Vitamin A resolves lineage plasticity to orchestrate stem cell lineage choices.

Lineage plasticity-a state of dual fate expression-is required to release stem cells from their niche constraints and redirect them to tissue compartments where they are most needed. In this work, we found that without resolving lineage plasticity, skin stem cells cannot effectively generate each lineage in vitro nor regrow hair and repair wounded epidermis in vivo. A small-molecule screen unearthed retinoic acid as a critical regulator. Combining high-throughput approaches, cell culture, and in vivo mouse genetics, we dissected its roles in tissue regeneration. We found that retinoic acid is made locally in hair follicle stem cell niches, where its levels determine identity and usage. Our findings have therapeutic implications for hair growth as well as chronic wounds and cancers, where lineage plasticity is unresolved.

Adult neural stem cells and neurogenesis are resilient to intermittent fasting.

Intermittent fasting (IF) is a promising strategy to counteract ageing shown to increase the number of adult-born neurons in the dentate gyrus of mice. However, it is unclear which steps of the adult neurogenesis process are regulated by IF. The number of adult neural stem cells (NSCs) decreases with age in an activation-dependent manner and, to counteract this loss, adult NSCs are found in a quiescent state which ensures their long-term maintenance. We aimed to determine if and how IF affects adult NSCs in the hippocampus. To identify the effects of every-other-day IF on NSCs and all following steps in the neurogenic lineage, we combined fasting with lineage tracing and label retention assays. We show here that IF does not affect NSC activation or maintenance and, that contrary to previous reports, IF does not increase neurogenesis. The same results are obtained regardless of strain, sex, diet length, tamoxifen administration or new-born neuron identification method. Our data suggest that NSCs maintain homeostasis upon IF and that this intervention is not a reliable strategy to increase adult neurogenesis.

Pregnancy-responsive pools of adult neural stem cells for transient neurogenesis in mothers.

Adult neural stem cells (NSCs) contribute to lifelong brain plasticity. In the adult mouse ventricular-subventricular zone, NSCs are heterogeneous and, depending on their location in the niche, give rise to different subtypes of olfactory bulb (OB) interneurons. Here, we show that multiple regionally distinct NSCs, including domains that are usually quiescent, are recruited on different gestation days during pregnancy. Synchronized activation of these adult NSC pools generates transient waves of short-lived OB interneurons, especially in layers with less neurogenesis under homeostasis. Using spatial transcriptomics, we identified molecular markers of pregnancy-associated interneurons and showed that some subsets are temporarily needed for own pup recognition. Thus, pregnancy triggers transient yet behaviorally relevant neurogenesis, highlighting the physiological relevance of adult stem cell heterogeneity.

Deciphering Adult Neural Stem Cells with Single-Cell Sequencing.

Adult neural stem cells (NSCs) are restricted to the two neurogenic regions of the mammalian brain, where they self-renew and generate progenies of multiple lineages, including neurons, astrocytes, and oligodendrocytes. Single-cell RNA sequencing technology, which reconstructs high-resolution transcriptional landscapes, provides valuable insights into cellular heterogeneity and developmental dynamics. In this review, we overviewed recent progress in the single-cell analyses of both conventional and unconventional NSCs. We discussed the heterogeneity among the stem cell pool and characterized the transcriptional alterations in aging and brain tumors. A comprehensive understanding of NSCs in physiological and pathological settings will provide insights for the rejuvenation of the aged brain and restoration of normal brain function in multiple neurological disorders.

Lysosomes at the Crossroads of Cell Metabolism, Cell Cycle, and Stemness.

Initially described as lytic bodies due to their degradative and recycling functions, lysosomes play a critical role in metabolic adaptation to nutrient availability. More recently, the contribution of lysosomal proteins to cell signaling has been established, and lysosomes have emerged as signaling hubs that regulate diverse cellular processes, including cell proliferation and cell fate. Deciphering these signaling pathways has revealed an extensive crosstalk between the lysosomal and cell cycle machineries that is only beginning to be understood. Recent studies also indicate that a number of lysosomal proteins are involved in the regulation of embryonic and adult stem cell fate and identity. In this review, we will focus on the role of the lysosome as a signaling platform with an emphasis on its function in integrating nutrient sensing with proliferation and cell cycle progression, as well as in stemness-related features, such as self-renewal and quiescence.

