T Cell Fate at the Single-Cell Level.

T cell responses display two key characteristics. First, a small population of epitope-specific naive T cells expands by several orders of magnitude. Second, the T cells within this proliferating population take on diverse functional and phenotypic properties that determine their ability to exert effector functions and contribute to T cell memory. Recent technological advances in lineage tracing allow us for the first time to study these processes in vivo at single-cell resolution. Here, we summarize resulting data demonstrating that although epitope-specific T cell responses are reproducibly similar at the population level, expansion potential and diversification patterns of the offspring derived from individual T cells are highly variable during both primary and recall immune responses. In spite of this stochastic response variation, individual memory T cells can serve as adult stem cells that provide robust regeneration of an epitope-specific tissue through population averaging. We discuss the relevance of these findings for T cell memory formation and clinical immunotherapy.

Tracing the origin of everything.

Highly potent adult stem cells fuel lifelong tissue homeostasis and regeneration in many aquatic invertebrates, yet their developmental backstories remain obscure. In this issue of Cell, Kimura and colleagues reveal the cellular origin of adult pluripotent stem cells and propose a molecular trajectory for their specification during acoel embryogenesis.

Embryonic origins of adult pluripotent stem cells.

Although adult pluripotent stem cells (aPSCs) are found in many animal lineages, mechanisms for their formation during embryogenesis are unknown. Here, we leveraged Hofstenia miamia, a regenerative worm that possesses collectively pluripotent aPSCs called neoblasts and produces manipulable embryos. Lineage tracing and functional experiments revealed that one pair of blastomeres gives rise to cells that resemble neoblasts in distribution, behavior, and gene expression. In Hofstenia, aPSCs include transcriptionally distinct subpopulations that express markers associated with differentiated tissues; our data suggest that despite their heterogeneity, aPSCs are derived from one lineage, not from multiple tissue-specific lineages during development. Next, we combined single-cell transcriptome profiling across development with neoblast cell-lineage tracing and identified a molecular trajectory for neoblast formation that includes transcription factors Hes, FoxO, and Tbx. This identification of a cellular mechanism and molecular trajectory for aPSC formation opens the door for in vivo studies of aPSC regulation and evolution.

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.

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.

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.

Beyond Casual Resemblance: Rigorous Frameworks for Comparing Regeneration Across Species.

The majority of animal phyla have species that can regenerate. Comparing regeneration across animals can reconstruct the molecular and cellular evolutionary history of this process. Recent studies have revealed some similarity in regeneration mechanisms, but rigorous comparative methods are needed to assess whether these resemblances are ancestral pathways (homology) or are the result of convergent evolution (homoplasy). This review aims to provide a framework for comparing regeneration across animals, focusing on gene regulatory networks (GRNs), which are substrates for assessing process homology. The homology of the wound-induced activation of Wnt signaling and of adult stem cells provides examples of ongoing studies of regeneration that enable comparisons in a GRN framework. Expanding the study of regeneration GRNs in currently studied species and broadening taxonomic sampling for these approaches will identify processes that are unifying principles of regeneration biology across animals. These insights are important both for evolutionary studies of regeneration and for human regenerative medicine.

Metabolic regulation of somatic stem cells in vivo.

Metabolism has been studied mainly in cultured cells or at the level of whole tissues or whole organisms in vivo. Consequently, our understanding of metabolic heterogeneity among cells within tissues is limited, particularly when it comes to rare cells with biologically distinct properties, such as stem cells. Stem cell function, tissue regeneration and cancer suppression are all metabolically regulated, although it is not yet clear whether there are metabolic mechanisms unique to stem cells that regulate their activity and function. Recent work has, however, provided evidence that stem cells do have a metabolic signature that is distinct from that of restricted progenitors and that metabolic changes influence tissue homeostasis and regeneration. Stem cell maintenance throughout life in many tissues depends upon minimizing anabolic pathway activation and cell division. Consequently, stem cell activation by tissue injury is associated with changes in mitochondrial function, lysosome activity and lipid metabolism, potentially at the cost of eroding self-renewal potential. Stem cell metabolism is also regulated by the environment: stem cells metabolically interact with other cells in their niches and are able to sense and adapt to dietary changes. The accelerating understanding of stem cell metabolism is revealing new aspects of tissue homeostasis with the potential to promote tissue regeneration and cancer suppression.

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.

Defining Adult Stem Cells by Function, not by Phenotype.

