Stem Cell Research

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.

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.

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

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.

Sickle cell disease (SCD) is caused by a point mutation in the {beta}-globin gene that promotes hemoglobin polymerization, leading to chronic hemolytic anemia, vaso-occlusive episodes, and progressive organ damage. The most efficacious therapies focus on reactivating fetal hemoglobin (HbF) expression to mitigate the pathological effects of sickle hemoglobin (HbS) polymerization. However, the predominantly used HbF inducer, hydroxyurea (HU), exhibits substantial interpatient variability in efficacy, and curative approaches such as gene therapy remain inaccessible to the vast majority of patients. Although all SCD patients share the same causative HBB glu7val mutation, differences in genetic background significantly influence disease severity and therapeutic response. We describe a SCD-specific induced pluripotent stem cell (iPSC) platform as a renewable and scalable preclinical model to interrogate treatment responses across the genetically diverse SCD patient population. By generating patient-specific iPSC-derived erythroblasts (iEry) representing distinct SCD genetic backgrounds, we demonstrate that this system faithfully recapitulates the heterogeneous HbF induction observed clinically in response to HU. Moreover, this platform enables the identification and evaluation of alternative therapeutic agents for HU non-responders and provides sufficient resolution to dissect drug-specific effects on erythroid differentiation and cellular phenotypes. Together, these findings support the use of iPSC-derived erythroid models as a versatile tool to advance precision therapeutic strategies for SCD. KEY POINTS- SCD iPSC-derived erythroid cells (iEry) reflect the diversity in HU-mediated HbF induction seen in SCD patients - SCD iEry recapitulate patient-specific treatment responses and can be used to identify therapeutic alternatives for HU non-responders - iEry provide a versatile platform to study the impact of novel HbF inducers on erythroid cell characteristics and differentiation parameters

This work aimed to establish a translationally viable, xeno-free, serum-free platform and protocol for the isolation and expansion of human salivary stem/progenitor cells (hS/PCs) suitable for regulatory qualification and future FDA-approved first-in-human autologous regenerative therapy trials for the treatment of hyposalivation disorders. Parotid gland specimens from non-cancerous regions/tissues were collected from consented surgical patients. Primary hS/PCs were isolated from tissue specimens, cultured in animal-component-free conditions, expanded to produce millions of cells, then enriched for CD44+ stem/progenitor cells by magnetic cell sorting. Normal epithelial purity was assessed using cytokeratins 5/14. Anti-CD133/PROM1 (cancer marker) and anti- fibroblast (clone TE-7) antibodies were used to demonstrate a lack of contaminating cells. Phenotype validation was performed by flow cytometry and immunocytochemistry on both CD44+ sorted and unsorted populations. Senescence-associated beta-galactosidase (SA-{beta}-gal) assays were performed across serial passages (P1-P6). Pluripotency was demonstrated by culture under conditions supporting lineage-specific differentiation. Primary hS/PCs demonstrated consistent expansion and epithelial morphology under serum-free conditions. CD44 expression remained high (>95%) throughout expansion, with negligible detection of CD133 or fibroblast markers, confirming epithelial purity and absence of tumorigenic or stromal contamination. Immunocytochemistry corroborated these expression profiles. SA-{beta}-gal staining revealed only a minor, passage-dependent increase (5-16%) in senescent cells from multiple donors, indicating retention of proliferative potential. Our defined, animal-free culture system supports stable expansion of pure low passage hS/PCs under conditions compatible with good manufacturing practice (GMP).

Deterioration of lung function is a major cause of long-term morbidity after hematopoietic stem cell transplantation (HSCT) and lung transplantation (LT). In both settings, obliterative bronchiolitis represents the most common final pathway, with bronchiolitis obliterans syndrome (BOS), representing its clinical correlate. Understanding of the pathophysiological mechanisms leading to BOS is limited by restricted access to human lung tissue and the imperfect relevance of animal models. We hypothesize that transplantation procedures cause bronchial epithelial damage that promotes the development of BOS. To investigate this, we established ex vivo human airway epithelia (HAE) cultures from bronchial biopsies of HSCT and LT recipients, collected prior to the development of BOS, and compared them with non-transplant controls. HAE from HSCT recipients exhibited reduced tissue differentiation ability, associated with defect in mucociliary clearance and impaired barrier integrity, most markedly in one patient who subsequently developed BOS. In contrast, LT-derived HAE showed normal mucociliary clearance and barrier integrity but displayed increased mucin secretion. Donor and recipient-derived cells were detected in both paraffin-embedded biopsies and reconstructed HAE derived from transplant recipients, demonstrating epithelial chimerism. Our data highlight specific modifications of the airway epithelium after LT and HSCT that may represent a first trigger for subsequent BOS development.

