Abstract
Growing evidence supports the view that in adult stem cells, the defining stem cell features of potency and self-renewal are associated with the quiescent state. Thus, uncovering the molecular logic of this reversibly arrested state underlies not only a fundamental understanding of adult tissue dynamics but also hopes for therapeutic regeneration and rejuvenation of damaged or aging tissue. A key question concerns how adult stem cells use quiescence to establish or reinforce the property of self-renewal. Since self-renewal is largely studied by assays that measure proliferation, the concept of self-renewal programs imposed during non-proliferating conditions is counterintuitive. However, there is increasing evidence generated by deconstructing the quiescent state that highlights how programs characteristic of this particular cell cycle exit may enhance stem cell capabilities, through both cell-intrinsic and extrinsic programs.
Toward this end, culture models that recapitulate key aspects of stem cell quiescence are useful for molecular analysis to explore attributes and regulation of the quiescent state. In this chapter, we review the different methods used to generate homogeneous populations of quiescent muscle cells, largely by manipulating culture conditions that feed into core signaling programs that regulate the cell cycle. We also provide detailed protocols developed or refined in our lab over the past two decades.
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References
Abdelalim EM (2013) Molecular mechanisms controlling the cell cycle in embryonic stem cells. Stem Cell Rev 9(6):764–773
Kapinas K et al (2013) The abbreviated pluripotent cell cycle. J Cell Physiol 228(1):9–20
Berthet C, Kaldis P (2007) Cell-specific responses to loss of cyclin-dependent kinases. Oncogene 26(31):4469–4477
Sachidanandan C et al (2002) Tristetraprolin and LPS-inducible CXC chemokine are rapidly induced in presumptive satellite cells in response to skeletal muscle injury. J Cell Sci 115(Pt 13):2701–2712
Dhawan J, Rando TA (2005) Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. Trends Cell Biol 15(12):666–673
Gray JV et al (2004) “Sleeping beauty”: quiescence in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 68(2):187–206
Dhawan J, Laxman S (2015) Decodoing the stem cell quiescence cycle: lessons from yeast for regenerative biology. J Cell Sci 128(24):4467–4474
Coller HA et al (2006) A new description of cellular quiescence. PLoS Biol 4(3):e83
Lemons JM et al (2010) Quiescent fibroblasts exhibit high metabolic activity. PLoS Biol 8(10):e1000514
Allen C et al (2006) Isolation of quiescent and nonquiescent cells from yeast stationary-phase cultures. J Cell Biol 174(1):89–100
Rubin H, Koide T (1973) Inhibition of DNA synthesis in chick embryo cultures by deprivation of either serum or zinc. J Cell Biol 56(3):777–786
Srivastava S et al (2010) Regulation of cellular chromatin state: insights from quiescence and differentiation. Organogenesis 6(1):37–47
Ellisen LW et al (2001) The Wilms tumor suppressor WT1 directs stage-specific quiescence and differentiation of human hematopoietic progenitor cells. EMBO J 20(8):1897–1909
Sang L et al (2008) Control of the reversibility of cellular quiescence by the transcriptional repressor HES1. Science 321(5892):1095–1100
Sousa-Victor P et al (2014) Geriatric muscle stem cells switch reversible quiescence into senescence. Nature 506(7488):316–321
Herman PK (2002) Stationary phase in yeast. Curr Opin Microbiol 5(6):602–607
Brejning J et al (2003) Genome-wide transcriptional changes during the lag phase of Saccharomyces cerevisiae. Arch Microbiol 179(4):278–294
Cheshier SH et al (1999) In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Natl Acad Sci U S A 96(6):3120–3125
Cotsarelis G et al (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61(7):1329–1337
Schultz E et al (1978) Satellite cells are mitotically quiescent in mature mouse muscle: an EM and radioautographic study. J Exp Zool 206(3):451–456
Schultz E (1996) Satellite cell proliferative compartments in growing skeletal muscles. Dev Biol 175(1):84–94
Fukada S et al (2011) Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers. Development 138(21):4609–4619
Arai F et al (2004) Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118(2):149–161
Mourikis P et al (2012) A critical requirement for notch signaling in maintenance of the quiescent skeletal muscle stem cell state. Stem Cells 30(2):243–252