MLL1 is required for maintenance of intestinal stem cells.

Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.

CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease.

Obesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as "brown" and "brite/beige" adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors.

Ex vivo expansion and functional activity preservation of adult hematopoietic stem cells by a diarylheptanoid from Curcuma comosa.

Hematopoietic stem cells (HSCs, CD34+ cells) have shown therapeutic efficacy for transplantation in various hematological disorders. However, a large quantity of HSCs is required for transplantation. Therefore, strategies to increase HSC numbers and preserve HSC functions through ex vivo culture are critically required. Here, we report that expansion medium supplemented with ASPP 049, a diarylheptanoid isolated from Curcuma comosa, and a cocktail of cytokines markedly increased numbers of adult CD34+ cells. Interestingly, phenotypically defined primitive HSCs (CD34+CD38-CD90+) were significantly increased under ASPP 049 treatment relative to control. ASPP 049 treatment also improved two functional properties of HSCs, as evidenced by an increased number of CD34+CD38- cells in secondary culture (self-renewal) and the growth of colony-forming units as assessed by colony formation assay (multilineage differentiation). Transplantation of cultured CD34+ cells into immunodeficient mice demonstrated the long-term reconstitution and differentiation ability of ASPP 049-expanded cells. RNA sequencing and KEGG analysis revealed that Hippo signaling was the most likely pathway involved in the effects of ASPP 049. These results suggest that ASPP 049 improved ex vivo expansion and functional preservation of expanded HSCs. Our findings provide a rationale for the use of ASPP 049 to grow HSCs prior to hematological disease treatment.

Diversity of Adult Neural Stem and Progenitor Cells in Physiology and Disease.

Adult neural stem and progenitor cells (NSPCs) contribute to learning, memory, maintenance of homeostasis, energy metabolism and many other essential processes. They are highly heterogeneous populations that require input from a regionally distinct microenvironment including a mix of neurons, oligodendrocytes, astrocytes, ependymal cells, NG2+ glia, vasculature, cerebrospinal fluid (CSF), and others. The diversity of NSPCs is present in all three major parts of the CNS, i.e., the brain, spinal cord, and retina. Intrinsic and extrinsic signals, e.g., neurotrophic and growth factors, master transcription factors, and mechanical properties of the extracellular matrix (ECM), collectively regulate activities and characteristics of NSPCs: quiescence/survival, proliferation, migration, differentiation, and integration. This review discusses the heterogeneous NSPC populations in the normal physiology and highlights their potentials and roles in injured/diseased states for regenerative medicine.

Drosophila, an Integrative Model to Study the Features of Muscle Stem Cells in Development and Regeneration.

Muscle stem cells (MuSCs) are essential for muscle growth, maintenance and repair. Over the past decade, experiments in Drosophila have been instrumental in understanding the molecular and cellular mechanisms regulating MuSCs (also known as adult muscle precursors, AMPs) during development. A large number of genetic tools available in fruit flies provides an ideal framework to address new questions which could not be addressed with other model organisms. This review reports the main findings revealed by the study of Drosophila AMPs, with a specific focus on how AMPs are specified and properly positioned, how they acquire their identity and which are the environmental cues controlling their behavior and fate. The review also describes the recent identification of the Drosophila adult MuSCs that have similar characteristics to vertebrates MuSCs. Integration of the different levels of MuSCs analysis in flies is likely to provide new fundamental knowledge in muscle stem cell biology largely applicable to other systems.

Delayed Double Treatment with Adult-Sourced Adipose-Derived Mesenchymal Stem Cells Increases Striatal Medium-Spiny Neuronal Number, Decreases Striatal Microglial Number, and Has No Subventricular Proliferative Effect, after Acute Neonatal Hypoxia-Ischemia in Male Rats.

