title: Murine muscle engineered from dermal precursors: an in vitro model for skeletal muscle generation, degeneration, and fatty infiltration.
abstract: Skeletal muscle can be engineered by converting dermal precursors into muscle progenitors and differentiated myocytes. However, the efficiency of muscle development remains relatively low and it is currently unclear if this is due to poor characterization of the myogenic precursors, the protocols used for cell differentiation, or a combination of both. In this study, we characterized myogenic precursors present in murine dermospheres, and evaluated mature myotubes grown in a novel three-dimensional culture system. After 5-7 days of differentiation, we observed isolated, twitching myotubes followed by spontaneous contractions of the entire tissue-engineered muscle construct on an extracellular matrix (ECM). In vitro engineered myofibers expressed canonical muscle markers and exhibited a skeletal (not cardiac) muscle ultrastructure, with numerous striations and the presence of aligned, enlarged mitochondria, intertwined with sarcoplasmic reticula (SR). Engineered myofibers exhibited Na(+)- and Ca(2+)-dependent inward currents upon acetylcholine (ACh) stimulation and tetrodotoxin-sensitive spontaneous action potentials. Moreover, ACh, nicotine, and caffeine elicited cytosolic Ca(2+) transients; fiber contractions coupled to these Ca(2+) transients suggest that Ca(2+) entry is activating calcium-induced calcium release from the SR. Blockade by d-tubocurarine of ACh-elicited inward currents and Ca(2+) transients suggests nicotinic receptor involvement. Interestingly, after 1 month, engineered muscle constructs showed progressive degradation of the myofibers concomitant with fatty infiltration, paralleling the natural course of muscular degeneration. We conclude that mature myofibers may be differentiated on the ECM from myogenic precursor cells present in murine dermospheres, in an in vitro system that mimics some characteristics found in aging and muscular degeneration.
authors: 1 Tissue Engineering Laboratory, Bioengineering Area, Instituto Biodonostia, Hospital Universitario Donostia , San Sebastian, Spain .
authors: Patricia
authors: P
authors: García-Parra
authors: Neia
authors: N
authors: Naldaiz-Gastesi
authors: Marcos
authors: M
authors: Maroto
authors: Juan Fernando
authors: JF
authors: Padín
authors: María
authors: M
authors: Goicoechea
authors: Ana
authors: A
authors: Aiastui
authors: José Carlos
authors: JC
authors: Fernández-Morales
authors: Paula
authors: P
authors: García-Belda
authors: Jaione
authors: J
authors: Lacalle
authors: Jose Iñaki
authors: JI
authors: Álava
authors: José Manuel
authors: JM
authors: García-Verdugo
authors: Antonio G
authors: AG
authors: García
authors: Ander
authors: A
authors: Izeta
authors: Adolfo
authors: A
authors: López de Munain
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D020405:Dermis / Q000166:cytology*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D015640:Ion Channel Gating / Q000187:drug effects; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D008954:Models, Biological; D024510:Muscle Development / Q000187:drug effects; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000473:pathology / Q000648:ultrastructure; D009132:Muscles / Q000473:pathology* / Q000503:physiopathology* / Q000648:ultrastructure; D018874:Spheroids, Cellular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D015415:Biomarkers; D008055:Lipids; D000109:Acetylcholine
references: PLoS One. 2012;7(5):e36049
references: 22574134
references: PLoS One. 2012;7(5):e38147
references: 22662278
references: Neurology. 1975 Jan;25(1):58-67
references: 46113
references: Aging Cell. 2012 Dec;11(6):986-95
references: 22882433
references: Stem Cells. 2009 May;27(5):1164-72
references: 19418448
references: Muscle Nerve. 2012 Jul;46(1):112-21
references: 22644824
references: Genesis. 2010 Jul;48(7):424-36
references: 20641127
references: Sci Transl Med. 2012 Jun 27;4(140):140ra89
references: 22745439
references: J Magn Reson Imaging. 2007 Feb;25(2):433-40
references: 17260395
references: Curr Opin Biotechnol. 2009 Oct;20(5):522-30
references: 19896826
references: Br J Pharmacol. 2008 Aug;154(8):1558-71
references: 18500366
references: Am J Pathol. 1999 Apr;154(4):1017-22
references: 10233840
references: In Vitro Cell Dev Biol Anim. 1997 Oct;33(9):659-61
references: 9358276
references: Biomaterials. 2012 Jan;33(2):428-43
references: 22014942
references: In Vivo. 2007 May-Jun;21(3):435-44
references: 17591352
references: Curr Opin Neurol. 2012 Oct;25(5):597-603
references: 22918488
references: Physiol Rev. 2013 Jan;93(1):23-67
references: 23303905
references: Muscle Nerve. 2005 Apr;31(4):418-30
references: 15704142
references: In Vitro Cell Dev Biol. 1990 Oct;26(10):955-61
references: 2243058
references: J Clin Invest. 2010 Jan;120(1):11-9
references: 20051632
references: Nature. 2012 Oct 18;490(7420):355-60
references: 23023126
references: Front Cell Neurosci. 2012 Mar 14;6:10
references: 22435050
references: BMC Neurosci. 2013 Apr 18;14:48
references: 23594371
references: Nat Commun. 2012 Jun 12;3:903
references: 22692546
references: Biogerontology. 2008 Aug;9(4):213-28
references: 18299960
references: Stem Cells Dev. 2010 Feb;19(2):259-68
references: 19594362
references: Biomaterials. 2011 Dec;32(36):9602-11
references: 21944471
references: Crit Rev Eukaryot Gene Expr. 2007;17(1):13-29
references: 17341181
references: Tissue Eng Part A. 2010 Oct;16(10):3251-9
references: 20528672
references: Tissue Eng. 2007 Jul;13(7):1431-42
references: 17561804
references: Curr Opin Neurol. 2012 Oct;25(5):586-7
references: 22892955
references: J Cell Biol. 2005 Sep 26;170(7):1135-46
references: 16186259
references: Physiol Rev. 1979 Jan;59(1):165-227
references: 375254
references: Acta Neuropathol. 2013 Feb;125(2):173-85
references: 23224362
references: Diabetes. 2003 Dec;52(12):2914-22
references: 14633851
references: Vet Rec. 2010 Nov 6;167(19):760
references: 21257518
references: In Vitro Cell Dev Biol. 1988 Mar;24(3):166-74
references: 3350785
references: Cell Stem Cell. 2012 May 4;10(5):610-9
references: 22560081
references: J Gen Physiol. 1988 Aug;92(2):145-59
references: 2459299
references: Invest Ophthalmol Vis Sci. 1991 Jan;32(1):106-11
references: 1987091
references: In Vitro Cell Dev Biol Anim. 1995 Nov;31(10):773-9
references: 8564066
references: Dev Biol. 2003 Oct 1;262(1):64-74
references: 14512018
references: Physiol Rev. 2009 Oct;89(4):1153-76
references: 19789379
references: Pflugers Arch. 1981 Aug;391(2):85-100
references: 6270629
references: J Cell Mol Med. 2010 Nov;14(11):2622-9
references: 21091904
references: Methods. 2001 Dec;25(4):402-8
references: 11846609
references: Biol Cell. 1986;57(1):17-22
references: 2945607
references: Cell Cycle. 2010 Jun 1;9(11):2045-6
references: 20505371
references: J Physiol. 2005 Oct 1;568(Pt 1):171-80
references: 16037088