprot.2015.110
Kann AP, Hung M, Wang W, Nguyen J, Gilbert PM, Wu Z, et al. An injury-responsive Rac-to-Rho GTPase switch drives activation of muscle stem cells through rapid cytoskeletal remodeling. Cell Stem Cell. 2022;29(6):933-947.e6.
pubmed: 35597234
pmcid: 9177759
doi: 10.1016/j.stem.2022.04.016
Machado L, Geara P, Camps J, Santos MD, Teixeira-Clerc F, Herck JV, et al. Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation. Cell Stem Cell. 2021;28(6):1125-1135.e7.
pubmed: 33609440
doi: 10.1016/j.stem.2021.01.017
van den Brink SC, Sage F, Vértesy Á, Spanjaard B, Peterson-Maduro J, Baron CS, et al. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nat Methods. 2017;14(10):935–6.
pubmed: 28960196
doi: 10.1038/nmeth.4437
Machado L, de Lima JE, Fabre O, Proux C, Legendre R, Szegedi A, Varet H, Ingerslev LR, Barrès R, Relaix F, Mourikis P. In situ fixation redefines quiescence and early activation of skeletal muscle stem cells. Cell Rep. 2017;21(7):1982–93.
pubmed: 29141227
doi: 10.1016/j.celrep.2017.10.080
van Velthoven CTJ, de Morree A, Egner IM, Brett JO, Rando TA. Transcriptional profiling of quiescent muscle stem cells in vivo. Cell Rep. 2017;21(7):1994–2004.
pubmed: 29141228
pmcid: 5711481
doi: 10.1016/j.celrep.2017.10.037
Almada AE, Horwitz N, Price FD, Gonzalez AE, Ko M, Bolukbasi OV, et al. FOS licenses early events in stem cell activation driving skeletal muscle regeneration. Cell Rep. 2021;34(4):108656.
pubmed: 33503437
pmcid: 9112118
doi: 10.1016/j.celrep.2020.108656
Jones NC, Tyner KJ, Nibarger L, Stanley HM, Cornelison DDW, Fedorov YV, et al. The p38α/β MAPK functions as a molecular switch to activate the quiescent satellite cell. J Cell Biol. 2005;169(1):105–16.
pubmed: 15824134
pmcid: 2171902
doi: 10.1083/jcb.200408066
Perdiguero E, Ruiz-Bonilla V, Gresh L, Hui L, Ballestar E, Sousa-Victor P, et al. Genetic analysis of p38 MAP kinases in myogenesis: fundamental role of p38α in abrogating myoblast proliferation. EMBO J. 2007;26(5):1245–56.
pubmed: 17304211
pmcid: 1817635
doi: 10.1038/sj.emboj.7601587
Troy A, Cadwallader AB, Fedorov Y, Tyner K, Tanaka KK, Olwin BB. Coordination of satellite cell activation and self-renewal by par-complex-dependent asymmetric activation of p38α/β MAPK. Cell Stem Cell. 2012;11(4):541–53.
pubmed: 23040480
pmcid: 4077199
doi: 10.1016/j.stem.2012.05.025
Charville GW, Cheung TH, Yoo B, Santos PJ, Lee GK, Shrager JB, et al. Ex vivo expansion and in vivo self-renewal of human muscle stem cells. Stem Cell Rep. 2015;5(4):621–32.
doi: 10.1016/j.stemcr.2015.08.004
Cosgrove BD, Gilbert PM, Porpiglia E, Mourkioti F, Lee SP, Corbel SY, et al. Rejuvenation of the aged muscle stem cell population restores strength to injured aged muscles. Nat Med. 2014;20(3):255–64.
pubmed: 24531378
pmcid: 3949152
doi: 10.1038/nm.3464
Ding S, Swennen GNM, Messmer T, Gagliardi M, Molin DGM, Li C, et al. Maintaining bovine satellite cells stemness through p38 pathway. Sci Rep. 2018;8(1):10808.
pubmed: 30018348
pmcid: 6050236
doi: 10.1038/s41598-018-28746-7
Segalés J, Islam ABMMK, Kumar R, Liu QC, Sousa-Victor P, Dilworth FJ, et al. Chromatin-wide and transcriptome profiling integration uncovers p38α MAPK as a global regulator of skeletal muscle differentiation. Skelet Muscle. 2016;6:9.