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

Haematopoietic stem cells (HSC), while usually quiescent, can rapidly divide following specific stimuli (mobilization). These HSC can seed additional niches, allowing for the swift generation of essential blood cells. However, studies in mice and humans have clearly demonstrated that cycling bone marrow (BM) HSC (cells in the G1/S/G2/M phases) engraft and reconstitute the haematopoietic system poorly compared with HSC in the G0 phase1. This raises the question why mobilized HSC, immediately following 3 or more cell divisions2, efficiently reconstitute the haematopoietic system. We studied this phenomenon in human HSC using scRNAseq analysis. We found that mobilized HSC rapidly start transcribing genes associated with quiescence, specific for the G0 phase of the cell cycle. We hypothesize that this rapid switch from actively dividing to quiescent cells combined with our extensive RNA expression data will allow us to better define pathways involved in this process.

Transfusion-ready red blood cells can be cultured ex vivo from hematopoietic progenitors. Despite its promising outlook, a cultured transfusion unit cannot be produced at competitive costs. Large media volumes are required to maintain a maximum erythroblast cell density of 1-2.106 cells/mL during the erythroblast proliferation stage. To identify the origin of the cell density limitation, we investigated the cellular support and metabolomic phenotype using different media formulations and feeding regimens. Media that were exposed to an increasing density of erythroblasts (termed spent media) displayed a proportional decrease in erythroblast proliferation support. A 1:1 combination of spent media with fresh media (not previously exposed to the cells) restored growth for all tested conditions. Filtering both fresh and spent media with a 3 kDa cut-off filter, and subsequent recombination of the two fractions, indicated that exhaustion of the small molecular weight fraction (<3 kDa) was primarily responsible for growth limitation. We performed targeted and untargeted metabolomics analysis, for both the intra- and extracellular compartments, following seeding in fresh medium (12, 24, 36 h). We observed degradation of nucleosides, depletion of amino acids, and a decrease in intermediates of the glutathione-ascorbate, {gamma}-glutamyl and cysteine-methionine cycles. The latter compounds suggested an increase in oxidative stress in high density erythroblast cultures. Elimination of nucleosides from the medium led to a lower accumulation of purine salvage intermediates, and a 30% increase in cell productivity. In conclusion, we demonstrate that high-density erythroid cultures are subject to metabolic stress, defining critical constraints for scalable culture expansion.

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.

BackgroundDirect conversion of human somatic cells into functional neurons could offer a faster way to generate patient-specific neurons for use in regenerative medicine, disease modelling, and drug development. Although it has been reported that neuronal direct reprogramming bypasses the intermediate pluripotent state, no reports have included time-lapse experiments, potentially overlooking transient intermediate states. Recent studies have shown that the conversion of human mesenchymal stromal cells (hMSCs) into neuron-like cells involves a transition through a transient intermediate state. Therefore, further research is needed to fully understand the process by which human somatic cells can become neurons without cell division. In this study we investigates whether direct neuronal reprogramming of human bone marrow-derived MSC (hBM-MSCs), dental pulp-derived MSC (hDP-MSCs), and adult human dermal fibroblasts (HDFa), involves a transient intermediate state, and sought to further validate the neuronal identity of hMSC-derived induced neurons. MethodsIn this study, we conducted time-lapse experiments to observe the transformation of hBM-MSCs, hDP-MSCs and HDFa, into neurons using a small-molecule-based direct reprogramming protocol. Cellular and ultrastructural changes were further characterized by confocal and electron microscopy. ResultsDirect conversion of hBM-MSCs, hDP-MSCs and HDFa into neuron-like cells occurred rapidly and in absence of cell division. Time-lapse analyses revealed that reprogramming proceeds through a transient intermediate state characterized by distinct morphological changes and dynamic nuclear remodelling. Furthermore, we found that neuron-like cells derived from hBM-MSCs and hDP-MSCs exhibit neuronal polarization, expressed specific neuronal and synaptic markers, formed interconnected cellular networks, and exhibited functional plasticity, providing further evidence that hMSCs can become functional neurons. ConclusionsThis study provides clear evidence that the direct neuronal reprogramming process involves a transition through an intermediate, transient state. Our findings also provide further evidence that hMSCs can become functional neurons. In summary, our work provides new insights into the direct neuronal reprogramming process, which is essential for advancing both developmental biology and regenerative medicine.

