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.
Quiescence In vitro G0 phase Stem cell Satellite cell
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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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