Adult neural stem cell activation in mice is regulated by the day/night cycle and intracellular calcium dynamics.

Neural stem cells (NSCs) in the adult brain transit from the quiescent state to proliferation to produce new neurons. The mechanisms regulating this transition in freely behaving animals are, however, poorly understood. We customized in vivo imaging protocols to follow NSCs for several days up to months, observing their activation kinetics in freely behaving mice. Strikingly, NSC division is more frequent during daylight and is inhibited by darkness-induced melatonin signaling. The inhibition of melatonin receptors affected intracellular Ca2+ dynamics and promoted NSC activation. We further discovered a Ca2+ signature of quiescent versus activated NSCs and showed that several microenvironmental signals converge on intracellular Ca2+ pathways to regulate NSC quiescence and activation. In vivo NSC-specific optogenetic modulation of Ca2+ fluxes to mimic quiescent-state-like Ca2+ dynamics in freely behaving mice blocked NSC activation and maintained their quiescence, pointing to the regulatory mechanisms mediating NSC activation in freely behaving animals.

Regulation of adult stem cell quiescence and its functions in the maintenance of tissue integrity.

Adult stem cells are important for mammalian tissues, where they act as a cell reserve that supports normal tissue turnover and can mount a regenerative response following acute injuries. Quiescent stem cells are well established in certain tissues, such as skeletal muscle, brain, and bone marrow. The quiescent state is actively controlled and is essential for long-term maintenance of stem cell pools. In this Review, we discuss the importance of maintaining a functional pool of quiescent adult stem cells, including haematopoietic stem cells, skeletal muscle stem cells, neural stem cells, hair follicle stem cells, and mesenchymal stem cells such as fibro-adipogenic progenitors, to ensure tissue maintenance and repair. We discuss the molecular mechanisms that regulate the entry into, maintenance of, and exit from the quiescent state in mice. Recent studies revealed that quiescent stem cells have a discordance between RNA and protein levels, indicating the importance of post-transcriptional mechanisms, such as alternative polyadenylation, alternative splicing, and translation repression, in the control of stem cell quiescence. Understanding how these mechanisms guide stem cell function during homeostasis and regeneration has important implications for regenerative medicine.

Skin in the Game: Stem Cells in Repair, Cancer, and Homeostasis.

The 2020 Canada Gairdner International Award has been awarded to Elaine Fuchs for her discovery of the role of adult skin stem cells in homeostasis, wound repair, inflammation, and cancer. These insights have established a foundation for basic knowledge on how adult stem cells form, maintain, and repair tissues and have provided the groundwork for additional exploration and discovery of pathways in other stem cell systems.

Snake Venom Gland Organoids.

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.

Progressive Pulmonary Fibrosis Is Caused by Elevated Mechanical Tension on Alveolar Stem Cells.

Fibrosis can develop in most organs and causes organ failure. The most common type of lung fibrosis is known as idiopathic pulmonary fibrosis, in which fibrosis starts at the lung periphery and then progresses toward the lung center, eventually causing respiratory failure. Little is known about the mechanisms underlying the pathogenesis and periphery-to-center progression of the disease. Here we discovered that loss of Cdc42 function in alveolar stem cells (AT2 cells) causes periphery-to-center progressive lung fibrosis. We further show that Cdc42-null AT2 cells in both post-pneumonectomy and untreated aged mice cannot regenerate new alveoli, resulting in sustained exposure of AT2 cells to elevated mechanical tension. We demonstrate that elevated mechanical tension activates a TGF-ß signaling loop in AT2 cells, which drives the periphery-to-center progression of lung fibrosis. Our study establishes a direct mechanistic link between impaired alveolar regeneration, mechanical tension, and progressive lung fibrosis.

The Lineage Before Time: Circadian and Nonclassical Clock Influences on Development.

