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Concise Review: Quiescence in Adult Stem Cells: Biological Significance and Relevance to Tissue Regeneration
Abstract
Adult stem cells (ASCs) are tissue resident stem cells responsible for tissue homeostasis and regeneration following injury. In uninjured tissues, ASCs exist in a nonproliferating, reversibly cell cycle-arrested state known as quiescence or G0. A key function of the quiescent state is to preserve stemness in ASCs by preventing precocious differentiation, and thus maintaining a pool of undifferentiated ASCs. Recent evidences suggest that quiescence is an actively maintained state and that excessive or defective quiescence may lead to compromised tissue regeneration or tumorigenesis. The aim of this review is to provide an update regarding the biological mechanisms of ASC quiescence and their role in tissue regeneration. Stem Cells 2015;33:2903—2912
Significance Statement
Stem cell quiescence is a novel area of research within the stem cell field. The traditional view of cell quiescence is an inactive cell state. However, this view is changing into a more complex picture. Cellular quiescence is active cellular state where myriad of molecular changes are taking place within the cells and that are relevant to stem cell functions and integrity. In the present review, we provide an updated information regarding the biological significance of cellular quiescence and the molecular mechanisms underlying this phenomenon as relevant to stem cell biology. Particular emphasis has been given to hematopoietic stem cells, muscle satellite cells and mesenchymal (stromal) stem cells.
Introduction
Quiescence is a reversible cell cycle arrested state characterized by the absence of cell proliferation but unlike terminally differentiated cells, quiescent cells maintain the ability to re-enter cell cycle and resume proliferation. As with the cell division cycle, much of our knowledge about quiescence has been derived from extensive studies in cultured cells from yeast to mammalian cells and recently from in vivo studies in animal models.
In baker's yeast, Saccharomyces cerevisiae, quiescence is often described as a “sleeping beauty” state, and is induced by nutrient limitation 1. Thus, quiescence represents a survival mechanism that promotes viability in adverse environmental conditions 2. In multicellular organisms, postnatal tissue homeostasis and regeneration following injury are mediated by a small population of cells known as adult (or tissue specific) stem cells (ASCs), with the capacity to proliferate and subsequently differentiate into lineage specific cell types. ASCs are maintained within uninjured tissues in a quiescent and undifferentiated state 3. Quiescent ASCs are activated upon tissue injury via soluble and mechanical signals emanating from the site of injury, leading to the production of transit amplifying progenitors that in turn differentiate into functional mature cells capable of tissue regeneration 3. A small population of transit amplifying ASCs exits the cell cycle and re-enters quiescence to maintain a reserve of quiescent ASCs that can respond to future demands 4. From an evolutionary perspective, quiescence may help to ensure a steady state number of ASCs available for tissue regeneration, and act as a protective mechanism against genotoxic stresses 5, 6.
In this review, we will focus on quiescence studies conducted in ASCs, notably mesoderm-derived ASCs (hematopoietic stem cells [HSCs], muscle satellite cells [MuSCs], and skeletal [mesenchymal] stem cells) that are clinically relevant for enhancing tissue regeneration. We will discuss current methodologies used to study the quiescent state and the molecular mechanisms regulating quiescence. In addition, we will evaluate evidences suggesting that quiescence is important for optimal functioning of ASCs, and that the inability of ASCs to maintain the quiescent state during ageing and under pathological conditions contributes to compromised tissue homeostasis and regeneration.
Defining Cellular Quiescence
Cellular quiescence or G0 is defined as a transient state, where cells exit the cell cycle in response to either growth-inhibiting signals or absence of growth-promoting signals. Cellular quiescence is characterized by an unreplicated genome or G1 DNA content, an altered cellular metabolism, increased autophagy, and distinct morphological changes such as decreased cell size and increased nucleus to cytoplasm ratio 7, 8. Cell cycle arrest observed during quiescence is reversible, and thus distinct from permanent growth arrest observed in terminally differentiated or senescent cells 9, 10
At the molecular level, ASCs entering the quiescent state in vitro exhibit altered expression of cell cycle regulatory genes, with downregulation of positive regulators of cell proliferation such as cyclins and cyclin dependent kinases (CDKs), and upregulation of negative regulators of cell cycle such as CDK inhibitors 10, 11. While similar changes are observed during ASC differentiation, differences have been reported in the type of negative cell cycle regulators associated with cell differentiation compared with quiescence-related cell cycle arrest 10. Quiescence in ASCs is also associated with reversible suppression of global RNA and protein synthesis 8, 10.
