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.

The nuclei of human adult stem cells can move within the cell and generate cellular protrusions to contact other cells.

BACKGROUND: The neuronal transdifferentiation of adult bone marrow cells (BMCs) is still considered an artifact based on an alternative explanation of experimental results supporting this phenomenon obtained over decades. However, recent studies have shown that following neural induction, BMCs enter an intermediate cellular state before adopting neural-like morphologies by active neurite extension and that binucleated BMCs can be formed independent of any cell fusion events. These findings provide evidence to reject the idea that BMC neural transdifferentiation is merely an experimental artifact. Therefore, understanding the intermediate states that cells pass through during transdifferentiation is crucial given their potential application in regenerative medicine and disease modelling. METHODS: In this study, we examined the functional significance of the variety of morphologies and positioning that cell nuclei of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) can adopt during neural-like differentiation using live-cell nuclear fluorescence labelling, time-lapse microscopy, and confocal microscopy analysis. RESULTS: Here, we showed that after neural induction, hBM-MSCs enter an intermediate cellular state in which the nuclei are able to move within the cells, switching shapes and positioning and even generating cellular protrusions as they attempt to contact the cells around them. These findings suggest that changes in nuclear positioning occur because human cell nuclei somehow sense their environment. In addition, we showed the process of direct interactions between cell nuclei, which opens the possibility of a new level of intercellular interaction. CONCLUSIONS: The present study advances the understanding of the intermediate stage through which hBM-MSCs pass during neural transdifferentiation, which may be crucial to understanding the mechanisms of these cell conversion processes and eventually harness them for use in regenerative medicine. Importantly, our study provides for the first time evidence that the nuclei of hBM-MSC-derived intermediate cells somehow sense their environment, generating cellular protrusions to contact other cells. In summary, human mesenchymal stromal cells could not only help to increase our understanding of the mechanisms underlying cellular plasticity but also facilitate the exact significance of nuclear positioning in cellular function and in tissue physiology.

Adult stem cells in the eye: Identification, characterisation, and therapeutic application in ocular regeneration - A review.

Adult stem cells, present in various parts of the human body, are undifferentiated cells that can proliferate and differentiate to replace dying cells within tissues. Stem cells have specifically been identified in the cornea, trabecular meshwork, crystalline lens, iris, ciliary body, retina, choroid, sclera, conjunctiva, eyelid, lacrimal gland, and orbital fat. The identification of ocular stem cells broadens the potential therapeutic strategies for untreatable eye diseases. Currently, stem cell transplantation for corneal and conjunctival diseases remains the most common stem cell-based therapy in ocular clinical management. Lens epithelial stem cells have been applied in the treatment of paediatric cataracts. Several early-phase clinical trials for corneal and retinal regeneration using ocular stem cells are also underway. Extensive preclinical studies using ocular stem cells have been conducted, showing encouraging outcomes. Ocular stem cells currently demonstrate great promise in potential treatments of eye diseases. In this review, we focus on the identification, characterisation, and therapeutic application of adult stem cells in the eye.

Clonal barcoding of endogenous adult hematopoietic stem cells reveals a spectrum of lineage contributions.

The hierarchical model of hematopoiesis posits that self-renewing, multipotent hematopoietic stem cells (HSCs) give rise to all blood cell lineages. While this model accounts for hematopoiesis in transplant settings, its applicability to steady-state hematopoiesis remains to be clarified. Here, we used inducible clonal DNA barcoding of endogenous adult HSCs to trace their contribution to major hematopoietic cell lineages in unmanipulated animals. While the majority of barcodes were unique to a single lineage, we also observed frequent barcode sharing between multiple lineages, specifically between lymphocytes and myeloid cells. These results suggest that both single-lineage and multilineage contributions by HSCs collectively drive continuous hematopoiesis, and highlight a close relationship of myeloid and lymphoid development.

Measures of genetic diversification in somatic tissues at bulk and single-cell resolution.