Diverse factors including metabolism, chromatin remodeling, and mitotic kinetics influence development at the cellular level. These factors are well known to interact with the circadian transcriptional-translational feedback loop (TTFL) after its emergence. What is only recently becoming clear, however, is how metabolism, mitosis, and epigenetics may become organized in a coordinated cyclical precursor signaling module in pluripotent cells prior to the onset of TTFL cycling. We propose that both the precursor module and the TTFL module constrain cellular identity when they are active during development, and that the emergence of these modules themselves is a key lineage marker. Here we review the component pathways underlying these ideas; how proliferation, specification, and differentiation decisions in both developmental and adult stem cell populations are or are not regulated by the classical TTFL; and emerging evidence that we propose implies a primordial clock that precedes the classical TTFL and influences early developmental decisions.

Comparing Sensory Organs to Define the Path for Hair Cell Regeneration.

Deafness or hearing deficits are debilitating conditions. They are often caused by loss of sensory hair cells or defects in their function. In contrast to mammals, nonmammalian vertebrates robustly regenerate hair cells after injury. Studying the molecular and cellular basis of nonmammalian vertebrate hair cell regeneration provides valuable insights into developing cures for human deafness. In this review, we discuss the current literature on hair cell regeneration in the context of other models for sensory cell regeneration, such as the retina and the olfactory epithelium. This comparison reveals commonalities with, as well as differences between, the different regenerating systems, which begin to define a cellular and molecular blueprint of regeneration. In addition, we propose how new technical advances can address outstanding questions in the field.

mTORC1 and SIRT1 Cooperate to Foster Expansion of Gut Adult Stem Cells during Calorie Restriction.

Longevity-promoting caloric restriction is thought to trigger downregulation of mammalian target of rapamycin complex 1 (mTORC1) signaling and upregulation of SIRT1 activity with associated health benefits. Here, we show that mTORC1 signaling in intestinal stem cells (ISCs) is instead upregulated during calorie restriction (CR). SIRT1 deacetylates S6K1, thereby enhancing its phosphorylation by mTORC1, which leads to an increase in protein synthesis and an increase in ISC number. Paneth cells in the ISC niche secrete cyclic ADP ribose that triggers SIRT1 activity and mTORC1 signaling in neighboring ISCs. Notably, the mTOR inhibitor rapamycin, previously reported to mimic effects of CR, abolishes this expansion of ISCs. We suggest that Paneth cell signaling overrides any direct nutrient sensing in ISCs to sculpt the observed response to CR. Moreover, drugs that modulate pathways important in CR may exert opposing effects on different cell types.

Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior.

The dentate gyrus of the mammalian hippocampus continuously generates new neurons during adulthood. These adult-born neurons become functionally active and are thought to contribute to learning and memory, especially during their maturation phase, when they have extraordinary plasticity. In this Review, we discuss the molecular machinery involved in the generation of new neurons from a pool of adult neural stem cells and their integration into functional hippocampal circuits. We also summarize the potential functions of these newborn neurons in the adult brain, their contribution to behavior, and their relevance to disease.

Astrocytes decrease adult neurogenesis during virus-induced memory dysfunction via IL-1.

Memory impairment following West Nile virus neuroinvasive disease (WNND) is associated with loss of hippocampal synapses with lack of recovery. Adult neurogenesis and synaptogenesis are fundamental features of hippocampal repair, which suggests that viruses affect these processes. Here, in an established model of WNND-induced cognitive dysfunction, transcriptional profiling revealed alterations in the expression of genes encoding molecules that limit adult neurogenesis, including interleukin 1 (IL-1). Mice that had recovered from WNND exhibited fewer neuroblasts and increased astrogenesis without recovery of hippocampal neurogenesis at 30 d. Analysis of cytokine production in microglia and astrocytes isolated ex vivo revealed that the latter were the predominant source of IL-1. Mice deficient in the IL-1 receptor IL-1R1 and that had recovered from WNND exhibited normal neurogenesis, recovery of presynaptic termini and resistance to spatial learning defects, the last of which likewise occurred after treatment with an IL-1R1 antagonist. Thus, 'preferential' generation of proinflammatory astrocytes impaired the homeostasis of neuronal progenitor cells via expression of IL-1; this might underlie the long-term cognitive consequences of WNND but also provides a therapeutic target.