Entry and Exit from Cell Cycle
The cell cycle proceeds through a sequence of coordinated events that are divided into phases (G1, S, G2, and M) based on landmark events of DNA Synthesis (S) and mitosis (M). Phase-specific cyclin-CDK complexes phosphorylate key targets to facilitate cell cycle progression. The G1 cyclins include cyclin D (CCND) that partners with CDK4 or CDK6, and cyclin E (CCNE) that partners with CDK2. G1 cyclin-CDK complexes drive cell cycle progression through the G1 phase and play a role in G1-S transition by phosphorylating and inhibiting retinoblastoma (Rb) protein. In S phase, CDK2 partners with cyclin A (CCNA) and promotes initiation of DNA replication whereas CCNA-CDK1 complex regulates S-phase to G2-phase transition. Cyclin B (CCNB)-CDK1 complex which is active in M-phase phosphorylates key molecules mediating chromosomal condensation, spindle formation, nuclear envelope disintegration and centriole separation (Fig. 1A).
(A): The cell cycle and the quiescence cycle. The cell cycle is divided into specific phases—G1, S, G2, and M phase. Each phase is regulated by a specific cyclin (CCN)–cyclin dependent kinase (CDK) complex (CCNA, cyclin A; CCNB, cyclin B; CCND, cyclin D; CCNE, cyclin E). The window of particular CCN-CDK activity is depicted as colored arcs within cell cycle. The restriction point (R) in late G1 phase depicts the point of commitment; once cell crosses the restriction point; mitogenic signals are no more required by the cell to complete the cell cycle. The “quiescence cycle” is a current working model. Molecular mechanisms regulating entry into quiescence (G0 entry) and exit from quiescence (G0 exit) are poorly described. p27, p130, and CDK2 can be used as markers of quiescent state, and based on recent data, p21 regulates G0 entry. (B): Quiescence cycle in adult stem cells (ASCs). ASCs oscillates between “deep” quiescent (G0) and quiescent “alert” (G(Alert)) states. Quiescent ASCs within uninjured tissues respond to distant tissue injury by transiting to a quiescent “alert” state with increased propensity for cell proliferation and tissue regeneration. The transition from G0 to G(Alert) state is under the control of mammalian target of rapamcin complex-1 (mTORC1) signaling. Downstream effectors of mTORC1 signaling in G(Alert) state is shown to be group of genes regulating mitochondrial metabolism. Abbreviations: CCNA, cyclin A; CCNB, cyclin B; CCND, cyclin D; CCNE, cyclin E; CDK, cyclin-dependent kinase.
The transition from one phase of cell cycle to the next is regulated by cell cycle checkpoint proteins that act as brakes when conditions are not favourable for cell proliferation 12. For example, for cells to transit from G1 to S-phase and initiate DNA replication, cells must assess the availability of nutrients and enzymes needed for DNA replication, availability of growth factors as well as the absence of DNA damage. The G1 check point proteins (ataxia telangiectasia mutated [ATM] and ataxia telangiectasia and rad3 related [ATR]) ensure that these criteria are fulfilled. Chemical agents or irradiation that cause DNA damage, result in G1 cell cycle arrest. Cell proliferation resumes after DNA damage is repaired. Similarly, to pass the G2 checkpoint, which is temporally located before the onset of mitosis, cells must ensure that DNA replication has been completed without any errors 13. Loss of checkpoint control proteins can lead to genomic instability, as observed frequently in a variety of cancers 14.
While the molecular mechanisms regulating cell cycle progression have been extensively studied, little is known about the mechanisms of entry into the quiescent state. Earlier studies suggested that a “restriction point” (R) temporally located in late G1 phase, governs the decision to enter S-phase 15. Extended periods in G1 arrest lead to quiescence or G0. The elucidation of Rb-mediated negative control of S-phase entry provides a molecular understanding of the restriction point, and supported the concept that integration of extrinsic cues with intrinsic parameters leads to quiescence. While this framework has adequately explained the behavior of synchronized cultured cell models, recent studies using asynchronous populations of cells indicate that the quiescence decision point may differ from the Rb-regulated restriction point.