Perinatal hypoxia-ischemia (HI) is a major cause of striatal injury. Delayed post-treatment with adult-sourced bone marrow-derived mesenchymal stem cells (BMSCs) increased the absolute number of striatal medium-spiny neurons (MSNs) following perinatal HI-induced brain injury. Yet extraction of BMSCs is more invasive and difficult compared to extraction of adipose-derived mesenchymal stem cells (AD-MSCs), which are easily sourced from subcutaneous tissue. Adult-sourced AD-MSCs are also superior to BMSCs in the treatment of adult ischemic stroke. Therefore, we investigated whether delayed post-treatment with adult-sourced AD-MSCs increased the absolute number of striatal MSNs following perinatal HI-induced brain injury. This included investigation of the location of injected AD-MSCs within the brain, which were widespread in the dorsolateral subventricular zone (dlSVZ) at 1 day after their injection. Cells extracted from adult rat tissue were verified to be stem cells by their adherence to tissue culture plastic and their expression of specific 'cluster of differentiation' (CD) markers. They were verified to be AD-MSCs by their ability to differentiate into adipocytes and osteocytes in vitro. Postnatal day (PN) 7/8, male Sprague-Dawley rats were exposed to either HI right-sided brain injury or no HI injury. The HI rats were either untreated (HI + Diluent), single stem cell-treated (HI + MSCs×1), or double stem cell-treated (HI + MSCs×2). Control rats that were matched-for-weight and litter had no HI injury and were treated with diluent (Uninjured + Diluent). Treatment with AD-MSCs or diluent occurred either 7 days, or 7 and 9 days, after HI. There was a significant increase in the absolute number of striatal dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32)-positive MSNs in the double stem cell-treated (HI + MSCs×2) group and the normal control group compared to the HI + Diluent group at PN21. We therefore investigated two potential mechanisms for this effect of double-treatment with AD-MSCs. Specifically, did AD-MSCs: (i) increase the proliferation of cells within the dlSVZ, and (ii) decrease the microglial response in the dlSVZ and striatum? It was found that a primary repair mechanism triggered by double treatment with AD-MSCs involved significantly decreased striatal inflammation. The results may lead to the development of clinically effective and less invasive stem cell therapies for neonatal HI brain injury.

Understanding the "SMART" features of hematopoietic stem cells and beyond.

Since the huge success of bone marrow transplantation technology in clinical practice, hematopoietic stem cells (HSCs) have become the gold standard for defining the properties of adult stem cells (ASCs). Here, we describe the "self-renewal, multi-lineage differentiation, apoptosis, rest, and trafficking" or "SMART" model, which has been developed based on data derived from studies of HSCs as the most well-characterized stem cell type. Given the potential therapeutic applications of ASCs, we delineate the key characteristics of HSCs using this model and speculate on the physiological relevance of stem cells identified in other tissues. Great strides are being made in understanding the biology of ASCs, and efforts are now underway to develop safe and effective ASC-based therapies in this emerging area.

Aging-related upregulation of the homeobox gene caudal represses intestinal stem cell differentiation in Drosophila.

The differentiation efficiency of adult stem cells undergoes a significant decline in aged animals, which is closely related to the decline in organ function and age-associated diseases. However, the underlying mechanisms that ultimately lead to this observed decline of the differentiation efficiency of stem cells remain largely unclear. This study investigated Drosophila midguts and identified an obvious upregulation of caudal (cad), which encodes a homeobox transcription factor. This factor is traditionally known as a central regulator of embryonic anterior-posterior body axis patterning. This study reports that depletion of cad in intestinal stem/progenitor cells promotes quiescent intestinal stem cells (ISCs) to become activate and produce enterocytes in the midgut under normal gut homeostasis conditions. However, overexpression of cad results in the failure of ISC differentiation and intestinal epithelial regeneration after injury. Moreover, this study suggests that cad prevents intestinal stem/progenitor cell differentiation by modulating the Janus kinase/signal transducers and activators of the transcription pathway and Sox21a-GATAe signaling cascade. Importantly, the reduction of cad expression in intestinal stem/progenitor cells restrained age-associated gut hyperplasia in Drosophila. This study identified a function of the homeobox gene cad in the modulation of adult stem cell differentiation and suggested a potential gene target for the treatment of age-related diseases induced by age-related stem cell dysfunction.

Self-renewal and differentiation of rat epididymal basal cells using a novel in vitro organoid model†.