pubmed: 26981231
pmcid: 4791895
doi: 10.1186/s13395-016-0074-x
Olson MF, Ashworth A, Hall A. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science. 1995;269(5228):1270–2.
pubmed: 7652575
doi: 10.1126/science.7652575
Ishii K, Sakurai H, Suzuki N, Mabuchi Y, Sekiya I, Sekiguchi K, et al. Recapitulation of extracellular laminin environment maintains stemness of satellite cells in vitro. Stem Cell Rep. 2018;10(2):568–82.
doi: 10.1016/j.stemcr.2017.12.013
Shaulian E, Karin M. AP-1 in cell proliferation and survival. Oncogene. 2001;20(19):2390–400.
pubmed: 11402335
doi: 10.1038/sj.onc.1204383
Zhang S, Yang F, Huang Y, He L, Li Y, Wan YCE, et al. ATF3 induction prevents precocious activation of skeletal muscle stem cell by regulating H2B expression. Nat Commun. 2023;14(1):4978.
pubmed: 37591871
pmcid: 10435463
doi: 10.1038/s41467-023-40465-w
Sambasivan R, Gayraud-Morel B, Dumas G, Cimper C, Paisant S, Kelly RG, et al. Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates. Dev Cell. 2009;16(6):810–21.
pubmed: 19531352
doi: 10.1016/j.devcel.2009.05.008
Prigge JR, Wiley JA, Talago EA, Young EM, Johns LL, Kundert JA, et al. Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei. Mamm Genome. 2013;4(9–10):389–99.
doi: 10.1007/s00335-013-9469-8
Murphy MM, Lawson JA, Mathew SJ, Hutcheson DA, Kardon G. Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development. 2011;138(17):3625–37.
pubmed: 21828091
pmcid: 3152921
doi: 10.1242/dev.064162
Buch T, Heppner FL, Tertilt C, Heinen TJAJ, Kremer M, Wunderlich FT, et al. A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration. Nat Methods juin. 2005;2(6):419–26.
doi: 10.1038/nmeth762
Song J, Willinger T, Rongvaux A, Eynon EE, Stevens S, Manz MG, et al. A mouse model for the human pathogen Salmonella typhi. Cell Host Microbe. 2010;8(4):369–76.
pubmed: 20951970
pmcid: 2972545
doi: 10.1016/j.chom.2010.09.003
Sambasivan R, Yao R, Kissenpfennig A, Van Wittenberghe L, Paldi A, Gayraud-Morel B, et al. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development. 2011;138(17):3647–56.
pubmed: 21828093
doi: 10.1242/dev.067587
Boldrin L, Neal A, Zammit PS, Muntoni F, Morgan JE. Donor satellite cell engraftment is significantly augmented when the host niche is preserved and endogenous satellite cells are incapacitated. Stem Cells. 2012;30(9):1971–84.
pubmed: 22730231
doi: 10.1002/stem.1158
Gattazzo F, Laurent B, Relaix F, Rouard H, Didier N. Distinct phases of post-natal skeletal muscle growth govern the progressive establishment of muscle stem cell quiescence. Stem Cell Rep. 2020;15(3):597–611.
doi: 10.1016/j.stemcr.2020.07.011
Ito H, Iwamoto I, Inaguma Y, Takizawa T, Nagata K, Asano T, et al. Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27. J Cell Biochem. 2005;95(5):932–41.
pubmed: 15864808
doi: 10.1002/jcb.20445
Ruan J, Qi Z, Shen L, Jiang Y, Xu Y, Lan L, et al. Crosstalk between JNK and NF-κB signaling pathways via HSP27 phosphorylation in HepG2 cells. Biochem Biophys Res Commun. 2015;456(1):122–8.
pubmed: 25446109
doi: 10.1016/j.bbrc.2014.11.045
Alfaro LAS, Dick SA, Siegel AL, Anonuevo AS, McNagny KM, Megeney LA, et al. CD34 promotes satellite cell motility and entry into proliferation to facilitate efficient skeletal muscle regeneration. Stem Cells. 2011;29(12):2030–41.