A landmark study using a sensor of CDK2 activity coupled with quantitative live cell imaging and automated tracking of successive cell divisions in culture has generated a new view of the mechanisms regulating entry into G0 16. This study identifies a quiescence decision point temporally located in late G2/M phase. In a mitogen-activated cell population, the cells that fail to achieve a threshold activity of CDK2, are committed to enter G0 arrest. This subset of cells is marked by higher levels of p21. Loss of p21 expression causes continuous cell proliferation even in absence of mitogenic signals 16. A detailed understanding of the regulation of the p21-CDK2 axis at G2/M should reveal new players in the quiescence decision point and the extent of its conservation in vivo.
Until recently, quiescence in ASCs in vivo has been considered to be a dormant cellular state with little metabolic activity. However, a recent study by Rodgers et al. 17 suggests that quiescent mammalian ASCs cycle between two molecularly distinct states: a sleeping or deeply quiescent (G0) state and a primed but still nondividing state (G(Alert)), induced in response to tissue injury 17 (Fig. 1B). Damage to skeletal muscle has long been known to activate resident stem cells to leave G0 and enter G1 18. The study by Rodgers et al. showed that even quiescent MuSCs in noninjured tissues respond to distant tissue injury by transiting to an alert or primed state (G(Alert)). Interestingly, not only MuSCs but also fibro-adipogenic progenitors (FAPs) and HSCs enter a G(Alert) state in response to muscle injury. Once the muscle regeneration process is complete, the primed cells slowly revert to the deeply quiescent (G0) state. In keeping with the original observations in yeast 1, this finding provides support for the notion of a “quiescence cycle” (by analogy to the cell division cycle). Furthermore, Rodgers et al. have demonstrated that the G(Alert) state is maintained through the TOR pathway, first identified in yeast as a central regulator of cell growth. The mammalian target of rapamycin complex 1 (mTORC1) is known to be sensitive to environmental and nutritional stimuli. In damaged muscle, mTORC1 is activated via signaling from hepatocyte growth factor/scatter factor (hepatocyte growth factor (HGF)/SF) that is stored in the extracellular matrix (ECM) and released in an active form by the action of serum protease during tissue injury. Once activated, HGF induces a signaling cascade through the PI3K-Akt pathway, resulting in activation of mTORC1. Interestingly, the primed (G(Alert)) MuSCs exhibit enhanced muscle regenerative capacity 17, strongly supporting the notion that the quiescence cycle contributes to stem cell function.
Identification of Quiescent Cells
Due to the paucity of information on quiescence-specific events, quiescent cells have traditionally been identified by the absence of markers associated with proliferation. Several techniques are available to identify quiescent cells in vivo and in vitro. Proliferating cells are identifiable by labeling of newly synthesized DNA using nucleotide analogues such as tritiated thymidine (3H-TdR), 5-bromo 2′-deoxyuridine (BrdU), and 5-ethynyl-2′-deoxyuridine (EdU). Once incorporated in proliferating cells, the labeled DNA can be detected by autoradiography (3H-TdR) or immunocytochemistry/immunohistochemistry (BrdU and EdU). Endogenous markers of proliferating cells such as proliferating cell nuclear antigen (PCNA, a DNA polymerase accessory protein which is expressed in S-phase), Ki67 (a protein associated with ribosomal RNA transcription and expressed in all phases except G0), minichromosome maintenance-2 (MCM-2, a protein that functions in replication origins and expressed in S phase), and phosphohistone H3 (an M-phase-specific histone modification) are extensively used to distinguish between proliferating and quiescent cells 19, 20. Cell cycle status can also be identified on the basis of DNA and RNA content, using DNA binding dyes such as propidium iodide (PI), DRAQ-5, and DAPI and RNA binding dyes such as pyronin Y and SYTO dyes. Although cells in either G1 or G0 phase possess an unreplicated genome (2N complement of DNA), quiescent (G0) cells are transcriptionally less active and possess lower total RNA content 21, which together readily distinguishes G0 cells from G1 cells in flow cytometry.
In experimental organisms, the identification of quiescent cells in vivo is based on their “label retaining” characteristics i.e., quiescent cells retain the incorporated DNA label due to infrequent cell division 22. BrdU is most commonly used label, taken up by cells during a period of BrdU exposure sufficient for the cell to cycle at least once. The label is retained within quiescent cells, but is diluted below detectable limits in proliferating cells due frequent cell divisions. Label-retaining cells (LRCs) have been detected in most adult mammalian tissues and have been shown to participate in homeostatic and regenerative repair 23. More recently, lineage-tracing techniques that enable fluorescent tagging of a particular cell type have been used to identify quiescent cells in transgenic mice 24. Histones are commonly tagged proteins as their incorporation is replication-dependent and the fusion proteins exhibit nuclear localization 25, 26.