Stem cell therapy represents a promising strategy for the replacement and functional restoration of damaged neural tissue in neurodegenerative conditions. Human dental pulp stem cells (hDPSCs) have emerged as potential candidates for neuroregeneration due to their ease of isolation, neural crest origin, neurotrophic and anti-inflammatory capacity, and demonstrated ability to differentiate in vitro into neuronal-like cells exhibiting electrophysiological activity. Although the immunomodulatory and neuroprotective properties of hDPSCs have been reported in multiple models of brain disease, their capacity to functionally integrate into host neuronal circuits remain poorly understood. In this study, we have grafted green fluorescent protein (GFP)-transduced, neural preconditioned hDPSCs into the CA1 region of the hippocampus of C57BL/6J mice. One month after transplantation, GFP+-hDPSCs survived in the brains of non-immunosuppressed mice and remained localized within the grafted area. Notably, the transplanted cells underwent in situ differentiation and exhibited a neuroblast-like phenotype, characterized by positive doublecortin expression and immature neuronal-like electrophysiological properties, like high membrane input resistance, low capacitance, and the ability to generate single action potentials after stimulation. Together, these findings provide the first evidence that hDPSCs can survive and integrate into the hippocampal network of the mouse brain at one-month post graft, supporting their potential use for future therapeutic applications in acute brain lesions and neurodegenerative disorders.

Currently, global conservation efforts for wildlife focus on a limited number of cell types and species. Although protocols from domestic and non-threatened related species have been applied to endangered species, cryopreservation techniques are species-specific and are constrained by a lack of understanding of reproductive biology in these species. Based on a review of 126 original studies from 27 countries, encompassing 160 species, we assess the current state of cryopreservation in wildlife, including gametes, embryos, somatic cells, and various tissues. Furthermore, we focused on the most homogeneous and frequently studied cell type in wildlife cryobanking: mammals sperm.. A meta-analysis of 27 studies was conducted to examine species-specific and protocol-dependent factors that affect post-thaw sperm quality. Our findings provide quantitative estimates of cryopreservation for various cell types and tissues in wildlife taxa. Furthermore, they serve as a crucial research roadmap, identifying major challenges in cryopreservation and proposing solutions.

The neural crest (NC) is a transient stem cell population which migrates throughout the developing embryo to contribute to diverse tissues dependent on axial origin. For example, cranial NC can give rise to bone and cartilage, while more posterior NC populations give rise to peripheral nervous system and neuroendocrine tissues. Perturbations in neural crest development can lead to severe congenital anomalies and cancers, with over 700 neurocristopathies reported. In humans, early NC development remains poorly understood due to the inaccessibility of tissue samples, thus necessitating the development of in vitro models. Currently, a limited number of NC organoid protocols are available, but these mainly focus on cranial NC and lack relevant tissue architecture. Here, we describe a novel bioengineered pipeline to derive human pluripotent stem cell (hPSC)-derived neuroepithelial organoids, "neurocrestoids" featuring physiologically-relevant tissue architecture. We show that neurocrestoids recapitulate the dynamics of induction, delamination, and migration of human neural crest cells (NCCs), and can be directly compared to murine NC explants for cross-species validation. Organoids express an array of HOX genes indicating the successful generation of cranial, vagal and trunk NCCs. Moreover, we have integrated our neurocrestoids with a customised micropatterned substrate suitable for live visualisation and guided separation of SOX10-positive migratory human NCCs. Our "NCC migration on-chip" are reproducible across multiple hPSC lines and should be scalable for future diagnostic and therapeutic applications, significantly improving our ability to study human NC pathologies.

