Stem Cells

Background: Human induced pluripotent stem cells (hiPSCs) hold promise for vascular regeneration, but preliminary research often relies on neonatal donors, whereas clinical applications will use cells derived from aged individuals. Although the impact of donor age on reprogramming efficiency has been studied, its effect on the functionality of hiPSC-derived endothelial progenitors (hiPSC-EPs) remains unclear. This question is the focus of the current study. Methods and Results: We derived EPs from iPSCs sourced from three neonatal donors (ND) and three mature donors (MD) matched 1:1 for sex and somatic cell origin. We assessed their functional, epigenetic, and transcriptomic characteristics. Despite higher CD34? yields from MD-iPSCs, MD-hiPSC-EPs formed poorly interconnected and non-lumenized vascular structures in 3D hydrogels, compared to neonatal donor (ND) lines. In 2D culture, MD-hiPSC-EPs exhibited reduced cell density and aberrant VE-Cadherin localization. DNA methylation analysis revealed that somatic cell origin was the dominant driver of variance, but consistent differences in methylation of mesoderm commitment, angiogenesis, ECM remodeling, and cytoskeleton-related genes were observed between age groups. Epigenetic age prediction showed MD-hiPSC-EPs had more developmentally advanced signatures, potentially explaining their shift away from vasculogenic competence. Our RNA-sequencing findings confirm trends seen in the DNA methylation data and show differential expression of pathways linked to mitochondrial regulation and nitric oxide signaling. Conclusions: Donor age significantly alters the vasculogenic function of hiPSC-EPs. These findings underscore the necessity of donor-specific considerations in hiPSC-based vascular engineering and highlight potential barriers to translating hiPSC-derived therapeutics into aged patient populations.

Background: Chronic and non-healing wounds remain a major clinical challenge with limited therapeutic options. Angiogenesis and inflammation are central to tissue repair, and mesenchymal stem cells (MSC) contribute to these processes through their trophic and immunomodulatory secretome. Cannabidiol (CBD) exhibits antioxidant and immunomodulatory properties. However, whether CBD-rich Cannabis sativa extract stimulate MSC toward a pro-angiogenic secretome remains unclear. Purpose: This study aims to determine whether purified CBD or a phytochemically CBD-rich full spectrum extract stimulate umbilical cord-derived human MSC (UC-hMSC) to secrete pro-angiogenic factors and enhance endothelial responses relevant to wound healing. Methods: UC-hMSC were preconditioned with either purified CBD or a CBD-rich full-spectrum extract. Transcriptional changes were assessed by qPCR. The functional impact of the resulting secretome was evaluated in vitro using HUVEC-based proliferation and tube formation assays, and in vivo through the chick chorioallantoic membrane assay. To explore underlying mechanisms, we examined HIF-1 stabilization and VEGFA release in UC-hMSC, and VEGFR-2/ERK signaling in HUVEC. Results: Purified CBD and full-spectrum CBD extract preconditioned UC-hMSC secretomes, increased HUVEC proliferation, tube formation, and enhanced vascular branching in the CAM assay. Mechanistic analyses indicated activation of the HIF-1/VEGF axis in UC-hMSC, and ERK1/2 activation in HUVEC that was sensitive to VEGFR-2 blockade. Conclusion: Purified CBD and CBD-rich full-spectrum extract prime UC-hMSC toward a pro-angiogenic secretome that promotes endothelial activation and neovascularization. These findings suggest that cannabinoid-based preconditioning of UC-hMSC involves the HIF-1/VEGF axis and VEGFR-2/ERK signaling pathways in endothelial cells, supporting further investigation of this approach in wound healing and regenerative therapies.

