Stem Cells

The ability to revascularize target tissues and organs through cell-based therapy would provide a novel approach for the treatment of a range of ischemic disorders including cardiovascular diseases, stroke and peripheral artery disease. Towards this goal, we have identified a human pluripotent stem cell (hPSC)-derived vascular progenitor (VP) population generated via an epicardial intermediate with functional engraftment properties. VP cells efficiently engraft the mammary fat pad and hind limb skeletal muscle of NSG recipient mice and form vessel-like structures that integrate with the host vasculature. In an ischemic hind limb mouse model, VPs generate extensive vascular grafts that improve perfusion, restore some function and preserve muscle integrity over a three-month period post-transplant. Single-cell transcriptomic and flow cytometric analyses show that the VP population, initially identified by the co-expression of CD140b, CD13 and KDR, displays an epicardial lineage signature and expresses a spectrum of genes and proteins indicative of vascular progenitor stage cells. Together, these findings demonstrate that it is possible to revascularize both normal and ischemic tissue through the transplantation of an appropriate hPSC-derived progenitor and in doing so, lay the foundation for developing cell-based therapy approaches to treat ischemic diseases. Graphical Abstract LegendHuman pluripotent stem cells are differentiated through an epicardial intermediate to generate vascular progenitor (VP) cells characterized by expression of CD140b, CD13 and KDR. These VP cells demonstrate the capacity to engraft both mammary fat pad and skeletal muscle tissue where they form stable perfused vascular networks. In a hindlimb ischemia model, VP cell transplantation restores blood flow and improves functional outcomes. eTOC BlurbFernandes et al. develop a protocol to generate engraftable vascular progenitors from human pluripotent stem cells through an epicardial intermediate. These cells form functional vessels in vivo, restore perfusion in ischemic tissue, and demonstrate tissue-specific adaptation while maintaining endothelial identity, providing a foundation for therapeutic revascularization. HighlightsO_LIA staged differentiation protocol generates vascular progenitors (VPs) from hPSCs via an epicardial intermediate. C_LIO_LIVP cells form stable, perfused vascular networks following transplantation into multiple tissue sites. C_LIO_LIVP cell therapy with or without VEGF nanoparticles restores perfusion and improves functional outcomes in hindlimb ischemia. C_LIO_LISingle-cell analysis reveals tissue-specific adaptation while maintaining endothelial identity. C_LI

BackgroundiPSCs generated from healthy individuals constitute an important control resource for disease modelling applications but existing biobanks are highly skewed towards populations of European ancestry while well characterized control lines from Indian populations remain limited. Given the extensive genetic diversity of the Indian subcontinent, the availability of ethnically relevant healthy control lines is important for developing accurate disease models and reducing population specific confounding effects. MethodologyWe used peripheral blood mononuclear cells (PBMNCs) of a healthy female donor of Eastern Indian origin for the generation a wild type iPSC line using non-integrating episomal reprogramming vectors. Established colonies were expanded and characterized through morphological assessment, expression of pluripotency and trilineage markers, episomal vector clearance analysis, and chromosomal stability evaluation and mycoplasma contamination analysis. ResultsThe line generated exhibited characteristic pluripotent stem cell morphology and also showed strong expression of pluripotency markers, was free from any contamination and free from the reprogramming vectors confirming an integration free system. The cells maintained a normal diploidy number during characterization. Expression of lineage specific markers associated with ectoderm, mesoderm and endoderm confirmed the developed iPSCs functional capacity to undergo trilineage differentiation. ConclusionWe have developed and validated an iPSC line from an underrepresented Indian population. This well characterized, ethnicity specific iPSC line provides a valuable cell line for establishing a high quality, well characterized control baseline, which is a major missing element in South Asian stem cell repositories and thus will provide a solid foundation for future disease specific modelling and screening.

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.

Pluripotent stem cells derived from livestock species represent valuable systems for studying early mammalian development and for establishing renewable, well-defined cell sources; however, direct comparative characterization of distinct pluripotent stem cell platforms in sheep remains limited. In this study, we established and evaluated two ovine pluripotent stem cell types: reprogrammed induced pluripotent stem cells (siPSCs) and embryonic disc-derived stem cells (sEDSCs). Both siPSCs and sEDSCs exhibited core features of pluripotency, including compact colony morphology, alkaline phosphatase activity, expression of key pluripotency-associated markers, and maintenance of a normal ovine karyotype. Flow cytometry and quantitative RT-PCR analyses revealed broadly overlapping yet distinguishable pluripotency marker expression profiles between the two cell types. Functional pluripotency was confirmed by embryoid body formation and in vitro differentiation into derivatives of all three germ layers. To further assess lineage-specific differentiation competence and compare functional outputs relevant to mesodermal differentiation, both pluripotent stem cell types were directed towards the adipogenic lineage. While siPSCs and sEDSCs were each capable of adipogenic differentiation, differences in differentiation efficiency and marker expression were observed. Together, these findings demonstrate that ovine siPSCs and sEDSCs share core pluripotency characteristics while retaining distinct molecular and functional properties, providing a robust comparative framework for studies of ovine pluripotency, lineage specification, and stem cell biology.