Quiescent stem cells can also been identified by eliminating the proliferating cell population. Studies in mice showed that intravenous administration of 5 fluorouracil (5-FU) eliminates cycling HSCs while sparing a small population of quiescent HSCs that can repopulate the bone marrow in serial transplantation studies 27. 5-FU is a pyrimidine analogue that irreversibly inhibits thymidylate synthase, the enzyme required for synthesis of the nucleotide thymidine monophosphate (dTMP), essential for DNA synthesis. Once taken up by cells, 5-FU induces apoptosis selectively in proliferating cells, while sparing quiescent cells. Similarly, quiescent MuSCs are less sensitive to a lethal dose of radiation which eliminates the proliferating cells in skeletal muscle 28.
Two newly developed fluorescent protein-based sensors have been used to identify quiescent cells directly. A CDK2 based sensor consisting of a fluorescent protein tagged to the C-terminal fragment of human DNA helicase B (DHB) can distinguish quiescent cells from actively proliferating cells 16. The subcellular localization of the sensor is cell cycle dependent. In quiescent cells, the sensor is primarily localized in the nucleus due to low CDK2 activity, whereas in proliferating cells, the sensor is progressively translocated out of the nucleus to the cytoplasm due to phosphorylation by CCNE-CDK2 and CCNA-CDK2 complexes. Thus, quiescent cells can be distinguished from proliferating cells by determining the relative distribution of the fluorescent sensor between nucleus and cytoplasm. Similarly, Okai et al. developed a fusion protein consisting of a defective mutant of p27 fused to a fluorescent tag 29. The fluorescent probe intensity is high in quiescent cells due to p27 accumulation and is rapidly lost due to degradation as quiescent cells enter the cell cycle. The tagged p27 probe has also been demonstrated to detect and isolate quiescent cells from various adult tissues in mice when expressed as transgene 29.
Ex Vivo Induction of Quiescence
Cellular quiescence can be modeled in ex vivo cell cultures using a variety of approaches. Mammalian cells can be induced to enter a quiescent state by manipulating a number of culture conditions including anchorage deprivation, growth to confluence and contact inhibition, mitogen deprivation and nutrient/amino acid limitation 30-32 (Fig. 2A). A key consideration concerns the type of growth arrest attained by the abrogation of mitogenic signaling in different cell types. While contact inhibition, mitogen deprivation and anchorage deprivation all induce reversible quiescence in fibroblasts, ASCs may respond differently to these culture conditions and enter different states of permanent arrest. For example, mitogen deprivation leads to differentiation in skeletal myoblasts 33 (Fig. 2B) and nonadherent culture leads to anoikis (cell death) in epithelial cells 34, which are both irreversible. Thus, alternative approaches are required to establish reversible quiescence in ASCs. In mesoderm-derived cells such as myoblasts and mesenchymal stem cells (MSCs), where attachment to a substrate is essential for cell growth, cells are effectively triggered into quiescence by suspension culture or culture on soft substrates in the presence of mitogens 35, 36 or by inhibiting adhesion-dependent signals using small molecule inhibitors of cellular contractility 37. Importantly, restoration of surface attachment/contractility leads to a synchronous return to the cell cycle.
(A): In vitro methods to induce quiescence (G0 state) in cultured mammalian cells. An illustration of current methods for inducing cellular quiescence in ex vivo cell cultures. The effector proteins and signaling pathways are mentioned. (B): Different types of cell cycle arrests are identified in mammalian cells. Different types of cell cycle arrests known in mammalian cells: quiescence, differentiation, senescence, and apoptosis. The effector molecules and signaling pathways mentioned refer to studies conducted mainly in myoblast cell cultures. Abbreviations: CDK, cyclin-dependent kinase; E2F, E2 transcription factor; ERK, extracellular signal regulated kinases; FAK, focal adhesion kinase; IGF, insulin-like growth factor; IP3, inositol triphosphate; MAPK, mitogen-activated protein kinases; MEK, mitogen activated protein kinase kinase; MSC, mesenchymal stem cell; PTPR, protein tyrosine phosphatase, receptor type.
Analysis of Quiescence in ASCs
Cellular quiescence has been studied in several tissue-specific ASCs and a growing body of evidence suggests that quiescence is an intrinsic property of ASCs in vivo, permitting these cells to persist in an undifferentiated state for prolonged periods of time. Defects in maintaining quiescence can lead to stem cell exhaustion and degenerative diseases 25, 38. Studies analyzing the quiescent state in different types of ASCs are presented below.
