mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert)

Abstract

A unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function. The extent to which stem cells can regulate quiescence is unknown. Here we show that the stem cell quiescent state is composed of two distinct functional phases, G0 and an 'alert' phase we term G(Alert). Stem cells actively and reversibly transition between these phases in response to injury-induced systemic signals. Using genetic mouse models specific to muscle stem cells (or satellite cells), we show that mTORC1 activity is necessary and sufficient for the transition of satellite cells from G0 into G(Alert) and that signalling through the HGF receptor cMet is also necessary. We also identify G0-to-G(Alert) transitions in several populations of quiescent stem cells. Quiescent stem cells that transition into G(Alert) possess enhanced tissue regenerative function. We propose that the transition of quiescent stem cells into G(Alert) functions as an 'alerting' mechanism, an adaptive response that positions stem cells to respond rapidly under conditions of injury and stress, priming them for cell cycle entry.

Figure 1. Satellite cells distant from the site of injury have different cell cycle kinetics than quiescent and activated satellite cells

(a) Schematic representation of the location of QSCs, CSCs, and ASCs in relation to muscle injury. (b) CSCs have greater propensity to cycle in vivo than do QSCs (n≥3; significance is versus QSCs). (c) Higher percentages of CSCs incorporate EdU after 40 hrs than QSCs. Data from a representative experiment is presented (n≥2; significance is versus QSCs). (d) CSCs require less time to compete the first division (n=3). Details on data presentation and sample size can be found in the Methods Summary and Supplemental Methods Sections.

Figure 2. Satellite cells that are distant from an injury have become ‘alert.’

(a) Representative images of QSCs, CSCs, ASCs immediately after isolation. (b) CSCs are larger than QSCs (n=3). (c) CSCs have a transcriptional profile that is intermediate between QSCs and ASCs (along PC1) as shown by PCA and Pearson’s r values (n=3). (d) Increased mitochondrial activity in CSCs compared to QSCs. (representative FACS plot, n=4). (e) CSCs have increased mtDNA content relative to QSCs (n≥3). (f) CSCs have more intracellular ATP then QSCs (n=4). (g) IF-IHC staining of TA muscle showing representative pS6⁻ and pS6⁺ SCs. (h) Quantification of IF-IHC staining for pS6 in SCs (n≥3; significance is versus noninjured).

Figure 3. Activation of mTORC1 is necessary and sufficient for the alert phenotype

TSC1 KO QSCs display characteristics of alert SCs: (a) increased propensity to cycle in vivo (n≥6); (b) reduced time to first division (n=3); and (c) increased mitochondrial activity (representative FACS plotn=3). Rptr KO suppresses induction of the alert state. Rptr KO CSCs show no differences in: (d) propensity to cycle in vivo (n≥6); (e) time to first division (n=3); and (f) mitochondrial activity (representative FACS plot, n=3). cMet KO CSCs show no injury-induce regulation of: (g) propensity to cycle in vivo (n≥4); (h) time to first division (n≥3); and (i) mitochondrial activity (representative FACS plot, n=3).

Figure 4. Stem cells in the alert state have enhanced functional properties

(a–b) CSCs have enhanced kinetics of myogenic differentiation ex vivo. They rapidly (a) express become MyoG⁺ and (b) fuse (n=3; significance is versus QSCs at same time point). (c) Schematic depiction of ‘alert’ regeneration experimental design. (d–e) A prior ‘alerting’ injury enhances the progress of muscle regeneration: (d) representative histological section and (e) quantification of nascent, centrally nucleated muscle fiber CSA (n≥3). (f–h) FAPs adopt characteristics of the alert state: (f) higher frequency of pS6⁺ FAPs in muscles contralateral to injury (representative IF-IHC staining); (g) quantification of pS6 staining (n=4); and (h) accelerated kinetics of cell cycle entry (n≥2). (i–j) LT-HSCs display characteristics of the alert state in response to muscle injury: (i) increased frequency of pS6 staining (n≥4); and (j) enhanced activation response to IFNγ (n≥3; *** p<0.001). (k) Model depicting quiescence cycle of G₀ and G_Alert phases.