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.

Unlike the conventional mature neutrophils, immature neutrophils have been investigated for their regenerative properties; however, their limited availability necessitates alternative generation strategies. Here, we used a combination of dimethylsulfoxide (DMSO) and 1,25-dihydroxyvitamin D3 (D3) to differentiate myeloid leukemia (HL-60) cells into immature neutrophil-like cells. Differentiated cells exhibited reduced cell size, loss of uniformity, decreased nuclear-to-cytoplasmic ratio, band-shaped nuclei, increased proportion of CD11b+CD14+ cells (indicative of immature neutrophils), decreased proportion of CD11b+CD16+ cells (indicative of mature neutrophils), higher levels of arginase 1, TGF{beta}1 (markers of immature neutrophils), and no expression of CD16, MRC1 (markers of mature neutrophils and M2 macrophages, respectively). Proteomic analysis revealed enrichment of proteins associated with immature neutrophils and wound healing. Functionally, these cells supported limbal stem cell growth and wound closure in vitro, indicating relevance for corneal regeneration. Administration of these cells to ex-vivo and in-vivo alkali-injured corneas, resulted in significant effect on promotion of wound healing, with epithelial regeneration and decreased fibrotic markers, proving that such cells hold promise for clinical translation as a therapeutic tool for tissue repair.

Advances in transcriptome profiling have revealed transcriptomic differences across different cellular states. However, functional interpretation requires precise perturbation tools and experimental frameworks. This study benchmarked two widely used modalities: CRISPR interference (CRISPRi) and Cas13d/CasRx. A standardized workflow was established to generate human pluripotent stem cells (PSCs) with inducible ZIM3-dCas9 or CasRx expression. The cell lines were subjected to flow cytometry, copy number, and immunocytochemical analyses. The knockdown performance was validated via robust OCT4 suppression and the expected downstream effects on pluripotency genes. Time-course measurements indicated that CRISPRi produced faster and stronger repression but slower recovery after inducer withdrawal. In contrast, CasRx yielded slower and typically weaker knockdown with rapid reversibility. Furthermore, a key limitation of CRISPRi was demonstrated using the ATF5-NUP62 locus, wherein CRISPRi could co-repress genes with overlapping promoter regions. In contrast, CasRx avoids these limitations and supports isoform-resolved targeting of circular and alternatively spliced transcripts, albeit with variable efficiency. These results provide practical guidance for selecting complementary knockdown tools to improve the interpretability of transcriptomic function studies. MOTIVATIONAdvances in transcriptome profiling have enabled the detection of subtle cell type-specific differences. However, mechanistic interpretation still depends on perturbation tools that can modulate transcripts with high precision and efficiency. Recent CRISPR-based modalities, CRISPRi and Cas13/CasRx, function as robust and orthogonal methods to achieve the knockdown of specific gene targets. However, a standardized approach for cell line preparation and comparative studies on their relative performances and limitations remains unclear. Consequently, this study presents a standardized workflow for generating cell lines that support high-efficiency knockdown using CRISPRi and CasRx. Moreover, it compares the trade-offs in potency, reversibility, and isoform resolution, along with a practical overview of method-specific pitfalls to guide tool selection and data interpretation in future studies. HIGHLIGHTSO_LIDoxycycline-inducible AAVS1 knock-in human PSC platforms for CRISPRi (ZIM3-dCas9) and CasRx (RfxCas13d) were generated to enable standardized RNA perturbation experiments. C_LIO_LIThe prepared cell lines demonstrated strong OCT4 knockdown, with expected downstream effects on the expression of another pluripotency gene, NANOG. C_LIO_LIA comparison of knockdown characteristics and their reversibility revealed rapid and sustained repression with CRISPRi, whereas slow but rapid recovery was observed with CasRx. C_LIO_LIA CRISPRi-specific off-target effect arising from TSS proximity/overlap (ATF5-NUP62) was identified, whereas CasRx achieved ATF5 knockdown without collateral repression of the neighboring NUP62 gene. C_LIO_LICasRx enables isoform-resolved knockdown of structural isoforms (circHIPK3 vs. linear HIPK3 mRNA) and splice isoforms (RAB6A-iso1 vs. RAB6A-iso2). C_LI

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.