The epididymis is composed of a pseudostratified epithelium that is comprised of various cell types. Studies have shown that rat basal cells share common properties with adult stem cells and begin to differentiate in vitro in response to fibroblast growth factor and 5α-dihydrotestosterone. The characterization of rat basal cells is therefore necessary to fully understand the role of these cells. The objectives of this study were to assess the ability of single basal cells to develop organoids and to assess their ability to self-renew and differentiate in vitro. We isolated basal cells from the rat epididymis and established three-dimensional cell cultures from the basal and nonbasal cell fractions. Organoids were formed by single adult epididymal basal cells. Organoids were dissociated into single basal cells, which were able to reform new organoids, and were maintained over 10 generations. Long-term culture of organoids revealed that these cells could be differentiated into cells expressing the principal cell markers aquaporin 9 and cystic fibrosis transmembrane conductance regulator. Electron microscopy demonstrated that organoids were composed of several polarized cell types displaying microvilli and the ability to form tight junctions. Additionally, organoids could be formed by basal cells from either the proximal or distal region of the epididymis and are able to secrete clusterin, a protein implicated in the maturation of spermatozoa. These data indicate that rat basal cells can be used to derive epididymal organoids and further support that notion that these may represent a stem cell population in the epididymis.

Regeneration, Rejuvenation, and Replacement: Turning Back the Clock on Tissue Aging.

While some animals, such as planaria and hydra, appear to be capable of seemingly endless cycles of regeneration, most animals experience a gradual decline in fitness and ultimately die. The progressive loss of cell and tissue function, leading to senescence and death, is generally referred to as aging. Adult ("tissue") stem cells maintain tissue homeostasis and facilitate repair; however, age-related changes in stem cell function over time are major contributors to loss of organ function or disease in older individuals. Therefore, considerable effort is being invested in restoring stem cell function to counter degenerative diseases and age-related tissue dysfunction. Here, we will review strategies that could be used to restore stem cell function, including the use of environmental interventions, such as diet and exercise, heterochronic approaches, and cellular reprogramming. Maintaining optimal stem cell function and tissue homeostasis into late life will likely extend the amount of time older adults are able to be independent and lead healthy lives.

Regulation of Adult Mammalian Neural Stem Cells and Neurogenesis by Cell Extrinsic and Intrinsic Factors.

Tissue-specific stem cells give rise to new functional cells to maintain tissue homeostasis and restore damaged tissue after injury. To ensure proper brain functions in the adult brain, neural stem cells (NSCs) continuously generate newborn neurons that integrate into pre-existing neuronal networks. Proliferation, as well as neurogenesis of NSCs, are exquisitely controlled by extrinsic and intrinsic factors, and their underlying mechanisms have been extensively studied with the goal of enhancing the neurogenic capacity of NSCs for regenerative medicine. However, neurogenesis of endogenous NSCs alone is insufficient to completely repair brains damaged by neurodegenerative diseases and/or injury because neurogenic areas are limited and few neurons are produced in the adult brain. An innovative approach towards replacing damaged neurons is to induce conversion of non-neuronal cells residing in injured sites into neurons by a process referred to as direct reprogramming. This review describes extrinsic and intrinsic factors controlling NSCs and neurogenesis in the adult brain and discusses prospects for their applications. It also describes direct neuronal reprogramming technology holding promise for future clinical applications.

Release of stem cells from quiescence reveals gliogenic domains in the adult mouse brain.

Quiescent neural stem cells (NSCs) in the adult mouse ventricular-subventricular zone (V-SVZ) undergo activation to generate neurons and some glia. Here we show that platelet-derived growth factor receptor beta (PDGFRß) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia. Selective deletion of PDGFRß in adult V-SVZ NSCs leads to their release from quiescence, uncovering gliogenic domains for different glial cell types. These domains are also recruited upon injury. We identify an intraventricular oligodendrocyte progenitor derived from NSCs inside the brain ventricles that contacts supraependymal axons. Together, our findings reveal that the adult V-SVZ contains spatial domains for gliogenesis, in addition to those for neurogenesis. These gliogenic NSC domains tend to be quiescent under homeostasis and may contribute to brain plasticity.

Direct reprogramming of somatic cells into induced hepatocytes: Cracking the Enigma code.