The protracted timing required for oligodendrocyte differentiation from human pluripotent stem cells (hPSCs) has limited their use in disease modeling, drug screening, and cell therapy. In particular, the signals that drive oligodendrocyte specification and maturation after neural induction and ventral patterning remain poorly understood. Here, we present a protocol to derive human oligodendrocytes from hPSCs that is based solely on extrinsic cues, and we identify dual inhibition of BMP and Notch signaling as critical drivers of oligodendrocyte commitment and maturation. By day 42 of differentiation, up to 70% of the cells are positive for the oligodendrocyte marker O4, with minimal astrocyte contamination, and show robust expression of mature myelin markers including MBP, MOG, and MAG. These hPSC-derived oligodendrocytes closely match the molecular identity of primary fetal human oligodendrocytes as assessed by single-cell RNA sequencing and are functional as shown by in vitro myelination assays. In addition to the rapid generation of myelinating oligodendrocytes, the new protocol can be modularly adapted for the efficient production of PDGFR+ oligodendrocyte precursors or mixed glial populations containing AQP4+ astrocytes, thereby providing a cellular toolbox for the study of human glial lineages in translational applications.

Axonal degeneration and insufficient neuronal survival remain major barriers to central nervous system repair. Stem cells from human exfoliated deciduous teeth (SHED) represent an accessible, developmentally immature, neural crest-derived mesenchymal stem cell population with potential relevance for neuroregenerative medicine. Here, we show that SHED display enhanced proliferative stability, preserved mesenchymal identity, and more sustained expansion capacity than adult dental pulp stem cells, supporting their suitability for scalable regenerative applications. Using embryonic chick retinal explants at neurogenic and post-neurogenic stages, we demonstrate that SHED robustly promote retinal ganglion cell axonogenesis, axonal regeneration, and neuronal survival. At embryonic day 5, SHED enhanced axonal outgrowth in both newly generated EdU/TUJ1 neurons and pre-existing EdU-/TUJ1 retinal ganglion cells. At embryonic day 13, when retinal neurons are post-mitotic and intrinsically less regenerative, SHED still significantly increased regenerative axonal extension and reduced developmental cell death. To investigate the molecular mechanisms underlying the neuroprotective and axogenic effects of SHED, proteomic profiling of SHED-retina co-culture secretomes was performed, revealing a highly enriched extracellular environment containing matrix-associated and neurodevelopmental proteins, including thrombospondin-1 (THBS1), galectin1 and 3, and multiple proteins associated with IGF2 pathway. Proteomic analysis of the SHED secretome, together with prior evidence implicating thrombospondin signaling in neuronal development and synaptogenesis, identified THBS1 as a strong candidate mediator of SHED-induced effects in chick retinal co-culture systems. Neutralization of THBS1, particularly in combination with gabapentin-mediated blockade of 2{delta}-1-dependent thrombospondin signaling, markedly reduced SHED-induced axonal growth and induced neuritic swellings consistent with impaired axonal integrity. In contrast, inhibition of THBS1 signaling did not significantly abolish the neuroprotective effect of SHED on neuronal survival, suggesting that distinct paracrine mechanisms independently regulate axonal regeneration and cell survival. Together, these findings demonstrate that SHED-derived combined secreted factors promote neuronal survival and axonal regeneration through partially divergent extracellular matrix-associated developmental pathways, positioning SHED and their secretome as promising candidates for cell-based and cell-free neuroregenerative strategies.