Bending gradients: how the intestinal stem cell gets its home.

We address the mechanism by which adult intestinal stem cells (ISCs) become localized to the base of each villus during embryonic development. We find that, early in gut development, proliferating progenitors expressing ISC markers are evenly distributed throughout the epithelium, in both the chick and mouse. However, as the villi form, the putative stem cells become restricted to the base of the villi. This shift in the localization is driven by mechanically influenced reciprocal signaling between the epithelium and underlying mesenchyme. Buckling forces physically distort the shape of the morphogenic field, causing local maxima of epithelial signals, in particular Shh, at the tip of each villus. This induces a suite of high-threshold response genes in the underlying mesenchyme to form a signaling center called the "villus cluster." Villus cluster signals, notably Bmp4, feed back on the overlying epithelium to ultimately restrict the stem cells to the base of each villus.

Developmentally regulated alternate 3' end cleavage of nascent transcripts controls dynamic changes in protein expression in an adult stem cell lineage.

Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3' cleavage site, resulting in the production of mRNA isoforms with different length 3' UTRs. Although widespread, the role of APA in the biology of cells, tissues, and organisms has been controversial. We identified >500 Drosophila genes that express mRNA isoforms with a long 3' UTR in proliferating spermatogonia but a short 3' UTR in differentiating spermatocytes due to APA. We show that the stage-specific choice of the 3' end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3' UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed >250 genes where the long 3' UTR versus short 3' UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3' end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.

Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair.

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.

The intestinal crypt, a prototype stem cell compartment.

Due to its intense self-renewal kinetics and its simple repetitive architecture, the intestinal epithelium has become a prime model for studying adult stem cells in health and disease. Transgenic mouse models allow in vivo visualization and genetic lineage tracing of individual intestinal stem cells and their offspring. Fluorescently marked stem cells can be isolated for molecular analyses or can be cultured to build ever-expanding "mini-guts" in vitro. These studies are filling in the outlines of a robust homeostatic self-renewal process that defies some of the classical definitions of stem cell behavior, such as asymmetric division, quiescence, and exhaustion.

Awakening adult neural stem cells: NOX signalling as a positive regulator of the quiescence-to-proliferation transition in the Xenopus retina.

A growing wealth of data suggest that reactive oxygen species (ROS) signalling might be crucial in conferring embryonic or adult stem cells their specific properties. However, how stem cells control ROS production and scavenging, and how ROS in turn contribute to stemness, remain poorly understood. Using the Xenopus retina as a model system, we first investigated the redox status of retinal stem cells (RSCs). We discovered that they exhibit higher ROS levels compared with progenitors and retinal neurons, and express a set of specific redox genes. We next addressed the question of ROS functional involvement in these cells. Using pharmacological or genetic tools, we demonstrate that inhibition of NADPH oxidase-dependent ROS production increases the proportion of quiescent RSCs. Surprisingly, this is accompanied by an apparent acceleration of the mean division speed within the remaining proliferating pool. Our data further unveil that such impact on RSC cell cycling is achieved by modulation of the Wnt/Hedgehog signalling balance. Altogether, we highlight that RSCs exhibit distinctive redox characteristics and exploit NADPH oxidase signalling to limit quiescence and fine-tune their proliferation rate.

Excess dietary sugar impairs Drosophila adult stem cells via elevated reactive oxygen species-induced JNK signaling.