Quiescence in HSCs
HSCs reside within the bone marrow and are responsible for renewal of mature blood cells throughout life. The quiescent nature of HSCs was first reported during studies of resistance of HSCs towards 5-FU-induced apoptosis 27. In adult mice, direct evidence for the presence of quiescent HSCs has been provided in studies showing that continuous in vivo administration of BrdU for more than 12 weeks is required for labelling HSCs 39 indicating their slow cycling nature. On the other hand, it is possible to isolate quiescent adult HSCs based on combinations of cell surface markers that select primitive undifferentiated HSCs. Examples include c-Kit+Sca-1+Lin−Tie2+ 5, c-Kit+Sca-1+Lin−CD150+CD48−CD34− 40, and c-Kit+Sca-1+Lin−CD48−CD150+ 26. Although these marker-defined populations may differ subtly in their stem cell function, they are all quiescent.
Quiescence in MuSCs
MuSCs are stem cells located between the plasma membrane of the muscle fibers and the surrounding basal lamina. MuSCs are undifferentiated progenitors that express the lineage determinant transcription factor, Pax7 41 but do not express transcription factors MyoD and Myogenin 42 which drive myogenic determination and differentiation respectively. MuSCs are key mediators of muscle repair and regeneration following muscle injury 43. MuSCs are quiescent in uninjured adult muscles, as demonstrated by studies where continuous administration of 3H-thymidine in mice for 9 days failed to label the majority of MuSCs 3. Also in mice, MuSCs identified on the basis of cell surface markers such as SMC2.6+CD45− 11, M-cadherin+ 44, and c-met+ 45 exhibit a quiescence phenotype as evidenced by 2N DNA content, low RNA content, no BrdU incorporation, and absence of MyoD/Myogenin expression. Quiescent MuSCs are resistant to sublethal doses of irradiation that eliminate the majority of proliferating cells 46. Molecular profiling of quiescent MuSCs revealed upregulation of cell cycle inhibitory genes such as Gas3, CDKN1C, CDKN1B, and Spry1 as well as negative regulators of myogenic differentiation such as BMP2, BMP4, BMP6, HEY1, and Notch3 11. Interestingly, novel surface markers of freshly isolated satellite cells including Caveolin-1, Calcitonin receptor, and integrin alpha 7 47 can be used to localize and purify undifferentiated MuSCs.
Quiescence in Skeletal Stem Cells
Skeletal stem cells or MSCs were originally isolated from bone marrow 48, but MSC-like cells have been isolated from the stromal compartment of several tissues including fat, lung, muscle, kidney, and skin 49. Although some studies suggest that MSCs are quiescent in their niche, limited information is available regarding their quiescence phenotype, primarily because specific markers of quiescent MSCs have not been identified 50, 51. Early studies of bone regeneration identified the presence of nonhematopoietic, label-retaining cells within bone marrow, suggesting the presence of quiescent MSCs capable of responding to bone injury and contributing to bone regeneration. For example, label-retaining MSCs were detected within bone marrow after injecting 3H-TdR label in rat, from day 9 of pregnancy till birth 52. In addition, label-retaining MSCs within murine bone marrow could be enriched by 5-FU treatment 53. MSCs purified from humans as well as from rodent bone marrow by using a combination of cell surface markers: PDGFRα+Sca-1+CD45−TER119− 50 and STRO1+VCAM1+ 51, contain a noncycling quiescent subpopulation as assessed by DNA and RNA content analysis. A recent lineage-tracing study in mice reports that MSC-like cells in the intestine are slow cycling, but quiescent bone marrow MSCs have not been described 54.
Molecular Mechanisms of ASC Quiescence
Given that quiescent ASCs display a distinct transcriptome and surface marker profile, it is evident that the quiescent state is not merely a consequence of reduced metabolism, but is actively achieved and maintained. Both cell intrinsic and cell extrinsic factors have been demonstrated to play a role in regulating ASCs quiescence 6, 41, 55. Mechanisms regulating entry, maintenance, and exit from the quiescent state are best described in yeast 56. In mammalian cells, the molecular mechanisms of quiescence have been studied extensively in ex vivo cell culture models of quiescence. Recent studies in genetically modified animals have demonstrated the relevance of these mechanisms for tissue regeneration under physiological conditions.
