Stem Cell Reports

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 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.

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

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

Rejuvenating aging human skin is a major therapeutic goal, but objective, quantitative measures of intrinsic aging are limited. We performed a cross-sectional histological study of UV-protected buttock and abdominal skin in adults spanning multiple decades of life to identify features that reliably track age. Epidermal thickness measured between rete ridges was unchanged, but rete ridge size declined linearly with age: ridges became shorter and thinner in both sites, though rete ridge number decreased only in the abdomen. Consistent with these structural changes, proliferative cells (Ki67+) per ridge and expression of integrin {beta}4 (ITGB4), a putative stem-cell marker, were reduced in aged skin. We combined these biomarkers into a predictive model that estimated skin age more accurately than any single marker. To test whether the model detects longitudinal change, we analyzed aged abdominal skin before and after xenografting onto young or aged mice, a procedure previously reported to rejuvenate human skin in young but not aged recipient mice. Both individual biomarkers and the imaging model indicated rejuvenation regardless of host age; however, notably, engraftment efficiency was lower in aged hosts, with surviving grafts showing younger histological phenotypes. These results provide quantitative criteria for assessing intrinsic skin aging and suggest that the process of engraftment itself is sufficient to induce rejuvenation-like changes.

Artery endothelial cells (ECs) arise through different pathways, including differentiation from mesodermal cells (vasculogenesis) or from already established vein or capillary plexus ECs (angiogenesis), the latter being most common during embryonic development and regeneration. Understanding the vein-to-artery (v2a) transition could improve revascularization therapies, but progress is limited by a lack of human models. Here, we develop a human pluripotent stem cell (hPSC) differentiation protocol that models the v2a EC conversion. Comparing v2a and mesoderm-to-artery (m2a) transcriptomes with publicly available single cell RNA sequencing (scRNA-seq) data from human embryos showed they reflected angiogenesis- and vasculogenesis-derived artery ECs, respectively. This reductionist system revealed that VEGF activation alongside PI3K inhibition was sufficient for vein ECs to acquire arterial identity within 48 hours. We model a critical step in vascular development and define the minimal signals required for artery differentiation from veins, providing a framework to promote this conversion in revascularization or therapeutic contexts.

The accelerating biodiversity crisis, compounded by emerging infectious diseases like African swine fever (ASF), necessitate innovative conservation and disease management. ASF susceptibility varies wildly across species, from near-100% mortality in Asian suids to asymptomatic carriage in African forest species. We report the first successful derivation of integration-free induced pluripotent stem cells (iPSCs) from four phylogenetically distinct species: wild boar (Sus scrofa), Bornean bearded pig (Sus barbatus), Babirusa (Babyrousa babyrussa), and Red river hog (Potamochoerus porcus). Using Sendai virus-mediated reprogramming, we achieved efficiencies between 0.003% and 0.26%. These iPSCs were successfully differentiated into CD14CD11b monocytes - the primary target cells for the ASF virus - establishing a renewable, comparative research platform. This system enables host-pathogen studies previously hindered by ethical and logistical constraints of wildlife sampling. Beyond disease research, these iPSC lines serve as vital genetic repositories for endangered suids. Our methodology provides a replicable framework for extending stem cell technology to other conservation-priority taxa, demonstrating how high-tech cellular tools can advance both fundamental research and biodiversity preservation against emerging pathogen threats.

Genetically diverse panels of human pluripotent stem cells enable genetic dissection of cellular phenotypes, but comparable induced pluripotent stem cell (iPSC) resources in model organisms remain limited. We generated a panel of iPSCs from the Diversity Outbred (DO) mouse population and established 288 genetically unique lines that retain the allele frequency distribution, heterozygosity, and low population structure of the source population. The lines exhibit consistent growth, pluripotent gene expression profiles, and capacity to form embryoid bodies. Transcriptomic profiling of the lines revealed significant variation in gene expression driven in part by genetic background. We used expression quantitative trait locus (eQTL) mapping to identify over 10,000 regulatory loci that influence gene expression variation, including multiple distal eQTL hotspots that are key gene regulatory hubs and are shared with DO embryonic stem cells (ESCs). The largest hotspot, mediated by Lifr, showed a shift in founder allele effects relative to ESCs, consistent with differences in cellular state and culture conditions. These results establish the DO iPSC panel as a genetically diverse, publicly accessible platform derived from a laboratory mouse genetic reference population, enabling integration of in vitro cellular phenotypes with in vivo traits within a closed, outbred population.