High-sugar diets (HSDs) often lead to obesity and type 2 diabetes, both metabolic syndromes associated with stem cell dysfunction. However, it is unclear whether excess dietary sugar affects stem cells. Here, we report that HSD impairs stem cell function in the intestine and ovaries of female Drosophila prior to the onset of insulin resistance, a hallmark of type 2 diabetes. Although 1 week of HSD leads to obesity, impaired oogenesis and altered lipid metabolism, insulin resistance does not occur. HSD increases glucose uptake by germline stem cells (GSCs) and triggers reactive oxygen species-induced JNK signaling, which reduces GSC proliferation. Removal of excess sugar from the diet reverses these HSD-induced phenomena. A similar phenomenon is found in intestinal stem cells (ISCs), except that HSD disrupts ISC maintenance and differentiation. Interestingly, tumor-like GSCs and ISCs are less responsive to HSD, which may be because of their dependence on glycolytic metabolism and high energy demand, respectively. This study suggests that excess dietary sugar induces oxidative stress and damages stem cells before insulin resistance develops, a mechanism that may also occur in higher organisms.

Planarians recruit piRNAs for mRNA turnover in adult stem cells.

PIWI proteins utilize small RNAs called piRNAs to silence transposable elements, thereby protecting germline integrity. In planarian flatworms, PIWI proteins are essential for regeneration, which requires adult stem cells termed neoblasts. Here, we characterize planarian piRNAs and examine the roles of PIWI proteins in neoblast biology. We find that the planarian PIWI proteins SMEDWI-2 and SMEDWI-3 cooperate to degrade active transposons via the ping-pong cycle. Unexpectedly, we discover that SMEDWI-3 plays an additional role in planarian mRNA surveillance. While SMEDWI-3 degrades numerous neoblast mRNAs in a homotypic ping-pong cycle, it is also guided to another subset of neoblast mRNAs by antisense piRNAs and binds these without degrading them. Mechanistically, the distinct activities of SMEDWI-3 are primarily dictated by the degree of complementarity between target mRNAs and antisense piRNAs. Thus, PIWI proteins enable planarians to repurpose piRNAs for potentially critical roles in neoblast mRNA turnover.

Kinase-inactivated CDK6 preserves the long-term functionality of adult hematopoietic stem cells.

ABSTRACT: Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which myc-associated zinc finger protein (MAZ) and nuclear transcription factor Y subunit alpha (NFY-A) are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.

Strategies for homeostatic stem cell self-renewal in adult tissues.

In adult tissues, an exquisite balance exists between stem cell proliferation and the generation of differentiated offspring. Classically, it has been argued that this balance is obtained at the level of a single stem cell, which divides strictly into a new stem cell and a progenitor. However, recent evidence suggests that balance can also be achieved at the level of the stem cell population. Some stem cells might be lost due to differentiation or damage, whereas others divide symmetrically to fill this gap. Here, we consider the general strategies for stem cell self-renewal and review the evidence for stochastic stem cell fate in adult tissues across a range of tissue types and organisms.

Single-molecule imaging of transcription dynamics in somatic stem cells.

Molecular noise is a natural phenomenon that is inherent to all biological systems1,2. How stochastic processes give rise to the robust outcomes that support tissue homeostasis remains unclear. Here we use single-molecule RNA fluorescent in situ hybridization (smFISH) on mouse stem cells derived from haematopoietic tissue to measure the transcription dynamics of three key genes that encode transcription factors: PU.1 (also known as Spi1), Gata1 and Gata2. We find that infrequent, stochastic bursts of transcription result in the co-expression of these antagonistic transcription factors in the majority of haematopoietic stem and progenitor cells. Moreover, by pairing smFISH with time-lapse microscopy and the analysis of pedigrees, we find that although individual stem-cell clones produce descendants that are in transcriptionally related states-akin to a transcriptional priming phenomenon-the underlying transition dynamics between states are best captured by stochastic and reversible models. As such, a stochastic process can produce cellular behaviours that may be incorrectly inferred to have arisen from deterministic dynamics. We propose a model whereby the intrinsic stochasticity of gene expression facilitates, rather than impedes, the concomitant maintenance of transcriptional plasticity and stem cell robustness.