Rodent models of kidney disease have been widely used to assess the efficacy, safety and mode of action of mesenchymal stromal cells (MSCs) as therapies. However, because kidney disease models, MSC type and the methods used to assess kidney injury tend to differ between research groups, it is difficult to obtain data that are sufficiently robust and reproducible to support clinical translation. We present here for the first time a side-by-side analysis of the performance of human MSCs derived from the most commonly used tissue sources, bone marrow (BM-), adipose- (A-) and umbilical cord (UC-), in a kidney ischaemia reperfusion injury (IRI) model in mice. For each animal, we performed a comprehensive assessment of kidney function and health by longitudinal transdermal measurements of sinistrin clearance, serum biomarker levels at the experimental endpoint, and histopathological scoring of sections from left and right kidneys. Furthermore, we tracked the MSCs by bioluminescence imaging in the injured mice to determine their viability over time and their capacity for homing to the damaged kidneys. Our results reveal that only modest if any beneficial effects of the MSC treatments were detectable on kidney function and histology, irrespective of cell type administered. Furthermore, all three MSC types were sequestered in the lungs without reaching the kidneys, and had completely disappeared within 7 days. Our data suggest that none of the MSC types has the capability to improve renal health following IRI to a meaningful extent, questioning their suitability as a clinical therapy. Significance StatementMSCs have been proposed as efficacious cell therapies in murine models of kidney disease, with potential for clinical translation. We compare efficacy of human MSCs of different tissue origins (adipose, bone marrow and umbilical cord) in a refined mouse model of renal IRI. Only modest if any beneficial effects on kidney function and histology were detectable for all three cell types, and cells did not reach the kidneys but sequestered in the lungs where they died.

Human pluripotent stem cell-derived islets (SC-islets) offer an excellent medium for human pancreatic disease modelling and mechanistic studies into diabetes. While substantial progress has been made in differentiation protocols, their implementation in different laboratories result in variable {beta}-cell proportions with contaminant non-endocrine and proliferative cell types. To date, no facility-level implementation exists for producing SC-islets that can be shipped and benchmarked across multiple sites. Here, we describe the scalable optimisation, standardization, and facility-level implementation of an established human stem cell differentiation strategy that consistently results in a high proportion of {beta}-cells, with up to 75% of cells co-expressing C-peptide and the pancreatic endocrine marker, ISL1. Functionally, SC-islets exhibit glucose-responsive calcium influx and insulin secretion, recapitulating key physiological {beta}-cell functions. Single-cell transcriptomic profiling reveals a simplified endocrine landscape dominated by {beta}-cells, with a striking transcriptional similarity to human primary {beta}-cells (Pearsons r2[~]0.9). We observe smaller fractions of - and enterochromaffin-like cells with very low levels of poly-hormonal or proliferating cell types (<3%). Taken together, we provide a well-defined, reproducible and accessible in vitro SC-islet platform benchmarked for functionality at multiple recipient sites.

Background Donation after circulatory death (DCD) is an increasingly accepted strategy to expand the adult heart donor pool, but its use in children remains limited and incompletely characterized. We compared national characteristics and post-transplant outcomes of pediatric DCD versus donation after brain death (DBD) heart transplantation. Methods We performed a retrospective cohort study of the Organ Procurement and Transplantation Network (OPTN) registry, including patients younger than 18 years who underwent primary isolated heart transplantation between January 1993 and March 2025. Recipients were stratified by donor type (DCD vs DBD). Continuous variables were compared with the Mann Whitney U test and categorical variables with the Fisher exact test. Survival was estimated by the Kaplan Meier method and compared using the log-rank test and Cox proportional hazards regression. Results Of 10,671 pediatric heart transplant recipients, 33 (approximately 0.3%) received DCD allografts. The first DCD transplant was recorded in 2004, with a marked increase in 2023 to 2024. Compared with DBD recipients, DCD recipients were more frequently infants (<1 year, 51.5% vs 28.4%) and more often had congenital heart disease (69.7% vs 47.6%; P=0.033); DCD donors were younger (median 0 vs 6 years; P=0.038) and more frequently died of anoxia (72.7% vs 37.0%; P<0.001). Donor and recipient left ventricular mass were lower in the DCD group (P<0.05), but predicted left ventricular mass matching was similar. DCD recipients had longer hospital stays (median 31.5 vs 19 days; P=0.023); rates of treated rejection, dialysis, stroke, and pacemaker implantation were comparable. Early survival did not differ (30-day, 90-day, and 1-year), and Kaplan Meier survival through 5 years was not significantly different (hazard ratio 1.17; 95% CI 0.49 to 2.81; log-rank P=0.73). More than 90% of DCD transplants were performed in four UNOS regions (11, 4, 5, and 8). Conclusions In this national analysis, pediatric DCD heart transplantation was uncommon but expanding rapidly, concentrated in a few regions, and used preferentially in infants and children with congenital heart disease. Early post-transplant outcomes were not significantly different from DBD, supporting cautious expansion of DCD as a means of enlarging the pediatric donor pool. The small number of DCD recipients and limited followup warrant confirmation in larger, longer-term studies. Keywords: pediatric heart transplantation; donation after circulatory death; donor pool; congenital heart disease; OPTN registry; organ allocation.