Intra-tissue genetic heterogeneity is universal to both healthy and cancerous tissues. It emerges from the stochastic accumulation of somatic mutations throughout development and homeostasis. By combining population genetics theory and genomic information, genetic heterogeneity can be exploited to infer tissue organization and dynamics in vivo. However, many basic quantities, for example the dynamics of tissue-specific stem cells remain difficult to quantify precisely. Here, we show that single-cell and bulk sequencing data inform on different aspects of the underlying stochastic processes. Bulk-derived variant allele frequency spectra (VAF) show transitions from growing to constant stem cell populations with age in samples of healthy esophagus epithelium. Single-cell mutational burden distributions allow a sample size independent measure of mutation and proliferation rates. Mutation rates in adult hematopietic stem cells are higher compared to inferences during development, suggesting additional proliferation-independent effects. Furthermore, single-cell derived VAF spectra contain information on the number of tissue-specific stem cells. In hematopiesis, we find approximately 2 × 105 HSCs, if all stem cells divide symmetrically. However, the single-cell mutational burden distribution is over-dispersed compared to a model of Poisson distributed random mutations. A time-associated model of mutation accumulation with a constant rate alone cannot generate such a pattern. At least one additional source of stochasticity would be needed. Possible candidates for these processes may be occasional bursts of stem cell divisions, potentially in response to injury, or non-constant mutation rates either through environmental exposures or cell-intrinsic variation.

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.

Sex-specific preservation of neuromuscular function and metabolism following systemic transplantation of multipotent adult stem cells in a murine model of progeria.

Onset and rates of sarcopenia, a disease characterized by a loss of muscle mass and function with age, vary greatly between sexes. Currently, no clinical interventions successfully arrest age-related muscle impairments since the decline is frequently multifactorial. Previously, we found that systemic transplantation of our unique adult multipotent muscle-derived stem/progenitor cells (MDSPCs) isolated from young mice-but not old-extends the health-span in DNA damage mouse models of progeria, a disease of accelerated aging. Additionally, induced neovascularization in the muscles and brain-where no transplanted cells were detected-strongly suggests a systemic therapeutic mechanism, possibly activated through circulating secreted factors. Herein, we used ZMPSTE24-deficient mice, a lamin A defect progeria model, to investigate the ability of young MDSPCs to preserve neuromuscular tissue structure and function. We show that progeroid ZMPST24-deficient mice faithfully exhibit sarcopenia and age-related metabolic dysfunction. However, systemic transplantation of young MDSPCs into ZMPSTE24-deficient progeroid mice sustained healthy function and histopathology of muscular tissues throughout their 6-month life span in a sex-specific manner. Indeed, female-but not male-mice systemically transplanted with young MDSPCs demonstrated significant preservation of muscle endurance, muscle fiber size, mitochondrial respirometry, and neuromuscular junction morphometrics. These novel findings strongly suggest that young MDSPCs modulate the systemic environment of aged animals by secreted rejuvenating factors to maintain a healthy homeostasis in a sex-specific manner and that the female muscle microenvironment remains responsive to exogenous regenerative cues in older age. This work highlights the age- and sex-related differences in neuromuscular tissue degeneration and the future prospect of preserving health in older adults with systemic regenerative treatments.

Sorting Technology for Mesenchymal Stem Cells from a Single Tissue Source.

Mesenchymal stem cells (MSCs) are adult stem cells that can be obtained, enriched and proliferated in vitro. They owned enormous potential in fields like regenerative medicine, tissue engineering and immunomodulation. However, though isolated from the same origin, MSCs are still essentially heterogeneous cell populations with different phenotypes and functions. This heterogeneity of MSCs significantly affects their therapeutic efficacy and brings obstacles to scientific research. Thus, reliable sorting technology which can isolate or purify MSC subpopulations with various potential and differentiation pathways is urgently needed. This review summarized principles, application status and clinical implications for these sorting methods, aiming at improving the understanding of MSC heterogeneity as well as providing fresh perspectives for subsequent clinical applications.

Haematopoietic development and HSC formation in vitro: promise and limitations of gastruloid models.