Central to the classical hematopoietic stem cell (HSC) paradigm is the concept that the maintenance of blood cell numbers is exclusively executed by a discrete physical entity: the transplantable HSC. The HSC paradigm has served as a stereotypic template in stem cell biology, yet the search for rare, hardwired professional stem cells has remained futile in most other tissues. In a more open approach, the focus on the search for stem cells as a physical entity may need to be replaced by the search for stem cell function, operationally defined as the ability of an organ to replace lost cells. The nature of such a cell may be different under steady state conditions and during tissue repair. We discuss emerging examples including the renewal strategies of the skin, gut epithelium, liver, lung, and mammary gland in comparison with those of the hematopoietic system. While certain key housekeeping and developmental signaling pathways are shared between different stem cell systems, there may be no general, deeper principles underlying the renewal mechanisms of the various individual tissues.

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.

Aged Stem Cells Reprogram Their Daily Rhythmic Functions to Adapt to Stress.

Normal homeostatic functions of adult stem cells have rhythmic daily oscillations that are believed to become arrhythmic during aging. Unexpectedly, we find that aged mice remain behaviorally circadian and that their epidermal and muscle stem cells retain a robustly rhythmic core circadian machinery. However, the oscillating transcriptome is extensively reprogrammed in aged stem cells, switching from genes involved in homeostasis to those involved in tissue-specific stresses, such as DNA damage or inefficient autophagy. Importantly, deletion of circadian clock components did not reproduce the hallmarks of this reprogramming, underscoring that rewiring, rather than arrhythmia, is associated with physiological aging. While age-associated rewiring of the oscillatory diurnal transcriptome is not recapitulated by a high-fat diet in young adult mice, it is significantly prevented by long-term caloric restriction in aged mice. Thus, stem cells rewire their diurnal timed functions to adapt to metabolic cues and to tissue-specific age-related traits.

Epithelial-Mesenchymal Micro-niches Govern Stem Cell Lineage Choices.

Adult tissue stem cells (SCs) reside in niches, which, through intercellular contacts and signaling, influence SC behavior. Once activated, SCs typically give rise to short-lived transit-amplifying cells (TACs), which then progress to differentiate into their lineages. Here, using single-cell RNA-seq, we unearth unexpected heterogeneity among SCs and TACs of hair follicles. We trace the roots of this heterogeneity to micro-niches along epithelial-mesenchymal interfaces, where progenitors display molecular signatures reflective of spatially distinct local signals and intercellular interactions. Using lineage tracing, temporal single-cell analyses, and chromatin landscaping, we show that SC plasticity becomes restricted in a sequentially and spatially choreographed program, culminating in seven spatially arranged unilineage progenitors within TACs of mature follicles. By compartmentalizing SCs into micro-niches, tissues gain precise control over morphogenesis and regeneration: some progenitors specify lineages immediately, whereas others retain potency, preserving self-renewing features established early while progressively restricting lineages as they experience dynamic changes in microenvironment.

Psat1-generated α-ketoglutarate and glutamine promote muscle stem cell activation and regeneration.

By satisfying bioenergetic demands, generating biomass, and providing metabolites serving as cofactors for chromatin modifiers, metabolism regulates adult stem cell biology. Here, we report that a branch of glycolysis, the serine biosynthesis pathway (SBP), is activated in regenerating muscle stem cells (MuSCs). Gene inactivation and metabolomics revealed that Psat1, one of the three SBP enzymes, controls MuSC activation and expansion of myogenic progenitors through production of the metabolite α-ketoglutarate (α-KG) and α-KG-generated glutamine. Psat1 ablation resulted in defective expansion of MuSCs and impaired regeneration. Psat1, α-KG, and glutamine were reduced in MuSCs of old mice. α-KG or glutamine re-established appropriate muscle regeneration of adult conditional Psat1 -/- mice and of old mice. These findings contribute insights into the metabolic role of Psat1 during muscle regeneration and suggest α-KG and glutamine as potential therapeutic interventions to ameliorate muscle regeneration during aging.

A developmental mechanism to regulate alternative polyadenylation in an adult stem cell lineage.

Alternative cleavage and polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3' UTRs from the same genetic locus, potentially impacting mRNA translation, localization, and stability. Developmentally regulated APA can thus make major contributions to cell type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, ∼500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of cleavage factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knockdown of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell type-specific APA at selected genes.

Hardwiring Stem Cell Communication through Tissue Structure.

Adult stem cells across diverse organs self-renew and differentiate to maintain tissue homeostasis. How stem cells receive input to preserve tissue structure and function largely relies on their communication with surrounding cellular and non-cellular elements. As such, how tissues are organized and patterned not only reflects organ function, but also inherently hardwires networks of communication between stem cells and their environment to direct tissue homeostasis and injury repair. This review highlights how different methods of stem cell communication reflect the unique organization and function of diverse tissues.

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.

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.