The specification of hematopoietic stem cells (HSCs) is tightly regulated by multiple transcription factors and signaling pathways. Inflammatory signaling is pivotal for embryonic HSC development, but the mechanisms that activate it in vivo remain poorly understood. Here, we show that Toll-like receptor 7 (TLR7) is essential for the emergence of embryonic HSC in both zebrafish and mouse embryos. TLR7 deficiency reduces HSC numbers but not primitive or definitive progenitors. Conversely, the TLR7 agonist R848 enhances embryonic HSC development. Mechanistically, TLR7 signaling acts through interferon regulatory factor 5 (IRF5) to induce the expression of inflammatory cytokines, which subsequently activate Notch signaling to promote HSC emergence through a non-cell-autonomous mechanism. Notably, we identify microRNA-146a (miR-146a) as a potential endogenous activator of TLR7, inducing inflammatory signaling and promoting HSC development. Pharmacological treatment with miR-146a significantly increases HSC numbers in zebrafish embryos. Together, our findings reveal a crucial role for miR-146a-TLR7-IRF5 signaling axis in HSC emergence, providing insights into the endogenous factors that drive tonic inflammatory signaling during normal hematopoiesis and suggesting the translational potential of TLR7 agonists and miR-146a for stem-cell-based therapeutics. Significance StatementThe embryonic origin of hematopoietic stem cells (HSCs) requires inflammatory signals, but the endogenous factor that triggers this process remains elusive. We identify microRNA-146a (miR-146a) as a natural activator of Toll-like Receptor 7 (TLR7) signaling, which is essential for HSC emergence. This miR-146a-TLR7 axis functions through IRF5 and inflammatory cytokines to activate the Notch signaling, specifically promoting embryonic HSC development. Our work addresses the critical question of endogenous ligands that mediate tonic inflammatory signaling in normal hematopoiesis, uncovers novel crosstalk between miRNAs and innate immunity in HSC specification, and identifies promising candidates for stem cell-based therapeutics.

Hematopoietic stem cells (HSCs) generated from induced pluripotent stem cells (iPSCs) offer a promising patient-specific alternative to allogeneic transplantation, yet current differentiation protocols fail to fully recapitulate in vivo HSC maturation. During mouse development, yolk sac (YS)-derived macrophages populate the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence, but the mechanisms by which they support ex vivo hematopoietic stem and progenitor cell (HSPC) generation remain poorly defined. Bulk RNA sequencing revealed that mature AGM CD206 macrophages upregulate pro-inflammatory cytokines and the adhesion molecule F4/80. Using F4/80 knockout embryos, we identify a previously unreported, niche-specific role for F4/80 in restraining the frequency and colony-forming activity of HSPC subsets in the AGM, while supporting endothelial cell maintenance; this effect was absent in the YS. Lineage-tracing with a Cdh5-CreERT2;Rosa26LSL-tdTomato pulse-chase system confirmed that both CD206 and CD206- AGM cells originate from early YS-derived endothelial precursors, with no evidence of local macrophage generation within the AGM. Functional co-culture assays further demonstrated that the ability of CD206 macrophages to enhance the progenitor potential of hemogenic endothelium is AGM-specific and not an intrinsic, ontogeny-determined property, as YS macrophages failed to confer the same benefit even when paired with AGM endothelial cells, and AGM macrophages were ineffective with YS endothelium. Differential expression and NicheNet ligand-receptor interaction analyses identified a small set of AGM-restricted macrophage genes - including Mmp2, Nrep, Ccl2, and Cxcl16 - which are predicted to interact with both endothelial and cluster cells during endothelial-to-hematopoietic transition. Together, these findings establish that AGM macrophages acquire niche-specific transcriptional and functional properties upon entry into the aortic microenvironment, independent of their YS origin, and identify candidate macrophage-derived factors and a novel regulatory role for F4/80 in shaping HSPC output. These insights may guide the refinement of iPSC-based HSC differentiation protocols through the targeted, temporally controlled addition of macrophage-associated signals.