Human cardiac microtissues are a promising model to study cardiac biology and disease, but their application is constrained by therapeutic remodelling strategies and limited knowledge of their functional protein expression profiles. Here, we define the use of human cardiac microtissue (hCMT) model generated by assembling iPSC-derived endothelial cells, cardiac fibroblasts, and cardiomyocytes to model ischemia-reperfusion injury (IRI) through a model of hypoxia and reoxygenation and nanovesicle-mediated functional remodelling. Engineered nanovesicles (NVs), generated directly from human stem cells, have been shown to influence cardiac tissue and cell repair, and provide a platform for scalable and reproducible cell free-mediated therapy. We show the functional regulation of the hCMT model and define that administration of NVs (from human induced pluripotent stem cell origin) during reoxygenation significantly increase cardiomyocyte survival and preserve contractility function (contractile duration, relaxation time, relaxation:contraction velocity). Quantitative proteomics was applied to decipher the cell proteome dynamics and molecular mechanisms of IRI in our in vitro model following NV treatment, linked with networks associated with cell survival, energy production, and stress response regulation. Conserved proteome dynamics in NVs from different iPSC source reveal conserved upregulation of cellular protein networks involved in tissue repair (HSP70, CYFIP1), cardiac function (XIRP1, SLMAP, MYH6, CTNNA1, NDUFS2, GPD2), response to stress (CANX, PDCD6,), pro-survival (MDH2, LRPPRC, NIPSNAP1) and pro-angiogenic (FARSA, ECE1, RRAS) relative to vehicle treatments in context of IRI. Finally, we show that NVs also mediate differential remodelling in hCMT in response to IRI based on their cell origin, including altered wound healing and tissue repair response. Our findings provide an advanced human stem cell-based platform to understand underlying mechanisms of IRI and assess cell-free therapeutic cardioprotective strategies. SummaryAdvanced human stem cell-based platform provides a cardiac microtissue model to understand nanovesicle-based function and proteome remodelling, with potential applications for disease modelling and therapeutic intervention.

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.

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.

Cryopreservation offers an option for long-term storage and global distribution of complex in vitro models, yet protocols for multicellular microphysiolgocial systems (MPS) such as brain organoids/spheroids remain limited. Here, we systematically compared three commercially available cryopreservation (mFreSR, CryoStorCS10, and 3dGRO) and two freezing time points, and established a robust workflow for freezing and recovering brain organoids. After defrosting, we assessed morphology and metabolic activity. We also evaluated electrophysiology, calcium transients, and neurite outgrowth. In addition, we measured astrocyte migration, apoptosis, mitochondrial integrity, microglia survival, and neural marker expression. We found that organoids require a 4-week recovery period to regain structural and functional stability. Although organoids frozen at week 6 showed higher metabolic activity after recovery, organoids cryopreserved at week 2 had clearly better functional outcomes. They exhibited stronger spontaneous network firing and maintained calcium transients. Finally, incorporated microglia-like cells survived the freezing and displayed comparable morphology to unfrozen controls. Across the endpoints measured here, 3dGRO showed the most favorable overall performance; formal ranking across media awaits harmonized normalization, single-organoid electrophysiology, and prespecified QC thresholds. Together, these results define a practical and reproducible cryopreservation strategy that preserves key physiological features of brain organoids and supports the establishment of ready-to-use organoid banks. The ability to reliably store and distribute complex brain-like tissues represents an essential step toward global standardization, scalable experimentation, and wider adoption of human-relevant microphysiological systems. Together, these results demonstrate recovery of key physiological features in the subset of organoids that remain viable after thaw and support the feasibility of brain organoid banking.

Bioengineers strive to recreate in vivo microenvironments in vitro to reduce our use of animal models and provide insights into human biology. While liver models show promise, sex differences in liver biology remain largely neglected in preclinical studies. Despite the 2014 EU mandate for the inclusion of women in clinical trials, decoupling of research data by sex is historically rare, with only 11% of papers disaggregating data by sex. This gap contributes to women being more susceptible to drug-induced liver injury (DILI) and being underserved in drug development, as well as to costly drug attrition levels. Here we present a novel approach to modelling sex differences in vitro. Human induced pluripotent stem cells (iPSCs) from both male (XY) and female (XX) donors, were differentiated into hepatocyte liver spheroids and exposed to in vivo-mimicking levels of testosterone, progesterone, and oestrogen in high-throughput microwell format. We successfully recapitulated sex-specific metabolic profiles and demonstrated significant differences in CYP1A2 and CYP3A4 drug metabolism and gene expression patterns consistent with reported in vivo observations, without compromising cell viability. These findings validate the utility of sex-differentiated microenvironments in early-stage research, offering a pathway to refine animal and clinical trials and improve therapeutic outcomes for all sexes.

Oligodendrocyte-enriched cortical organoids (OCOs) are a powerful platform for modeling oligodendrogenesis in a human cellular context. However, neuronal activity is impaired in conventional culture media, limiting assessment of neuronal function in conjunction with oligodendrocyte biology. To address this, we used a modified BrainPhys medium termed neuronal activity medium (NAM) and defined the optimal developmental window for NAM exposure to generate OCOs with robust neuronal activity (NAM-OCOs). Stage-specific exposure to NAM, prior to oligodendrocyte expansion, leads to enhanced structural maturation, as evidenced by increased organoid size, heightened synaptogenesis, and upregulation of transcripts associated with neuronal complexity. Further, NAM-OCOs display increased cellular heterogeneity, including greater representation of GABAergic interneurons while preserving oligodendrocyte development and maturation. Altogether, our studies demonstrate that stage-specific exposure to an activity-permissive environment enhances neuronal activity, establishing an OCO model which integrates neuronal activity with oligodendrocyte development and maturation. HighlightsO_LIIncreased neuronal activity in oligodendrocyte-enriched cortical organoids (OCOs) C_LIO_LIStage-specific Neuronal Activity Medium (NAM) optimizes activity C_LIO_LINAM-OCOs display increased cellular heterogeneity and neuronal maturation C_LIO_LIOligodendrogenesis is preserved in NAM-OCOs C_LI eTOC blurbIn this article, Chung et al enhance neuronal activity in oligodendrocyte-enriched cortical organoids (OCOs) through stage-specific exposure to Neuronal Activity Medium (NAM). OCOs exposed to NAM display elevated cellular heterogeneity, structural maturation, and synaptogenesis, while preserving oligodendrocyte development and maturation. These results establish an increasingly comprehensive OCO model for studying neuronal function and oligodendrogenesis.