Cell Intrinsic Mechanisms Regulating Quiescence
Tumor suppressor genes (TSGs) inhibit cell division and loss of function of TSGs leads to uncontrolled cell proliferation. TSGs function either directly or indirectly by suppressing genes required for cell cycle progression 57. In ASCs, quiescence is associated with induction of TSGs and transgenic animal models deficient for TSGs exhibit impaired self-renewal of ASCs compartment and compromised tissue regeneration 38, 58. Evidence for TSG association with quiescent ASCs is discussed below.
Rb Family
The Rb or pocket protein family consists of three members: Rb (Rb protein), p107 (Rb-like protein-1) and p130 (Rb-like protein-2). The founding member of this family, Rb acts as the gate keeper of the G1/S transition and is a key target for the CCND-CDK4/6 pathway 59. Rb restrains cell cycle progression via control of S-phase transcriptional activators—particularly the E2F transcription factor family 59.
In HSCs, all three Rb family proteins are expressed and loss of an individual Rb family protein does not perturb quiescence in these cells 60. However, in mice, deficient for all three Rb family proteins, the HSC compartment is severely defective and mutant HSCs exhibit enhanced proliferation and impaired marrow repopulation ability upon serial transplantation 61, 62. This phenotype is consistent with the loss of ability of pocket protein-deficient HSCs to maintain quiescence. By contrast, mice deficient for Rb alone exhibit an increased number of myoblasts within uninjured muscles as compared to wild type animals. When cultured in vitro, myoblasts from Rb-deficient mice exhibit accelerated cell cycle entry, loss of myogenic differentiation as well as increased cell death and autophagy 63, 64. Thus, Rb is essential for maintaining MuSC quiescence in vivo as well as cell survival during myogenic differentiation. Interestingly, p130 is highly expressed in quiescent MuSCs and overexpression of p130 in proliferating myoblasts leads to cell cycle arrest. Also, p130 inhibits myogenic differentiation by suppressing myogenic genes 65. Thus, p130 maintains MuSCs quiescence by a dual mechanism: blocking cell cycle progression and suppressing the myogenic differentiation program.
Cyclin Dependent Kinase Inhibitors
CKIs regulate quiescence through inhibition of CDKs. p21Cip1, p27Kip1, and p57Kip2 inhibit CDK2, CDK4, and CDK6 blocking cell cycle progression 66. Loss of CKIs abolishes the ability of ASCs to maintain cellular quiescence. Targeted ablation of p21 in mice is associated with increased HSC proliferation, greater susceptibility to cell cycle-specific myelotoxic injury and poor bone marrow reconstitution ability during serial transplantation, suggesting that increased proliferation is associated with compromised stemness 38. Similarly, p57-deficient mice exhibit reduced numbers of quiescent HSCs within bone marrow as well as reduced HSC self-renewal 58. Furthermore, HSCs deficient for both, p57 and p27 exhibit higher proliferative capacity and lower bone marrow reconstitution ability 67. Similarly, MuSCs deficient for both p21 and p57, display increased proliferation in vivo and fail to undergo myogenic differentiation 68. While CKIs would be expected to participate in slowing the cell cycle, it appears that distinct constellations of cell cycle inhibitors distinguish reversibly quiescent myoblasts from permanently arrested myotubes 10. Thus, the precise networks governed by the individual CKIs in different quiescent cell types remain to be uncovered.
Cell Extrinsic Factors Regulating Quiescence
The local microenvironment of ASCs known as the stem cell “niche” plays an important role in regulating quiescence. The dynamic interaction between ASCs and their niche which includes different cell types, blood vessels, ECM proteins and nerve fibers, is critical for optimal stem cell function 5, 69, 70. Communication between cells within the niche is mediated through secreted paracrine factors and their cognate receptors, cell–cell and cell–ECM interactions that stimulate key signaling pathways in ASCs, particularly Wnt, Notch, and FGF pathways (Table 1).