In mammals, a small population of spermatogonial stem cells (SSCs) is established shortly after birth. These cells self-renew and produce sperm for the entirety of a males reproductive lifespan, passing the genome on to the next generation. Thus, establishment of a population of SSCs with high genomic integrity is essential. SSCs are derived from a much larger precursor population of male germ cells (MGCs) that differentiate during fetal life. During the last third of gestation, MGCs undergo a prolonged period of G0 cell cycle arrest during which they sustain high levels of transcription and acquire epigenetic programming for SSC fate. Although these differentiation steps can cause cellular and genomic damage, it has been unclear whether selection for germ cell quality occurs during G0 arrest since no classic markers of cell death have been detected. In this study, we utilize a mouse model to characterize a population of MGCs that begin to accumulate markers if cell death, such as AnnexinV (AnV) and propidium iodide (PI), at E16.5. The AnV- and PI-positive MGC population is characterized by low expression of the RNA-binding protein, Dead End 1 (DND1), and exhibit dsDNA breaks and mitochondrial dysfunction. Interestingly, we do not see evidence of an active cell death cascade until the time of birth, where we see phosphorylation of MLKL, a hallmark of a necroptotic cell death mechanism. Based on these findings, we propose that variable cellular health is an important basis for selection of the SSC precursors. Significance StatementSpermatogonial stem cells (SSCs) are essential for reproductive fitness, yet how their precursors are selected during development is not known. Utilizing a mouse model, this study describes high levels of cellular damage within a subset of male germ cells (MGCs) during G0 arrest. The damaged MGC population was marked by low expression of the RNA-binding protein, DND1, and was strongly associated with mitochondrial dysfunction and dsDNA breaks. We observed signs of non-apoptotic cell death by embryonic day (E)16.5 and the appearance of necroptotic markers in MGCs at the time of birth. This study uncovers previously unknown heterogeneity in the MGC pool and points to MGC health as an important source of selection during G0 arrest.

Leukemia inhibitory factor (LIF)/STAT3 signaling is central to maintaining naive pluripotency in mouse embryonic stem cells (mESCs). We identify Asgard, a previously uncharacterized long non-coding RNA, as a direct STAT3 target required for efficient self-renewal. Asgard is rapidly induced by LIF, enriched in the epiblast, and its depletion reduces alkaline phosphatase-positive colony formation while enhancing differentiation. Mechanistically, Asgard fulfils a dual role: it acts as the primary transcript for the differentiation-promoting microRNA Odin, while also functioning as a sponge to sequester Odin and related miRNAs. This dual mechanism enables Asgard to both generate and buffer pro-differentiation signals, thereby stabilizing the pluripotent state while preserving responsiveness to lineage cues. Our work reveals a new paradigm in RNA-mediated control of stem cell identity, where a single STAT3-regulated lncRNA couples microRNA production with competitive inhibition to safeguard naive pluripotency.

During early development of the placenta, a subset of murine trophectoderm stem cells (TSCs) undergo endoreplication, an unusual form of cell division cycle that decouples DNA synthesis from cytokinesis, resulting in physiological polyploidy. Oscillations in CDK2 activity are essential for the orderly progression of the cell cycle to ensure replicated DNA is accurately partitioned into two daughter cells. However, it remains underexplored how the dynamics of CDK2 activity regulate endoreplication in the context of TSCs differentiation. To address this question, we leveraged the variability in cell fate decisions in an established in vitro system of TSCs differentiation that relies on removal of a growth factor, FGF4, to induce endoreplication. Using quantitative single-cell live confocal microscopy of a precise CDK2 biosensor, DHB-Venus, we identified at least three different outcomes upon FG4 removal: self-renewal, endoreplication, and migration. Our quantitative analyses showed high levels of Cdk2 activity in self-renewing cells whereas intermediate DHB-Venus turnover is linked to increased nuclear and cell size, indicating a shift to endoreplication. Importantly, we also characterize a third class of differentiating TSCs with migratory characteristics that correlate with low levels Cdk2 activity without a change in nuclear size. In sum, our results demonstrated a correlation between different fate outcomes and specific thresholds of CDK2 activity. Our findings show that TSCs can distinguish between different outcomes through modulating the central kinase of the cell cycle, CDK2, positioning it as a key regulator of early trophoblast differentiation. Summary StatementThis study investigates the oscillatory behavior of CDK2 activity during murine trophectoderm differentiation and its potential role in guiding cell fate decisions.

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.

Certain aspects of lipid metabolism are known to regulate hematopoietic stem cell (HSC) function, including fatty acid oxidation and lipid uptake, but there is a limited understanding of the contribution of de novo fatty acid synthesis to HSC homeostasis. Here, we show that endogenous fatty acid synthesis is essential for HSC function. Conditional deletion of Acaca, the gene that encodes the rate-limiting enzyme for de novo fatty acid synthesis, acetyl-CoA carboxylase 1 (ACC1), in hematopoietic cells profoundly reduces HSC function, marked by a reduced ability to reconstitute irradiated mice after competitive transplantation. ACC1 deficiency reduced quiescence, increased uptake of extracellular lipids, and increased reactive oxygen species in HSCs. The loss of HSC function is partly caused by increased fatty acid oxidation as deletion of CPT1a, which is required for long-chain fatty acid oxidation, partially rescued HSC function. A balance between fatty acid synthesis and fatty acid oxidation is thus critical for the maintenance of HSC function.