Rossi DJ et al (2007) Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature 447(7145):725–729
Relaix F, Zammit PS (2012) Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage. Development 139(16):2845–2856
Subramaniam S et al (2014) Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest. PLoS One 8(6):e65097
Evertts AG et al (2013) H4K20 methylation regulates quiescence and chromatin compaction. Mol Biol Cell 24(19):3025–3037
Sebastian S et al (2009) MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation. Proc Natl Acad Sci U S A 106(12):4719–4724
Jin K et al (2009) Mirk regulates the exit of colon cancer cells from quiescence. J Biol Chem 284(34):22916–22925
Buttitta LA, Edgar BA (2007) Mechanisms controlling cell cycle exit upon terminal differentiation. Curr Opin Cell Biol 19(6):697–704
Subramaniam S et al (2013) Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest. PLoS One 8(6):e65097
Ben-Ze’ev A et al (1980) Protein synthesis requires cell-surface contact while nuclear events respond to cell shape in anchorage-dependent fibroblasts. Cell 21(2):365–372
Fukada S et al (2007) Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 25(10):2448–2459
Pallafacchina G et al (2010) An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells. Stem Cell Res 4(2):77–91
Bonavaud S et al (2002) Preparation of isolated human muscle fibers: a technical report. In Vitro Cell Dev Biol Anim 38(2):66–72
Cornelison DD, Wold BJ (1997) Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191(2):270–283
Rosenblatt JD et al (1995) Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 31(10):773–779
Milasincic DJ et al (1996) Anchorage-dependent control of muscle-specific gene expression in C2C12 mouse myoblasts. In Vitro Cell Dev Biol Anim 32(2):90–99
Yoshida N et al (1998) Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’. J Cell Sci 111(Pt 6):769–779
Dhawan J, Helfman DM (2004) Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation. J Cell Sci 117(Pt 17):3735–3748
Kitzmann M et al (1998) The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells. J Cell Biol 142(6):1447–1459
Sambasivan R et al (2009) The small chromatin-binding protein p8 coordinates the association of anti-proliferative and pro-myogenic proteins at the myogenin promoter. J Cell Sci 122(Pt 19):3481–3491
Sambasivan R et al (2008) A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts. J Biosci 33(1):27–44
Sellathurai J et al (2013) A novel in vitro model for studying quiescence and activation of primary isolated human myoblasts. PLoS One 8(5):e64067
Cheedipudi S et al (2015) A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene. Nucleic Acids Res 43(13):6236–6256
Seale P et al (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102(6):777–786
Beauchamp JR et al (2000) Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol 151(6):1221–1234
Gopinath SD et al (2007) The RhoA effector mDiaphanous regulates MyoD expression and cell cycle progression via SRF-dependent and SRF-independent pathways. J Cell Sci 120(Pt 17):3086–3098
Yaffe D, Saxel O (1977) A myogenic cell line with altered serum requirements for differentiation. Differentiation 7(3):159–166
Acknowledgments
We thank past and present members of the Dhawan lab, especially Chetana Sachidanandan, Ramkumar Sambasivan, and Sindhu Subramaniam for developing and refining the protocols presented here, and Lamuk Zaveri and Hardik Gala for the comparative analysis of the cell cycle in ESC and ASC. RA was supported by a DST Start up grant for young scientists and an NCBS-inStem Career Development Fellowship; MR and HG were supported by doctoral fellowships from CSIR and NV by a doctoral fellowship from DBT. The Dhawan lab is supported by core funds from the Dept. of Biotechnology to InStem, core funds from the Council of Scientific and Industrial Research to CCMB, and grants from the Dept. of Biotechnology Indo-Danish Strategic Fund, Indo-Australia Biotechnology Fund, and the Indo-French Center for the Promotion of Advanced Research.
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Arora, R., Rumman, M., Venugopal, N., Gala, H., Dhawan, J. (2017). Mimicking Muscle Stem Cell Quiescence in Culture: Methods for Synchronization in Reversible Arrest. In: Perdiguero, E., Cornelison, D. (eds) Muscle Stem Cells. Methods in Molecular Biology, vol 1556. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-6771-1_15
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DOI: https://doi.org/10.1007/978-1-4939-6771-1_15
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