采用荧光激活细胞分选法从营养不良骨骼肌中分离成纤维成脂前体（FAP）细胞

Nicoletta Cordani; Viviana Pisa; Laura Pozzi; Clara Sciorati

doi:10.21769/BioProtoc.1292

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Peer-reviewed

Isolation of FAP Cells from Mouse Dystrophic Skeletal Muscle Using Fluorescence Activated Cell Sorting

采用荧光激活细胞分选法从营养不良骨骼肌中分离成纤维成脂前体（FAP）细胞

Nicoletta Cordani

VP Viviana Pisa

LP Laura Pozzi

Clara Sciorati email

发布: 2014年11月20日第4卷第22期 DOI: 10.21769/BioProtoc.1292 浏览次数: 14631

评审: Anonymous reviewer(s)

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Abstract

A population of muscle resident CD45^-, CD31^- cells expressing the mesenchymal PDGF receptor alpha (PDGFRα) as well as Sca-1 was first isolated in healthy mouse muscles in Uezumi et al. (2010). In the same year, Joe et al. (2010) identified and purified fibro-adipogenic precursors (FAPs), cells located into the interstitial space between myofibers close to vessels, negative for CD45, CD31,α7-Integrin, but expressing CD34, Sca-1.

Both groups demonstrated that these cells are not myogenic in vitro or in vivo, but they are capable of differentiating in vitro towards both fibrogenic and adipogenic lineage (Uezumi et al., 2011). Further marker analysis indicates that the two groups identified independently the same cell population (Natarajan et al., 2010).

FAPs are an important source of fibrosis and adipogenesis in dystrophic skeletal muscle (Natarajan et al., 2010; Cordani et al., 2014). We have recently demonstrated that Nitric Oxide regulates FAP fate inhibiting in vitro their differentiation into adipocytes. In mdx mice, an animal model of DMD, fed with a diet containing the nitric oxide donating drug, Molsidomine, the number of PDGFRα⁺ cells was reduced as well as the deposition of both skeletal muscle fat and connective tissues (Cordani et al., 2014). Here we described a method to isolate in both wild type and in mdx dystrophic muscle pure population of FAPs by double selection for SCA-1 and PDGFRα positivity in absence of the satellite cell markers SM/C2.6 and α7integrin as well of the pan-lymphocytes marker CD45 or endothelial marker CD31.

Keywords: Fibroadypose precursors (fibroadypose前兆)

Skeletal muscle (骨骼肌)

Fluorescence cell sorting (荧光细胞分选)

Mouse dystrophy model (营养不良模型小鼠)

Satellite cells (卫星细胞)

Materials and Reagents

8-10 weeks old mice C57BL/6J wild-type mice (Charles River Laboratories International, http://www.criver.com) and mdx-4cv mice (B6Ros.Cg-Dmdmdx-4cv/J, crossed on C57/BL/6 background; Jackson ImmunoResearch Laboratories)
Note: Animals were treated in accordance with European Community guidelines and with the approval of the Institutional Ethical Committee.
Collagenase II (Worthington Biochemical, catalog number: CLSS2 )
Dulbecco's modified high glucose Eagle's medium (DMEM high glucose) (EuroClone, catalog number: ECB7501L )
100 U/ml penicillin and 100 μg/ml streptomycin (EuroClone)
L-glutammine (EuroClone)
Recombinant human basic fibroblast growth factor (b-FGF) (Pepro Tech, catalog number: 100-18B )
Growth factor reduced BD Matrigel^TM Matrix (BD Biosciences, catalog number: 356230 )
Foetal Bovine Serum (FBS) (EuroClone)
Sterile phosphate buffered saline (PBS) w/o Ca⁺⁺Mg⁺⁺ (EuroClone)
Antibodies
1. Anti-CD31-phycoerythrin/Cy7 (anti-CD31-PE/Cy7, clone 390) (eBioscience, catalog number: 25-0311 )
2. Anti-CD45-PECy7 (clone 30-F11) (eBioscience, catalog number: 15-0451 )
3. Anti-SM/C2.6-Biotin (kindly provided by Dr. Fukada) (Fukada et al., 2004)
4. Streptavidin-PE (BioLegend)
5. Anti-α7-Integrin-PE (clone R2F2) (AbLab Laboratorio di Istologia e Citologia Patologica Veterinaria, catalog number: AB10RS24MW215 )
6. Anti-PDGFRα-allophycocyanin (APC, CD140a, clone APA5) (BioLegend, catalog number: 135907 )
7. Anti-LY-6A/E SCA-1-allophycocyanin/Cy7 (APC/Cy7, clone B7) (BD Biosciences, catalog number: 560654 )
8. 7-aminoactinomycin D (7-AAD) (Life Technologies, catalog number: A1310 )
Growth medium (GM) (see Recipes)
Wash buffer (WB) (see Recipes)
Collagenase II solution (see Recipes)
Erythrocytes lysis buffer (see Recipes)
Note: Use 1 ml of this solution for approximately 1 g of muscle.
Sorting buffer (see Recipes)
Matrigel solution (see Recipes)
b-FGF solution (see Recipes)

Equipment

Scissors and tweezers
Cell culture plastic dishes (Corning, Costar^®)
Six multiwells (Corning, Costar^®)
Centrifuge
50 and 15 ml plastic tubes
18G-10 ml syringes
70 μm and 40 μm cell strainer caps (BD Biosciences)
Beckam Coulter Cell Sorter MoFlo^TM XDP (Beckman Coulter, catalog number: ML99030 )
Cell culture incubator at 37 °C and 5% CO₂
Microscope or cell counter

Procedure

English

中文翻译

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如何引用

Cordani, N., Pisa, V., Pozzi, L. and Sciorati, C. (2014). Isolation of FAP Cells from Mouse Dystrophic Skeletal Muscle Using Fluorescence Activated Cell Sorting. Bio-protocol 4(22): e1292. DOI: 10.21769/BioProtoc.1292.