Haematopoietic stem cells (HSCs) are the most extensively studied adult stem cells. Yet, six decades after their first description, reproducible and translatable generation of HSC in vitro remains an unmet challenge. HSC production in vitro is confounded by the multi-stage nature of blood production during development. Specification of HSC is a late event in embryonic blood production and depends on physical and chemical cues which remain incompletely characterised. The precise molecular composition of the HSC themselves is incompletely understood, limiting approaches to track their origin in situ in the appropriate cellular, chemical and mechanical context. Embryonic material at the point of HSC emergence is limiting, highlighting the need for an in vitro model of embryonic haematopoietic development in which current knowledge gaps can be addressed and exploited to enable HSC production. Gastruloids are pluripotent stem cell-derived 3-dimensional (3D) cellular aggregates which recapitulate developmental events in gastrulation and early organogenesis with spatial and temporal precision. Gastruloids self-organise multi-tissue structures upon minimal and controlled external cues, and are amenable to live imaging, screening, scaling and physicochemical manipulation to understand and translate tissue formation. In this review, we consider the haematopoietic potential of gastruloids and review early strategies to enhance blood progenitor and HSC production. We highlight possible strategies to achieve HSC production from gastruloids, and discuss the potential of gastruloid systems in illuminating current knowledge gaps in HSC specification.

Suspension-Induced Stem Cell Transition: A Non-Transgenic Method to Generate Adult Stem Cells from Mouse and Human Somatic Cells.

Adult stem cells (ASCs) can be cultured with difficulty from most tissues, often requiring chemical or transgenic modification to achieve adequate quantities. We show here that mouse primary fibroblasts, grown in suspension, change from the elongated and flattened morphology observed under standard adherent culture conditions of generating rounded cells with large nuclei and scant cytoplasm and expressing the mesenchymal stem cell (MSC) marker (Sca1; Ly6A) within 24 h. Based on this initial observation, we describe here a suspension culture method that, irrespective of the lineage used, mouse fibroblast or primary human somatic cells (fibroblasts, hepatocytes and keratinocytes), is capable of generating a high yield of cells in spheroid form which display the expression of ASC surface markers, circumventing the anoikis which often occurs at this stage. Moreover, mouse fibroblast-derived spheroids can be differentiated into adipogenic and osteogenic lineages. An analysis of single-cell RNA sequence data in mouse fibroblasts identified eight distinct cell clusters with one in particular comprising approximately 10% of the cells showing high levels of proliferative capacity expressing high levels of genes related to MSCs and self-renewal as well as the extracellular matrix (ECM). We believe the rapid, high-yield generation of proliferative, multi-potent ASC-like cells via the process we term suspension-induced stem cell transition (SIST) could have significant implications for regenerative medicine.

Comparative Analysis of Somatic Stem Cells With Emphasis on Osteochondral Tissue Regeneration.

Congenital anomalies, diseases, and injuries may result in osteochondral damage. Recently, a big hope has been given to somatic stem cells (SSCs) which are characterized as undifferentiated cells with an ability of long-term self-renewing and plasticity. They are adherent with a fibroblast-like morphology in vitro and express various surface markers (e.g. CD29, CD73, CD90, and CD105), but they are negative for CD31, CD34, CD45, and HLA-DR. SSCs secrete various bioactive molecules, which are involved in processes of regeneration. The main goal of the present study was the characterization and comparison of biological properties of SSCs obtained from adipose tissue, dental pulp, and urine concerning osteochondral regeneration. SSCs were maintained in an appropriate growth medium up to the third passage and were analyzed by light and electron microscope. The immunophenotype was analyzed by flow cytometry. The kinetics of proliferation was measured by MTT assay. Human Cytokine/Chemokine Multiplex Assay was used, and SSCs secretory profile was measured by Luminex MAGPIX® Instrument. Pellet cultures and a chondrogenic medium were used to induce chondrogenic differentiation. Osteogenic differentiation was induced by the osteogenic medium. Chondrogenic and osteogenic differentiation was analyzed by real-time PCR. SSCs had similar fibroblast-like morphology. They have similar kinetics of proliferation. SSCs shared the expression CD29, CD44, CD73, CD90, and CD105. They lack expression of CD29 and CD34. SSCs secerned similar levels of IL10 and IL18 while differing in IFN-gamma, IL6, IL8, MCP-1, and RANTES production. SSCs possess a similar capacity for chondrogenic differentiation but slightly differ in osteogenic differentiation. In conclusion, it can be emphasized that SSCs from adipose tissue, dental pulp, and urine share the majority of cellular characteristics typical for SSCs and have great potential to be used in osteochondral tissue regeneration.