BackgroundPremature ovarian insufficiency (POI) affects approximately 1% of women under 40 and is characterized by elevated levels of gonadotropins, reduced estradiol, impaired folliculogenesis, and infertility. Bone marrow-derived mesenchymal stem cell (BM-MSC)-based therapy has emerged as a promising regenerative strategy in preclinical POI models. This systematic review and meta-analysis evaluated BM-MSC-based interventions, including cell transplantation and secretome/extracellular vesicle administration, in animal models of POI. MethodsA systematic review and meta-analysis was conducted following PRISMA guidelines. PubMed, Web of Science, Scopus, ScienceDirect, and the Cochrane Library were searched from inception to February 19, 2025. Preclinical studies assessing BM-MSC-based interventions in animal models of POI were included. ResultsThirty-four studies comprising 1,357 animals were included. Compared with controls, BM-MSC-based therapy increased serum estradiol (standardized mean difference [SMD] 3.11; 95% confidence interval [CI] 2.38-3.84) and anti-Mullerian hormone (SMD 1.86; 95% CI 1.03-2.69), while reducing follicle-stimulating hormone (SMD -3.54; 95% CI -4.37 to -2.71) and luteinizing hormone (SMD -3.44; 95% CI -5.17 to -1.70). Follicular counts increased across developmental stages, with fewer atretic follicles. Reproductive outcomes improved, including normal estrous cycles (risk ratio [RR] 7.80; 95% CI 3.15-19.34), pregnancy occurrence (RR 3.72; 95% CI 2.14-6.44), and offspring number (SMD 1.57; 95% CI 1.04-2.09). ConclusionBM-MSC-based therapy consistently improved hormonal, follicular, and reproductive outcomes in preclinical POI models. More well-designed, standardized, and adequately controlled studies to confirm these findings are warranted. Systematic review registration: CRD42023449053

Human pluripotent stem cells (hPSCs) provide a powerful platform for modeling early human embryonic development. Here, we investigate the mechanisms underlying mesodermal heterogeneity using a minimal directed differentiation system that simultaneously generates paraxial (PXM), intermediate (IM) and lateral plate mesoderm (LPM) populations. Single-cell RNA sequencing across defined time points during hPSC differentiation revealed a temporal sequence of lineage specification with LPM emerging first, followed by PXM and IM differentiation. Ligand-receptor and differential gene expression analyses identified BMP4 as a key regulator enriched in LPM-associated clusters versus mesoderm progenitors (MPs) that hold PXM and IM precursors. Whereas LPM cells cluster with an early BMP4 signal, IM clusters are associated with later BMP4. Moreover, these early and late BMP4 signals regulate this lineage specification potentially through distinct downstream pathways. Leveraging this insight, we established a stepwise protocol combining early BMP inhibition with subsequent BMP4 supplementation, suppressing initial LPM fate to efficiently induce IM from a mixed MP population. Longer culture of these selective IM progenitors promotes more mature nephrogenesis. Moreover, we demonstrate that during early differentiation high levels of BMP4 can still redirect MPs to more lateroventral fates, illustrating a degree of plasticity within the mesoderm lineage. Together, our results define a temporal framework for BMP4 signaling in mesoderm fate determination and provide a strategy for selective mesoderm differentiation from hPSCs. HIGHLIGHTSO_LIDevelopment of a minimal 2D differentiation platform allows for heterogenous mesoderm formation. C_LIO_LITemporal BMP4 signaling differentially directs mesoderm fates, with early exposure favoring LPM and late exposure promoting IM identity. C_LIO_LILPM cells arise first while later mesoderm progenitors hold both IM and PXM-fated cells. C_LIO_LISequential BMP modulation promotes IM and enhances nephrogenesis. C_LI

