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Single Cell Gene Expression Profiling of Skeletal Muscle-Derived Cells

  • Sole Gatto
  • Pier Lorenzo Puri
  • Barbora MalecovaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1556)

Abstract

Single cell gene expression profiling is a fundamental tool for studying the heterogeneity of a cell population by addressing the phenotypic and functional characteristics of each cell. Technological advances that have coupled microfluidic technologies with high-throughput quantitative RT-PCR analyses have enabled detailed analyses of single cells in various biological contexts. In this chapter, we describe the procedure for isolating the skeletal muscle interstitial cells termed Fibro-Adipogenic Progenitors (FAPs ) and their gene expression profiling at the single cell level. Moreover, we accompany our bench protocol with bioinformatics analysis designed to process raw data as well as to visualize single cell gene expression data. Single cell gene expression profiling is therefore a useful tool in the investigation of FAPs heterogeneity and their contribution to muscle homeostasis.

Key words

Fibro-Adipogenic Progenitors (FAPs) Muscle regeneration Single cell gene expression Fluorescence Activated Cell Sorting (FACS) Flow cytometry Data analysis Fluidigm® technology Principal component analysis Hierarchical clustering heatmap Violin plots Bar plots 

Notes

Acknowledgment

This work was supported by NIH P30 pilot grant P30AR061303 and CIRM training grant TG2-01162 to B.M., and NIH grants R01AR056712, R01AR052779, and P30 AR061303 to P.L.P. We thank Thomas C. Roberts and Alessandra Dall’Agnese for critical reading of the manuscript.

Supplementary material

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References

  1. 1.
    Joe AWB, Yi L, Natarajan A et al (2010) Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nat Cell Biol 12:153–163CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Heredia JE, Mukundan L, Chen FM et al (2013) Type 2 innate signals stimulate fibro/adipogenic progenitors to facilitate muscle regeneration. Cell 153:376–388CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Mozzetta C, Consalvi S, Saccone V et al (2013) Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice. EMBO Mol Med 5:626–639CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Saccone V, Consalvi S, Giordani L et al (2014) HDAC-regulated myomiRs control BAF60 variant exchange and direct the functional phenotype of fibro-adipogenic progenitors in dystrophic muscles. Genes Dev 28:841–857CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Uezumi A, Fukada S-I, Yamamoto N et al (2010) Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nat Cell Biol 12:143–152CrossRefPubMedGoogle Scholar
  6. 6.
    Uezumi A, Ito T, Morikawa D et al (2011) Fibrosis and adipogenesis originate from a common mesenchymal progenitor in skeletal muscle. J Cell Sci 124:3654–3664CrossRefPubMedGoogle Scholar
  7. 7.
    Dulauroy S, Di Carlo SE, Langa F et al (2012) Lineage tracing and genetic ablation of ADAM12(+) perivascular cells identify a major source of profibrotic cells during acute tissue injury. Nat Med 18:1262–1270CrossRefPubMedGoogle Scholar
  8. 8.
    Lemos DR, Babaeijandaghi F, Low M, et al (2015) Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors, Nature medicine.Google Scholar
  9. 9.
    Pannerec A, Formicola L, Besson V et al (2013) Defining skeletal muscle resident progenitors and their cell fate potentials. Development 140:2879–2891CrossRefPubMedGoogle Scholar
  10. 10.
    B.Malecova and P.L.Puri (2012) “Mix of Mics”- Phenotypic and Biological Heterogeneity of “Multipotent” Muscle Interstitial Cells (MICs), Journal of Stem Cell Research & Therapy.Google Scholar
  11. 11.
    Judson RN, Zhang R-H, Rossi FMA (2013) Tissue-resident mesenchymal stem/progenitor cells in skeletal muscle: collaborators or saboteurs? FEBS J 280:4100–4108CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Natarajan A, Lemos DR, Rossi FMV (2010) Fibro/adipogenic progenitors: a double-edged sword in skeletal muscle regeneration. Cell Cycle 9:2045–2046CrossRefPubMedGoogle Scholar
  13. 13.
    Giordani L, Puri PL (2013) Epigenetic control of skeletal muscle regeneration: Integrating genetic determinants and environmental changes. FEBS J 280:4014–4025CrossRefPubMedGoogle Scholar
  14. 14.
    Yi L, Rossi F (2011) Purification of progenitors from skeletal muscle. Journal of visualized experiments, JoVEGoogle Scholar
  15. 15.
    Sacco A, Doyonnas R, Kraft P et al (2008) Self-renewal and expansion of single transplanted muscle stem cells. Nature 456:502–506CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Livak KJ, Wills QF, Tipping AJ et al (2013) Methods for qPCR gene expression profiling applied to 1440 lymphoblastoid single cells. Methods 59:71–79CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Core Team R (2013) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria http://www.R-project.org Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Open Access This chapter is distributed under the terms of the Creative Commons Attribution Noncommercial License, which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Authors and Affiliations

  • Sole Gatto
    • 1
  • Pier Lorenzo Puri
    • 1
    • 2
  • Barbora Malecova
    • 1
    • 3
    Email author
  1. 1.Development, Aging and Regeneration ProgramSanford Burnham Prebys Medical Discovery InstituteLa JollaUSA
  2. 2.IRCCS Fondazione Santa LuciaRomeItaly
  3. 3.Regulus Therapeutics10614 Science Center Dr.San DiegoUSA

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