Engineered adult stem cells: Current clinical trials status of disease treatment.

Regenerative medicine is an interdisciplinary field involving the process of replacing and regenerating cells/tissues or organs by integrating medicine, science, and engineering principles to enhance the intrinsic regenerative capacity of the host. Recently, engineered adult stem cells have gained attention for their potential use in regenerative medicine by reducing inflammation and modulating the immune system. This chapter introduces adult stem cell engineering and chimeric antigen receptor T cells (CAR T) gene therapy and summarises current engineered stem cell- and extracellular vesicles (EVs)-focused clinical trial studies that provide the basis for the proposal of a personalised medicine approach to diseases diagnosis and treatment.

m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency.

Embryos across metazoan lineages can enter reversible states of developmental pausing, or diapause, in response to adverse environmental conditions. The molecular mechanisms that underlie this remarkable dormant state remain largely unknown. Here we show that N6-methyladenosine (m6A) RNA methylation by Mettl3 is required for developmental pausing in mouse blastocysts and embryonic stem (ES) cells. Mettl3 enforces transcriptional dormancy through two interconnected mechanisms: (1) it promotes global mRNA destabilization and (2) it suppresses global nascent transcription by destabilizing the mRNA of the transcriptional amplifier and oncogene N-Myc, which we identify as a crucial anti-pausing factor. Knockdown of N-Myc rescues pausing in Mettl3-/- ES cells, and forced demethylation and stabilization of Mycn mRNA in paused wild-type ES cells largely recapitulates the transcriptional defects of Mettl3-/- ES cells. These findings uncover Mettl3 as a key orchestrator of the crosstalk between transcriptomic and epitranscriptomic regulation during developmental pausing, with implications for dormancy in adult stem cells and cancer.

Mesenchymal Stem Cell-Derived Extracellular Vesicles: An Emerging Diagnostic and Therapeutic Biomolecules for Neurodegenerative Disabilities.

Mesenchymal stem cells (MSCs) are a type of versatile adult stem cells present in various organs. These cells give rise to extracellular vesicles (EVs) containing a diverse array of biologically active elements, making them a promising approach for therapeutics and diagnostics. This article examines the potential therapeutic applications of MSC-derived EVs in addressing neurodegenerative disorders such as Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). Furthermore, the present state-of-the-art for MSC-EV-based therapy in AD, HD, PD, ALS, and MS is discussed. Significant progress has been made in understanding the etiology and potential treatments for a range of neurodegenerative diseases (NDs) over the last few decades. The contents of EVs are carried across cells for intercellular contact, which often results in the control of the recipient cell's homeostasis. Since EVs represent the therapeutically beneficial cargo of parent cells and are devoid of many ethical problems connected with cell-based treatments, they offer a viable cell-free therapy alternative for tissue regeneration and repair. Developing innovative EV-dependent medicines has proven difficult due to the lack of standardized procedures in EV extraction processes as well as their pharmacological characteristics and mechanisms of action. However, recent biotechnology and engineering research has greatly enhanced the content and applicability of MSC-EVs.

Maintenance of adult stem cells from human minor salivary glands via the Wnt signaling pathway.

