title: Isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal muscle.
abstract: The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56(+) and later as CD56(+)/desmin(+) cells and (ii) muscle-derived fibroblasts, identified as CD56(-) and TE-7(+). Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (>95% myogenic cells) and good yield (~2.8 x 10(6) ± 8.87 x 10(5) cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56(+) cells bound to microbeads are retained by the field whereas CD56(-) cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.
authors: Centre of Human and Aerospace Physiological Sciences, King's College London; Wellcome Trust-Medical Research Council, Cambridge Stem Cell Institute; chibeza.agley@kcl.ac.uk.
authors: Chibeza C
authors: CC
authors: Agley
authors: Centre of Human and Aerospace Physiological Sciences, King's College London.
authors: Anthea M
authors: AM
authors: Rowlerson
authors: Centre of Human and Aerospace Physiological Sciences, King's College London.
authors: Cristiana P
authors: CP
authors: Velloso
authors: Centre of Human and Aerospace Physiological Sciences, King's College London.
authors: Norman L
authors: NL
authors: Lazarus
authors: Centre of Human and Aerospace Physiological Sciences, King's College London.
authors: Stephen D R
authors: SD
authors: Harridge
mesh_terms: D002454:Cell Differentiation / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003893:Desmin / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology* / Q000378:metabolism*
chemical_list: D003893:Desmin
references: Physiol Rev. 2013 Jan;93(1):23-67
references: 23303905
references: Cytometry A. 2010 Jul;77(7):643-7
references: 20583279
references: Dev Biol. 1986 Oct;117(2):628-35
references: 3758484
references: J Biophys Biochem Cytol. 1961 Feb;9:493-5
references: 13768451
references: Nat Methods. 2005 Dec;2(12):910-9
references: 16299476
references: Cell Stem Cell. 2009 Sep 4;5(3):298-309
references: 19733541
references: Biotechniques. 2000 May;28(5):884-6, 888
references: 10818693
references: J Histochem Cytochem. 2012 Jun;60(6):428-38
references: 22511600
references: Cell. 2007 Jun 1;129(5):999-1010
references: 17540178
references: J Cell Biol. 2003 Dec 8;163(5):1133-43
references: 14662751
references: J Tissue Eng. 2013;4:2041731412472690
references: 23440031
references: J Cell Sci. 2007 May 15;120(Pt 10):1703-5
references: 17502480
references: Cytometry. 1990;11(2):231-8
references: 1690625
references: Exp Cell Res. 2008 Jan 1;314(1):193-203
references: 17963748
references: J Cell Biol. 2009 Jun 29;185(7):1135-48
references: 19564400
references: PLoS One. 2014 Feb 28;9(2):e90398
references: 24587351
references: Mol Biol Cell. 2012 Mar;23(5):754-7
references: 22379119
references: Prog Histochem Cytochem. 2009;44(3):125-72
references: 19822255
references: Exp Cell Res. 1988 Jan;174(1):252-65
references: 3335226
references: J Vis Exp. 2013 Mar 22;(73):e50074
references: 23542587
references: J Anat. 1981 Jun;132(Pt 4):557-79
references: 7298498
references: Aging Cell. 2013 Jun;12(3):333-44
references: 23374245
references: Histochem Cell Biol. 2001 Jul;116(1):63-8
references: 11479724
references: Development. 2011 Sep;138(17):3625-37
references: 21828091
references: Stem Cell Reports. 2014 Jan 14;2(1):92-106
references: 24678452
references: J Cell Physiol. 2003 Jul;196(1):70-8
references: 12767042
references: J Appl Physiol (1985). 2001 Aug;91(2):534-51
references: 11457764
references: Methods Mol Biol. 2012;798:21-52
references: 22130829
references: J Cell Sci. 2013 Dec 15;126(Pt 24):5610-25
references: 24101731
references: J Microsc. 2006 Dec;224(Pt 3):213-32
references: 17210054
references: Muscle Nerve. 2009 Sep;40(3):455-65
references: 19705426
references: J Cell Biol. 2004 Jul 5;166(1):11-5
references: 15240566