There is an unmet need for functional primary human hepatocytes to support the pharmaceutical and (bio)medical demand. The unique discovery, a decade ago, that somatic cells can be drawn out of their apparent biological lockdown to reacquire a pluripotent state has revealed a completely new avenue of possibilities for generating surrogate human hepatocytes. Since then, the number of papers reporting the direct conversion of somatic cells into induced hepatocytes (iHeps) has burgeoned. A hepatic cell fate can be established via the ectopic expression of native liver-enriched transcription factors in somatic cells, thereby bypassing the need for an intermediate (pluripotent) stem cell state. That said, understanding and eventually controlling the processes that give rise to functional iHeps remains challenging. In this review, we provide an overview of the state-of-the-art reprogramming cocktails and techniques, as well as their corresponding conversion efficiencies. Special attention is paid to the role of liver-enriched transcription factors as hepatogenic reprogramming tools and small molecules as facilitators of hepatic transdifferentiation. To conclude, we formulate recommendations to optimise, standardise and enrich the in vitro production of iHeps to reach clinical standards, and propose minimal criteria for their characterisation.

Biomechanical Strain Promotes the Differentiation of Murine Oogonial Stem Cells.

Cells within tissues are routinely subjected to physiological stress and strain, arising from direct interactions with neighboring cells as well as with extracellular matrix components. Accordingly, there is tremendous interest in deciphering how cells sense, and respond to, changes in biomechanical forces. In this study, we explored the effects of mechanostimulation on the differentiation of mouse female germline or oogonial stem cells (OSCs) as a model for adult stem cell function. We report that increasing levels, or repeated application of a subthreshold fixed level, of radial strain to OSCs in culture significantly increased rates of in vitro oocyte formation as a measure of stem cell differentiation. These responses involved changes in F-actin-mediated cytoskeletal tension as well as in activation of intracellular signaling by Rho-associated protein kinase (ROCK) and Yes-associated protein (YAP) phosphorylation. In addition, application of mechanical strain to OSCs enhanced association of YAP with muscle-specific cytidine-adenosine-thymidine (MCAT) response elements in the promoter stimulated by retinoic acid gene 8 (Stra8), the transcriptional activation of which is required for germline meiotic commitment. These data indicate that biomechanical strain directly promotes the differentiation of adult female germline stem cells through a signaling pathway involving F-actin, ROCK, YAP, and Stra8.

Merlin and expanded integrate cell signaling that regulates cyst stem cell proliferation in the Drosophila testis niche.

The Drosophila testis is a model organism stem cell niche in which two stem cell populations coordinate together to produce sperm; thus, these stem cells must be balanced in the niche. Merlin, a tumor-suppressor and human disease gene required for contact inhibition of proliferation, is known to limit the proliferation of the somatic cyst stem cells in the testis niche. Expanded encodes a protein that is structurally similar to Merlin in Drosophila, and is semi-redundant with Merlin in multiple tissues. We found that expanded depletion caused similar cyst lineage cell over-proliferation as observed with Merlin, and double mutants showed more severe phenotypes than either gene individually. Thus, these genes have partially redundant functions in the cyst lineage cells of this niche. We also expressed non-phosphorylatable constitutively "tumor suppressing" alleles of Merlin in cyst lineage cells, and surprisingly, we observed a similar cyst lineage over-proliferation phenotype. Merlin is known to impact multiple different signaling pathways to exert its effect on proliferation. We found that the Merlin loss of function phenotype was associated with an increase in MAPK/ERK signaling, consistent with Merlin's established role in transmembrane receptor inhibition. Constitutive Merlin displayed a reduction in both MAPK/ERK signaling and PI3K/Tor signaling. PI3K/Tor signaling is required for cyst cell differentiation, and inhibition of this pathway by Merlin activation phenocopied the Tor cyst lineage loss of function phenotype. Thus, Merlin impacts and integrates the activity of multiple signaling pathways in the testis niche. The ability of Merlin to dynamically change its activity via phosphorylation in response to local contact cues provides an intriguing mechanism whereby the signaling pathways that control these stem cells might be dynamically regulated in response to the division of a neighboring germ cell.