Hematopoietic stem cells (HSCs) in the adult mouse can be prospectively isolated to near-purity through phenotypic markers, enabling detailed analysis of stem cell function. Homeobox B5 (Hoxb5) was previously identified as a definitive marker of long-term (LT) HSCs in adult bone marrow1. In contrast, fetal HSCs have not been purified to the same extent. Here, we show that Hoxb5 is expressed in fetal liver (FL) HSCs at embryonic day (E) 12.5-16.5 using a single-color tri-mCherry reporter driven by endogenous Hoxb5 regulation. Prospective purification by stringent multiparameter flow cytometry revealed Hoxb5 FL-HSCs to exhibit robust, multilineage reconstitution upon serial transplantation. Quantitative assays reveal that Hoxb5 enriches FL-HSCs to near-single-cell purity, analogous to its role in the adult bone marrow, underscoring its reliability in distinguishing LT-HSCs throughout hematopoietic ontogeny. Notably, Hoxb5 expression is not exclusive to FL-HSCs, as it is also detected across the fetal liver hematopoietic hierarchy and in fetal liver endothelial cells, suggesting developmental stage-specific regulation of its expression. In addition, single-cell RNA sequencing of FL-HSCs identified distinct transcriptional states defined by Hoxb5 expression. These findings establish Hoxb5 as a robust marker for enhancing the purification of fetal liver phenotypic HSCs (pHSC) and provide a framework for dissecting the molecular regulation of HSC ontogeny.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold tremendous promise for disease modeling, drug discovery, and cardiac regenerative therapies. However, the immature phenotype of hPSC-CMs remains a major barrier limiting their translational utility. Here, we performed integrated multi-omic profiling to identify molecular pathways and regulatory programs associated with hPSC-CM maturation during long-term culture. hPSC-CMs were cultured for 113 days and analyzed using metabolomics, proteomics, and transcriptomics across progressive stages of maturation. Long-term culture induced widespread multi-omic remodeling, including significant changes in 142/934 metabolites, 550/3,556 proteins, and 2,892/23,309 transcripts from Day 30 to Day 113. Metabolomic analyses revealed early increases in phospholipid biosynthesis and mitochondrial beta oxidation of fatty acids from Day 30 to Day 60, suggesting metabolic priming precedes later maturation events. In contrast, proteomic remodeling was more prominent during later stages of maturation and was characterized by enhanced calcium handling and cell cycle exit. Transcriptomic analyses demonstrated progressive increases in ion channel expression, t-tubule organization, fatty acid metabolism, creatine shuttle pathways, and cell cycle arrest programs. Transcriptomic and integrative multi-omic pathway analyses identified coordinated suppression of TGF{beta}, MAPK, Wnt, and Hedgehog signaling together with activation of integrin-related, respiratory electron transport, muscle contraction, and Slit-Robo signaling pathways during maturation. Moreover, multi-omic transcription factor activity analysis prioritized a GATA4-centered network of putative cardiomyocyte maturation regulators including SOX7, SOX18, TBX2, and ZFPM2 (FOG2). Together, these findings elucidate the degree and pace of hPSC-CM maturation during long-term culture and establish an integrated multi-omic framework for identifying strategies to accelerate hPSC-CM maturation.

Mesenchymal Stromal Cells (MSC) face several heterogeneity challenges hindering clinical and commercial success. Employing a multiple response model, interplay between donor heterogeneity, and critical processing parameters (CPPs), effects on MSC potency and cell expansion attributes were investigated through computed composite attribute scores. Twelve unique CPP combinations were tested in thirteen marrow-derived MSC(M) and five adipose-tissue MSC(AT) training and test datasets, respectively. Donor heterogeneity and select CPP conditions affected a curated gene panel (surrogate for MSC potency); while MSC expansion was primarily influenced by CPPs. Model performances were evaluated against clinical effectiveness data from a previously deployed clinical trial; top-performing model predicted donor rankings coincided with clinical effectiveness data, validating the modeling approach used. Our model predicted that only 8% of tested donors were agnostic to CPPs; a majority (62%) of donors showed CPP-dependent optimal composite quality attributes, with MSC seeding density as a key driver; medium supplementation and oxygen preferences were highly donor dependent. Approximately 30% of donors performed poorly at all conditions tested and may be prospectively identified using a subset of genes (TGFB, VEGF, PDCD1LG1, PDCD1LG2, IDO). Model predicted optimal parameters worked for 69% of tested donors, while sub-optimal parameters worked for only 23% of donors and were confirmed in an independent CD14+ macrophage assay. Our integrated computational and experimental framework predictably identified interactive effects of donor heterogeneity and CPP conditions to optimize MSC potency attributes.