BACKGROUND: Xerostomia is a salivary gland dysfunction that negatively impacts the life quality of patients; however, there is no effective treatment for xerostomia. Bioengineered organs, generated using stem cells obtained from newborn salivary glands and ligated injury models, are a new organ transplantation strategy that could be feasible for xerostomia treatment. Reconstruction of salivary gland organoids by seed cells obtained from human minor salivary glands will offer theoretical fundaments and technology support for clinical application and organ regeneration research. Herein, we aimed to propose a new method for culturing and enriching adult human minor salivary gland stem cells in vitro in a three-dimensional (3D) environment via Wnt signaling activation. METHODS: Obtained and characterized human minor salivary gland stem cells (hMSGSCs) with self-organization ability were 3D-cultured to generate organoids. We examined hMSGSCs proliferation and colony formation using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Telomerase reverse transcriptase staining, flow cytometry, immunofluorescence assay, RNA isolation, RT-PCR, and qPCR were performed to assess hMSGSCs structure and the function of reconstructive organoids in vitro. RESULTS: hMSGSCs showed typical epithelial-like characteristics, such as positive for CD49f and cell KRT expression. hMSGSCs served as adult stem cells in salivary glands and could differentiate into acinar and duct cells. Upon the addition of Noggin, CHIR99021, and Wnt3A to the 3D culture system, hMSGSCs showed higher LGR5 expression and decreased AMY1B and MUC5B expression. Therefore, the Wnt and bone morphogenetic protein (BMP) pathways are important in regulating hMSGSCs self-organization and differentiation. CONCLUSIONS: We showed that the stem cell properties of hMSGSCs in a 3D culture system can be maintained by activating the Wnt signaling pathway and inhibiting the BMP signaling pathway. Our findings contribute new insights on salivary gland organoid generation in vitro.

Gap junction-transported cAMP from the niche controls stem cell progeny differentiation.

The niche has been shown to control stem cell self-renewal in different tissue types and organisms. Recently, a separate niche has been proposed to control stem cell progeny differentiation, called the differentiation niche. However, it remains poorly understood whether and how the differentiation niche directly signals to stem cell progeny to control their differentiation. In the Drosophila ovary, inner germarial sheath (IGS) cells contribute to two separate niche compartments for controlling both germline stem cell (GSC) self-renewal and progeny differentiation. In this study, we show that IGS cells express Inx2 protein, which forms gap junctions (GJs) with germline-specific Zpg protein to control stepwise GSC lineage development, including GSC self-renewal, germline cyst formation, meiotic double-strand DNA break formation, and oocyte specification. Germline-specific Zpg and IGS-specific Inx2 knockdowns cause similar defects in stepwise GSC development. Additionally, secondary messenger cAMP is transported from IGS cells to GSCs and their progeny via GJs to activate PKA signaling for controlling stepwise GSC development. Therefore, this study demonstrates that the niche directly controls GSC progeny differentiation via the GJ-cAMP-PKA signaling axis, which provides important insights into niche control of stem cell differentiation and highlights the importance of GJ-transported cAMP in tissue regeneration. This may represent a general strategy for the niche to control adult stem cell development in various tissue types and organisms since GJs and cAMP are widely distributed.

CD133/Prom1 marks proximal mouse oviduct epithelial progenitors and adult epithelial cells with a low generative capacity.

The epithelium lining the oviduct or fallopian tube consists of multiciliated and secretory cells, which support fertilization and preimplantation development, however, its homeostasis remains poorly understood. CD133/Prom1 expression has been used as a marker to identify adult stem cell populations in various organs and often associated with cancer cells that have stem-like properties. Using an antibody targeted to CD133 and a Cre recombinase-based lineage tracing strategy, we found that CD133/Prom1 expression is not associated with a stem/progenitor population in the oviduct but marked predominantly multiciliated cells with a low generative capacity. Additionally, we have shown that CD133 is disparately localised along the oviduct during neonatal development, and that Prom1 expressing secretory cells in the ampulla rapidly transitioned to multiciliated cells and progressively migrated to the ridge of epithelial folds.

Kidins220 sets the threshold for survival of neural stem cells and progenitors to sustain adult neurogenesis.

In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.

One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids.

Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.