pubmed: 21997891
doi: 10.1002/stem.759
Deterding LJ, Williams JG, Humble MM, Petrovich RM, Wei SJ, Trempus CS, et al. CD34 antigen: determination of specific sites of phosphorylation in vitro and in vivo. Int J Mass Spectrom. 2011;301(1–3):12–21.
pubmed: 21499536
pmcid: 3077033
doi: 10.1016/j.ijms.2010.05.027
Gioftsidi S, Relaix F, Mourikis P. The Notch signaling network in muscle stem cells during development, homeostasis, and disease. Skelet Muscle. 2022;12(1):9.
pubmed: 35459219
pmcid: 9027478
doi: 10.1186/s13395-022-00293-w
De Micheli AJ, Spector JA, Elemento O, Cosgrove BD. A reference single-cell transcriptomic atlas of human skeletal muscle tissue reveals bifurcated muscle stem cell populations. Skelet Muscle. 2020;10(1):19.
pubmed: 32624006
pmcid: 7336639
doi: 10.1186/s13395-020-00236-3
Garcia SM, Tamaki S, Lee S, Wong A, Jose A, Dreux J, et al. High-yield purification, preservation, and serial transplantation of human satellite cells. Stem Cell Rep. 2018;10(3):1160–74.
doi: 10.1016/j.stemcr.2018.01.022
Bustos F, de la Vega E, Cabezas F, Thompson J, Cornelison DDW, Olwin BB, et al. NEDD4 regulates PAX7 levels promoting activation of the differentiation program in skeletal muscle precursors. Stem Cells. 2015;33(10):3138–51.
pubmed: 26304770
doi: 10.1002/stem.2125
Montecino F, González N, Blanco N, Ramírez MJ, González-Martín A, Alvarez AR, et al. c-Abl kinase is required for satellite cell function through Pax7 regulation. Front Cell Dev Biol. 2021;9:606403.
pubmed: 33777928
pmcid: 7990767
doi: 10.3389/fcell.2021.606403
Dick SA, Chang NC, Dumont NA, Bell RAV, Putinski C, Kawabe Y, et al. Caspase 3 cleavage of Pax7 inhibits self-renewal of satellite cells. Proc Natl Acad Sci U S A. 2015;112(38):E5246-5252.
pubmed: 26372956
pmcid: 4586827
doi: 10.1073/pnas.1512869112
González N, Moresco JJ, Cabezas F, de la Vega E, Bustos F, Iii JRY, et al. Ck2-dependent phosphorylation is required to maintain pax7 protein levels in proliferating muscle progenitors. PLoS ONE. 2016;11(5):e0154919.
pubmed: 27144531
pmcid: 4856311
doi: 10.1371/journal.pone.0154919
Nethe M, De Kreuk BJ, Tauriello DVF, Anthony EC, Snoek B, Stumpel T, et al. Rac1 acts in conjunction with Nedd4 and dishevelled-1 to promote maturation of cell-cell contacts. J Cell Sci. 2012;125:3430–42.
pubmed: 22467858
pmcid: 3516380
Palacios D, Mozzetta C, Consalvi S, Caretti G, Saccone V, Proserpio V, et al. TNF/p38α/polycomb signaling to Pax7 locus in satellite cells links inflammation to the epigenetic control of muscle regeneration. Cell Stem Cell. 2010;7(4):455–69.
pubmed: 20887952
pmcid: 2951277
doi: 10.1016/j.stem.2010.08.013
Hu P, Nebreda AR, Hanenberg H, Kinnebrew GH, Ivan M, Yoder MC, et al. P38α/JNK signaling restrains erythropoiesis by suppressing Ezh2-mediated epigenetic silencing of Bim. Nat Commun. 2018;9:3518.
pubmed: 30158520
pmcid: 6115418
doi: 10.1038/s41467-018-05955-2
Van Velthoven CTJ, De Morree A, Egner IM, Brett JO, Rando TA. Transcriptional profiling of quiescent muscle stem cells in vivo. Cell Rep. 2017;21(7):1994–2004.
pubmed: 29141228
pmcid: 5711481
doi: 10.1016/j.celrep.2017.10.037
Eberhardt W, Doller A, Akool ES, Pfeilschifter J. Modulation of mRNA stability as a novel therapeutic approach. Pharmacol Ther avr. 2007;114(1):56–73.
doi: 10.1016/j.pharmthera.2007.01.002