The adrenal cortex is a critical for life endocrine system. It manages metabolic homeostasis, electrolyte balance, stress response, and sex development. This is accomplished through the release of various steroids from a dynamically changing landscape of concentric cellular zones/layers. Adrenocortical dysfunction is implicated in pathologies ranging from adrenal insufficiency to hypertension. The in-depth investigation of adrenal gland biology, pathology and drug discovery has been hampered by a lack of human, experimentally tractable models. Particularly missing are models that recapitulate the cellular diversity of the adrenal cortex with representation of all fetal and adult cell layers and the capsule. Here, we employ human pluripotent stem cells (hPSCs) to generate cells of all three cortex zones alongside capsular cells in a single 2D platform. This platform mimics the cellular diversity expected from an organoid, yet it provides the simplicity of monolayer cultures, that are better suited for drug discovery and high-throughput settings. These cultures secrete zone-specific steroids (cortisol, aldosterone, and DHEA-S) and exhibit robust, physiologically relevant ACTH stimulation responses. Transcriptomic analysis revealed sequential acquisition of profiles consistent with adrenocortical development, zonation, and signaling programs consistent with zone maintenance. Our platform is validated by a literature meta-analysis that defines transcriptomic signatures for each human cortical cell type, spanning fetal and adult stages. Together, this novel adrenocortical platform enables investigation of adrenal development, disease mechanisms, and therapeutic strategies. Significance StatementWe describe a hPSC-based 2D differentiation strategy with the cell type complexity of an organoid and the technical simplicity necessary for high-throughput assays. The platform contains all cortical subtypes that mediate electrolyte regulation, stress response, sex development and self-maintenance of the tissue. This co-differentiation offers a unique opportunity to study human adrenal development, biology, pathology and enables drug discovery.

The complexity of stem cell differentiation programs remains incompletely understood across stem cell types, including for human bone marrow mesenchymal stromal/stem (BM-MSCs) cells, a heterogeneous cell population orchestrating bone formation and establishing a functional hematopoietic niche in the bone marrow. BM-MSCs form and repair bone through the evolutionarily conserved process of endochondral ossification (EO), initiated by deposition of a transient cartilage template subsequently remodeled into bone and bone marrow tissues. Despite their considerable potential for skeletal regeneration, the early molecular and cellular events underlying BM-MSCs commitment to endochondral ossification remain elusive. To overcome donor-dependent variability in chondrogenic potential that limits mechanistic studies, we here exploit OssiGel as a potent chondro-inductive extracellular matrix offering robust recapitulation of endochondral ossification by BM-MSCs. Through multi-omics profiling of OssiGel-primed BM-MSCs, we identify rapid chromatin remodeling at chondrogenic enhancer regions as a prerequisite for lineage commitment. The emergence of a chondro-progenitor population is detected as early as 3 days in vivo, and correlates with successful EO recapitulation. Mechanistically, we identify LINC02511 as a novel enhancer-associated element involved in the onset of EO. We confirm presence of LINC02511 in human skeletal atlases, and its CRISPR-mediated silencing was shown to significantly impair EO. By integrating tissue engineering with single cell multi-omics profiling, our study provides a framework for deciphering BM-MSCs fate decisions, highlighting the role of enhancers and non-coding elements as key determinants of early lineage specification. These findings advance our understanding of BM-MSCs biology and will prompt their translational exploitation in regenerative medicine.

Senescence human bone marrow mesenchymal stem cells (BMSCs), vulnerable to age-related defects, is poor in tissue regeneration. Cells in bone marrow accumulated senescent contributing to the development of metabolic energy regulation hold prospects for therapeutic advances. This study aimed to evaluate energy metabolic changes in male bone marrow mesenchymal stem cells senescence process. Our research established cell specific surface marker and enzymes expression level changes, as well as ECAR and OCR resonance. Notably, CD14, HLA-DRB1 and CD90 upregulated, glycolysis-related genes are increased, tricarboxylic acid cycle-related genes are decreased. We firstly identified links between time-dependent cell aging process and energy metabolism in BMSCs.