The rapid rise of stem cell-based human embryo models has reignited interest in studying early human development while offering a promising platform to de-risk drugs. Among these, three-dimensional human gastruloids provide a tractable system to model symmetry breaking, germ layer specification and axial organization. However, existing gastruloid protocols remain expensive, specialized, variable and evaluated in a limited number of human pluripotent stem cell (hPSC) lines, restricting broader adoption. Here, we present a simple, robust, standardized gastruloid protocol achieving greater than 90% elongation efficiency with low inter- and intra-experimental variability, developed primarily in BJNhem20, a well-characterized Indian-origin human embryonic stem cell line. Further, we show that the protocol is applicable in a diverse set of hPSC lines. Using a TBXT (Brachyury)-GFP reporter in BJNhem20, we optimized cell seeding density, induction medium and Wnt activation strength, guided by real-time, quantitative assessment of mesoderm induction and symmetry breaking, allowing precise titration of CHIR99021. Comparative testing identified an in-house "Essential 6" medium formulation as the most consistent condition for robust TBXT induction. Optimization of aggregation density produced reproducible gastruloids with polarized TBXT expression and consistent axial elongation, within 72 hours. Single-cell RNA sequencing of individual gastruloids confirmed high transcriptional reproducibility and conserved lineage clusters, aligned with developmental trajectories. Cell line-specific CHIR99021 titration was sufficient to successfully transfer the optimized protocol to two additional lines, BJNhem19 and RUES2-GLR. This simplified and robust protocol reduces costs and improves accessibility, enabling broader application of stem cell-based human embryo models.

Zebrafish combines the power of genetics and unparalleled in vivo imaging for investigating the dynamics of vertebrate hematopoietic development. Across species, the transcription factor Runx1 is essential for definitive hematopoiesis. We generated a zebrafish runx1-2A-creERT2 CRISPR knock-in for tamoxifen-regulated Cre recombinase Runx1 lineage tracing and characterized its activity using the ubi:Switch recombinase-dependent fluorescence reporter, microscopic live imaging and flow cytometry. Tamoxifen treatment beginning at gastrula stage labeled all expected Runx1 lineages in the early embryo, including neuroectodermal olfactory placode and Rohan-Beard neurons, primitive hematopoietic blood cells, and nascent hematopoietic stem and progenitor cells (HSPCs) in the dorsal aorta. Runx1 HSPCs colonized the larval caudal hematopoietic tissue and thymus from three to five days of development. Timed tamoxifen induction of Cre activity allowed separation of Runx1 primitive hematopoiesis from definitive HSPC emergence and larval stem cell niche colonization. Flow cytometry of kidney marrow and peripheral blood from adults treated with tamoxifen at gastrula stage revealed Runx1 embryonic hematopoietic cells contributed to adult hematopoietic precursors, myeloid, lymphoid, and peripheral blood lineages. Labeling of all blood lineages was also effective by tamoxifen treatment of 5-month-old adults. The zebrafish runx1-2A-creERT2 line provides a powerful tool for precise spatial and temporal analysis of Runx1 progenitor mechanisms in developmental and adult hematopoiesis. Key PointsO_LIzebrafish endogenous runx1-2A-creERT2 provides inducible Cre recombinase genetic analysis in all runx1 neuromesodermal and blood lineages C_LIO_LIzebrafish runx1-2A-creERT2 line enables in vivo spatial and temporal analysis of embryonic and adult hematopoiesis C_LI