IntroductionReliable generation of hepatocyte-like cells (HLCs) from pluripotent stem cells remains limited by heterogeneity and incomplete maturation of the cells. Derivation of induced pluripotent- and embryonic stem cells into hepatocytes typically relies on complex, and costly reagent-intensive protocols, with inconsistent reporting of differentiation efficiencies and functional maturation criteria. Variability in protocol designs highlights the need for optimisation, particularly in mouse embryonic stem cells (mESCs) systems that can be more comparable with mouse models for underpinning translational and toxicological studies. Here, we developed and evaluated two cytokine-based strategies: an advanced hepatic-inducing cocktail (A-HIC) and a simplified hepatic-inducing cocktail (HIC), both designed to reduce complexity while increasing functional maturation. MethodsHepatic differentiation and maturation were assessed by morphology, immunofluorescence, flow cytometry, and qRT-PCR. Functional competence was evaluated via urea production, glutathione synthesis, indocyanine green handling, cytochrome P450 inducibility, and impedance-based cell layer integrity monitoring. ResultsMorphological, molecular and phenotypic analyses confirmed that both protocols supported hepatic lineage progression, generating heterogeneous populations of hepatoblast-like and more mature HLCs. Gene expression confirmed the loss of pluripotency, transient endoderm induction, and subsequent hepatic specification. Functionally, cells exhibited glycogen storage, inducible urea production, glutathione depletion, and active ICG uptake and clearance, with stable monolayer formation by day 21. A-HIC-derived HLCs demonstrated enhanced maturation, with higher ASGR1 expression and stronger Cyp1a1 induction. DiscussionThese findings suggest that both protocols generate functional HLCs; however, A-HIC yields a higher proportion of functionally mature cells with reduced variability. This approach enables a simple, cost-effective, and time-efficient generation of HLCs, supported by improved functional characterisation with potential applicability to more complex pluripotent systems, including human iPSC-based models for disease modelling and toxicology.

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.

Human in vitro hepatic models that accurately recapitulate liver function are essential for fundamental and translational research; however, currently utilised models for disease modelling and drug discovery lack physiological fidelity and require prolonged culture time. Here, we present a streamlined 10-day protocol for efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte like cells (HLCs) and hepatic liver organoids (HLOs). Both models exhibited mature hepatocyte differentiation, as evidenced by albumin secretion and CYP3A4 metabolic activity. Interestingly, HLOs display enhanced multicellular complexity, incorporating endothelial, stellate, and macrophage populations along with hepatocytes, thereby more closely recapitulating the native liver microenvironment than HLCs. Here, steatosis was induced in both platforms, which resulted in triglyceride accumulation and upregulated lipogenic markers (DGAT1, DGAT2). However, only HLOs recapitulated advanced disease characteristics, including inflammatory (IL-10) and fibrotic (alpha SMA, COL1A1) responses. Resmetirom, a thyroid hormone receptor-{beta} agonist, significantly reduced steatosis and restored molecular signatures in both models. Additionally, transplanted organoids demonstrated prolonged survival and displayed host-derived vascularization, thereby validating in vivo maturation. Collectively, this platform provides a rapid and physiologically relevant liver model, with 3D HLOs offering superior utility for disease modelling, therapeutic evaluation, and regenerative applications due to their enhanced functional and physiological relevance.

Pluripotent stem cells (PSCs) have remarkable capacity for unlimited self-renewal and differentiation into all somatic lineages. Although translational regulation has been implicated in the maintenance of PSC identity, the specific mechanisms involved remain poorly understood. Here, we identified EIF3H, a conserved subunit of the eIF3 translation initiation complex, as an essential regulator of human primed PSC proliferation and differentiation. CRISPR interference-mediated knockdown of EIF3H markedly reduced colony size, impaired proliferation, and diminished differentiation potential in all three germ layers. Integrated transcriptomic and translatomic profiling revealed that EIF3H loss decreased the translation of metal ion-related genes. Notably, the targeted suppression of metallothionein genes encoding metal-binding proteins recapitulated the proliferative defects observed in EIF3H-deficient PSCs, demonstrating a functional requirement for EIF3H-mediated translation of this gene family. Taken together, these findings establish EIF3H as a critical translational regulator that sustains PSC self-renewal and differentiation by maintaining the expression of key metabolic and stress-response genes, providing new insights into the molecular basis of pluripotency.

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