| Signaling Pathways associated with ASC quiescence | Niche components that affect signaling in ASCs | References |
|---|---|---|
| ERK signaling | Osteoblastic cells in bone marrow niche and perivascular and interstitial cells within muscles regulate ERK signaling in HSCs and MuSCs respectively through secreted ligands. | 5, 71 |
| Wnt signaling | Osteoblastic cells in bone marrow niche and endothelial cells within muscles regulate Wnt signaling in HSCs and MuSCs respectively. | 69, 72-74 |
| Notch signaling | Jagged I expressing osteoblastic cells thai are in close contact with HSCs regulate Notch signaling in HSCs. | 75 |
| FGF signaling | Over expression of FGF2 in aged muscle induces proliferation in mouse MuSCs and prevents establishment of quiescent MuSCs. | 25 |
| HGF signaling | HGF present in the extracellular matrix of various tissues is shown to regulate quiescence in ASCs. | 17, 76 |
Wnt signaling is known to play a context-dependent role in ASCs. In HSCs, inhibition of Wnt signaling by ectopic expression of the secreted Wnt inhibitor Dkk1 results in loss of HSC quiescence and self-renewal ability, whereas overexpression of a constitutively active form of β-catenin (the transcriptional mediator of canonical Wnt signaling) increases self-renewal of HSCs while blocking their differentiation 69, 77. Wnt signaling appears to regulate HSC fate through regulation of p21 77. In the HSC niche, the Wnt pathway is regulated by osteoblastic cells that are in close contact with HSCs 69. In MuSCs, levels of Wnt signaling mark different cellular states: moderate levels of Wnt signaling is required for the quiescent state 10, as either enhancing or inhibiting Wnt signaling alters the quiescence program.
The Notch pathway plays a critical role in cell fate decision and stem cell homeostasis 78. Notch signaling is commonly associated with inhibition of differentiation and maintenance of a self-renewing state. In adult murine HSCs, ectopic expression of activated Notch (notch intracellular domain [NICD]) enhances self-renewal and proliferation in vitro 79. Conversely, loss of Notch signaling leads to increased HSCs differentiation in vitro and HSC depletion in vivo 80. Thus, Notch signaling induces expansion of HSCs but preserves self-renewal ability. Unlike the soluble Wnt ligands, Notch ligands are often cell-surface molecules and mediate signaling via direct cell–cell interaction. Interestingly, like Wnt signaling, Notch signaling in HSCs is also regulated by osteoblastic cells 75, 81. In MuSCs, Notch signaling maintains cellular quiescence 55, 82. Over-expression of NICD in mouse MuSCs, suppresses proliferation and preserves self-renewal ability while inhibiting myogenic differentiation in vitro 83, whereas loss of RBP-J, a downstream transcriptional effector of Notch, leads to loss of quiescence and induction of precocious differentiation 6, 84.
Extracellular signal regulated kinase (ERK) signaling is involved in regulating quiescence via multiple receptors. Receptor tyrosine kinases (RTKs) are cell surface receptors which upon extracellular ligand binding induce kinase activity in their cytoplasmic domains. Secreted growth factors such as HGF, insulin-like growth factor, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) are potent activators of RTKs 85. Quiescent MuSCs express tyrosine kinase receptors for such ligands. However, several intracellular inhibitors that bind to and inactivate specific growth factors signaling molecules are highly expressed in quiescent MuSCs. Quiescent MuSCs express Sprouty1 (Spry1), an inhibitor of RTKs, which binds and inhibits the kinase activity of these receptors even in the presence of their cognate ligands. Spry1 expression is lost in activated MuSCs, and re-expressed in their progeny re-entering quiescence. Targeted deletion of Spry1 in mice leads to enhanced ERK signaling and failure of MuSCs to re-enter quiescence after repair of injury 4. Similarly, sustained FGF2 signaling is shown to disrupt MuSCs quiescence in vivo 25.
The downstream effectors of ERK pathways are mitogen-activated protein kinases (MAPK) such as p38α/β MAPK. In MuSCs, p38α/β MAPK induces myogenic determination factor MyoD and subsequent proliferation. In transgenic mice, constitutively active p38α/β MAPK leads to precocious myoblast differentiation 86, 87. Some evidence exists for asymmetric division as a contributor to quiescence in MuSCs, wherein one daughter cell commits to proliferation and eventual differentiation while the other daughter cell retains the stem cell characteristics 88. During activation of MuSCs, p38α/β MAPK is shown to localize asymmetrically in one of the daughter cell that becomes the transit amplifying progenitor whereas the other daughter cell contributes toward replenishment of quiescent cells 89. The upstream regulators of this asymmetric distribution of p38α/β MAPK are partitioning defective 3 and protein kinase C 89.