AimsWhile the muscle-enriched microRNA-378a (miR-378a) has been implicated in cardiac hypertrophy and stress responses, its role in maintaining cardiomyocyte metabolic homeostasis, mitochondrial function, and angiogenic paracrine signaling under physiological and post-injury conditions remains unclear. This study addresses these gaps by examining the molecular and functional consequences of miR-378a deficiency in murine heart and human cardiomyocytes. Methods and ResultsCardiac structure and function were analyzed in miR-378a-deficient (miR-378a-/-) and wild-type (miR-378a+/+) mice at 12 weeks and 17 months of age, revealing that miR-378a loss promoted myocardial fibrosis, altered IGF1R-AKT signaling, and impaired cardiac performance, with age-dependent effects. Integrated transcriptomic and proteomic analyses in miR-378a-/- and control mice, as well as in human iPSC-derived cardiomyocytes (hiPSC-CM) of both genotypes, revealed deregulated pathways related to translation, metabolism, and cardiomyopathy-associated signaling. In hiPSC-CM, miR-378a knockout (KO) impaired mitochondrial respiration, disrupted mitochondrial morphology, and reduced mitochondrial DNA content, accompanied by altered mitophagy and biogenesis. KO cells also showed increased glucose uptake but reduced glycogen storage, accompanied by changes in key metabolic regulators, and displayed diminished angiogenic potential. Finally, hiPSC-CM overexpressing miR-378a were delivered in a mouse model of acute myocardial infarction, but overexpression did not further enhance their therapeutic effect. ConclusionsThis study broadens our understanding of miR-378as physiological role in murine hearts and human cardiomyocytes, demonstrating its impact on contractility, mitochondrial integrity, glucose metabolism, and angiogenic paracrine signaling. However, overexpression of miR-378a in hiPSC-CM offers limited additional benefit in cell therapy for acute myocardial infarction.

Adult long-term hematopoietic stem cells (LT-HSCs) are classically defined by self-renewal, multilineage regenerative capacity, and relative quiescence, but how and when lifelong LT-HSCs are established during development remains unclear. Here, we demonstrate that Hoxb5 fetal liver HSCs exhibit bona fide LT-HSC activity, including long-term multilineage reconstitution and serial transplantation capacity, whereas Hoxb5- fetal liver HSCs display limited regenerative potential. Embryonic lineage tracing further demonstrates that E14.5 Hoxb5-expressing hematopoietic cells contribute broadly to adult hematopoiesis, including the adult HSC compartment, and give rise to functional adult LT-HSCs. Across developmental stages, single-cell transcriptional profiling revealed that fetal Hoxb5 HSCs remain highly proliferative while maintaining canonical LT-HSC transcriptional programs and superior repopulating activity relative to predominantly quiescent adult Hoxb5 HSCs. Fetal Hoxb5 HSCs also exhibited elevated ITGA4-mediated adhesion programs, and disruption of the ITGA4-VCAM1 axis impaired engraftment following transplantation. Together, these findings establish a developmental continuum linking fetal and adult LT-HSCs and identify enhanced ITGA4-mediated adhesion as a defining feature of fetal LT-HSCs.

Hematopoietic stem and progenitor cell (HSPC) niches support lifelong production of blood and immune cells. Recently, we identified a gene signature unique to HSPC niche endothelial cells that is highly conserved across species and developmental time, and includes the scavenger receptors, stab1/2 and mrc1a. Whether these receptors support HSPC development in the niche remains unclear. To investigate this, we used chemical inhibition and CRISPR mutagenesis in zebrafish and found that loss of stab2, and to a lesser degree stab1, reduced the number of embryonic HSPCs labeled by runx1:mCherry. Subsequent analyses of runx1:mCherry; cd41:GFP double transgenic embryos revealed an imbalance in the HSPC pool in stab2 mutants, with reductions in sub-populations containing stem cells and erythroid progenitors, the latter of which was most decreased. Our findings suggest stabilin scavenger receptors support HSPC development in the fetal niche, which could inform clinical strategies for culturing and expanding HSPCs.