Role Of Quiescence in ASC Function
Loss of quiescence is associated with defective ASC function. Quiescence in HSCs preserves the stem cell compartment and a balance between proliferation, quiescence, and differentiation ensures persistence of regenerative cells throughout life 5, 69. Reconstitution of bone marrow after lethal irradiation or other myelotoxic injury is dependent on stem cell quiescence, as this process is impaired when quiescence in HSCs is abolished through ablation of Rb proteins 61, 70. As mentioned above, disruption of quiescence regulators such as p21 or p57 also decreases bone marrow reconstitution ability of HSC 38, 90. Targeted deletion of the chromatin remodeler Satb1 leads to loss of quiescence in HSCs in vivo, causing over-proliferation and precocious differentiation resulting in gradual depletion of functional stem cells 91. Thus in HSCs, quiescence helps maintaining the stem cell phenotype and preserves self-renewal ability.
Disruption of quiescence in MuSCs leads to impaired muscle regeneration and repair. In mice MuSCs, loss of quiescence regulators such as Spry1 (downstream of RTKs) and RBPJ (downstream of Notch) leads to depletion of MuSCs due to over-proliferation and precocious differentiation. Age-dependent changes in the muscle microenvironment affect MuSCs function by disrupting quiescence. For example, increased FGF2 expression in aged muscles leads to the persistent activation of MuSCs and prevents the restoration of the quiescent compartment leading to impaired muscle regeneration 25. Sousa-Victor et al. found that continuous expression of p16 in aged MuSCs causes a shift from quiescence-associated to senescence-associated cell cycle arrest. MuSCs from aged mice thus undergo senescence upon injury induced activation signals 92. The translational machinery also regulates quiescence in MuSCs. Compromising the RNAi pathway by targeted inactivation of Dicer in mice disrupts quiescence in myoblasts, and leads to loss of muscle regeneration 93. As a corollary, transplantation of quiescent MuSCs has an enhanced muscle regeneration capacity as compared to culture expanded MuSCs 94
Finally, cellular quiescence appears to protect ASCs from oxidative stress. Reactive oxygen species (ROS) encompass a variety of partially reduced metabolites of oxygen (e.g., superoxide anions, hydrogen peroxide, and hydroxyl radicals) that are generated intracellularly. At high and/or sustained levels, ROS can cause severe damage to DNA, protein, and lipids. Recent studies suggest that quiescent ASCs are protected from ROS, through upregulation of genes that mitigate the toxic effect of free radicals. For example, genes implicated in response to oxidative stress such as glutathione peroxidase 3 (GPX3), sulfiredoxin (SRXN) and thioredoxin reductase 1 (TXNRD1) are highly expressed in quiescent ASCs 95. Also, cell surface transporters Abcb1a, Abca5 and Abcc9 that mediate efflux of toxic substances from the cell, are upregulated in quiescent stem cells 95. Quiescent HSCs also possess mechanisms enhancing cell survival under adverse conditions such as hypoxia. Under hypoxic conditions, HIF1α is upregulated in quiescent HSCs, translocates to the nucleus where it binds with HIF1β and transcriptionally regulates prosurvival genes 96.
Concluding Remarks and Future Prospects
Cellular quiescence is emerging as an actively maintained state playing an important role in regulating ASC functions. The quiescent state protects ASCs from proliferation-associated genotoxic stresses as well as from damaging environmental conditions. Thus, quiescent ASCs exhibit better survival ability under adverse conditions of tissue injury. In addition, there is accumulating evidence that loss of quiescence in ASCs leads to compromised tissue regeneration. It is plausible that stem cell therapies using quiescent ASCs might prove beneficial over current approaches that focus on transplanting proliferating cells following ex vivo expansion. With the establishment of new quiescence culture models as well as the introduction of valuable new reagents based on quiescence sensors in vivo, a more detailed understanding of the biological role of cellular quiescence will be uncovered. Integrating global studies of quiescence-specific transcriptomes, epigenomes, proteomes, and secretomes will establish a detailed description of the biology of quiescent state, with the hope of specifically targeting quiescent stem cells in vivo to enhance tissue regeneration.
Acknowledgments
This work was supported by an Indo-Denmark Collaborative Grant from the Danish Strategic Research Council and the Government of India, Department of Biotechnology (to M.K. and J.D.), core funds from the Institute for Stem Cell Biology and Regenerative Medicine (to J.D. and M.R.), a (doctoral) Senior Research Fellowship from the Government of India, Council for Scientific and Industrial Research (to M.R.), and grants from the NovoNordisk Foundation and the Lundbeck Foundation (to M.K.).
Author Contributions
M.R.: manuscript writing; J.D. and M.K.: conception and design, manuscript writing, and final approval of manuscript.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.