Hypoxia is a potent stressor and a major cause of {beta}-cell failure and loss after islet transplantation. Autophagy is a critical homeostatic mechanism that preserves organelle integrity and metabolic balance in cells under stress, but whether it supports {beta}-cell adaptation to sustained oxygen deprivation is unclear. Here, we used {beta}-cell-specific Atg5 knockout together with hypoxia and transplantation models, to demonstrate that autophagy is a major determinant of {beta}-cell survival during oxygen limitation and supports islet graft function. However, prolonged hypoxia suppressed autophagic flux, reduced lysosomal activity, and led to autophagosome accumulation, indicating failure of the lysosomal clearance pathway. This was accompanied by a marked reduction in transcription factor EB (TFEB) and its lysosomal target genes. Genetic and pharmacological activation of TFEB restored lysosomal gene expression and cathepsin B activity and improved {beta}-cell viability under hypoxia, implicating TFEB decline as a contributor to autophagy-lysosome dysfunction. Together, these findings outline a sequence in which autophagy initially safeguards {beta}-cells but becomes ineffective under sustained hypoxia as TFEB levels fall, identifying TFEB as a potential target to strengthen {beta}-cell resilience and survival in islet transplantation.

Stem cells proliferate rapidly to maintain fast tissue turnover during regeneration. However, the feedback mechanisms in stem cells that prevent hyperproliferation remain unclear, and their dysregulation can lead to organ failure and cancer. Here, we identified nuclear factor-Y (NF-Y) as the transcriptional repressors to maintain the stem cell quiescence during intestinal homeostasis. We found that NF-Y negatively regulates intestinal stem cell (ISC) proliferation through preferentially occupying the promoters of EGFR signaling pathway components Egfr/Mkp3/Raf/Ras/pointed, via the action of histone acetyltransferase Nejire (Nej)/p300 dependent transcription regulation. While the loss of NF-Y enhances ISC proliferation, cell death and sensitivity to stress and tumor induced mortality. Moreover, NF-Y acts together with Nej to restrict Egfr expression and suppress ISC hyperproliferation. Together, these results demonstrate NF-Y acts with Nej serve as a key negative feedback module to orchestrate transcription initiation and termination of growth signaling in the control of stem cell activity in homeostatic and disease conditions.

Zebrafish (Danio rerio) possess remarkable regenerative capacity, making them an ideal model for studying the molecular mechanisms underlying tissue regeneration. In this article we report the identification of blastema linked exosome like extracellular vesicles (EVs) in zebrafish, that to the vesicles were plausibly being translocated in the proximo-distal axis through filipodia. We further thoroughly examined the exosome like EVs isolated from regenerating tissues of zebrafish caudal fins to characterize their nucleic acid cargo and evaluate their potential regulatory functions in regeneration. Caudal fins were amputated and allowed to regenerate and exosome like EVs isolated from blastema tissues displayed increased abundance compared to non-amputated controls. RNA sequencing identified a dynamic cluster of EV linked microRNAs (miRs). These differentially expressed miRs, including dre-miR-21, dre-miR-200b, dre-miR-218a and dre-let-7e were upregulated and associated with promoting proliferation, migration, differentiation, and tumour suppression pathways. Moreover, dre-miR-100, dre-miR-146a and dre-miR-200c regulated osteogenic differentiation, inflammatory signalling, epithelial-mesenchymal transition, and cell adhesion. Regeneration is generally believed to be coordinated only by local morphogen diffusion. Through this study it is indicative that filipodia bound EVs might have a pivotal role in long-range communication between blastema and the proximal tissues during the regeneration process. A detailed analyses of the miR targets and their validation could potentially lead to novel advancement and solutions in the field of regeneration and regenerative medicine in the near future.