Some long non-coding RNAs (lncRNAs) are known to regulate gene expression. However, the underlying temporal dynamics of lncRNAs influencing gene and epigenetic regulation and mechanisms of lncRNA regulation in trans are less understood. To investigate this, we genetically engineered 17 doxycycline-inducible lncRNA transgenes for ectopic expression at the H11 safe harbor locus in human pluripotent stem cells (hiPSCs), and we generated high-density temporal RNA-seq and ATAC-seq profiles. Most lncRNA transgenes were induced at 2 hours and maintained expression through the 96-hour time course. Surprisingly, when we sought to identify gene expression changes due to the lncRNAs, we found that the global transcriptional landscape was dominated by a strong systemic response triggered by doxycycline exposure. We rigorously defined this cohort of genes as a Doxycycline-Responsive Gene Signature (DRGS). The DRGS was also present in at least 28 public datasets from dox-inducible transgene studies involving diverse cell types. Next, we determined which lncRNAs exhibited trans-regulatory events. We identified DANCR, FENDRR, LINC00667, LINC00847, LNCPRESS1, and PNKY as lncRNAs that regulate specific transcript expression in trans. The downstream target genes encoded 53 mRNAs and 10 lncRNAs. None of the target lncRNAs altered gene expression proximal to their own loci (i.e., triggering secondary cis-effects). Surprisingly, the target genes of LINC00847 (transcribed from chromosome 22) were substantially enriched on chromosome 19, with a preponderance of target genes encoding RNA metabolism and RNA splicing factors. Collectively, our study provides a resource to discern artifacts in the doxycycline-inducible system and identifies temporally regulated targets of 6 lncRNAs for future mechanistic studies.

Manufacturing red blood cells (RBCs) from human induced pluripotent stem cells (iPSCs) can improve our understanding of embryonic erythropoiesis, foster innovative treatments for RBC-related diseases, and ultimately address clinical blood supply shortages. However, existing systems face low efficiency, enucleation failure, and uncertainty about the develop-mental wave of cultured RBCs. We successfully used self-organized hemanoids to improve iPSC-derived RBC generation. Based on the hypothesis that cellular interactions and 3D organization promote hematopoietic cell fate, we aimed to thoroughly characterize hemanoids. We visualized the spatiotemporal emergence of hematopoiesis by generating a CD43-GFP reporter iPSC line. Imaging and spatial transcriptomics analysis provided de-tailed insight into the hemanoid architecture, identifying stromal cells and hepatoblasts as potential erythropoiesis-supportive elements. The developmental stage mirrors extraembryonic hematopoiesis. Given the difficulties of accessing these early stages in vivo, our system offers a platform not only for further clinical translation but also for exploring hu-man embryonic blood wave dynamics.

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.

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.

Mesenchymal stromal cells exhibit potent immunomodulatory properties and are under active investigation for the treatment of immune-mediated disorders. However, their clinical translation is hindered by the lack of standardized potency assays. Here, we established a reproducible mixed lymphocyte reaction platform by systematically optimizing peripheral blood mononuclear cell donor composition, culture conditions, and co-culture ratios to define a robust activation window. Using this system, we compared bone marrow and adipose derived Mesenchymal stromal cells across independent donor batches. Both sources effectively suppressed T cell proliferation, with the adipocyte derived source consistently showing greater inhibitory activity, while a conserved lower threshold of suppression was observed across both sources. Mesenchymal stromal cells reduced early (CD25+) and late (CD25+HLA-DR+) T cell activation, with downregulation of these markers emerging as a sensitive correlate of functional potency. Notably, bone marrow derived mesenchymal stromal cells exerted stronger suppression on late-stage activation and preferentially suppressed CD8+ T cell expansion. Mechanistically, this immunosuppression was associated with modulation of the PD-1 pathway, characterized by decreased soluble PD-1, increased PD-L1, and induction of mesenchymal stromal cells derived PD-L2. PD-L2 levels inversely correlated with T cell proliferation, identifying a PD-1/PD-L2 regulatory axis linked to the cells potency. These findings define a standardized and mechanistically informed potency assay framework for assessing mesenchymal stromal cell immunomodulatory function.

The ability to generate functional B cells from human pluripotent stem cells (hPSCs) would open new opportunities to develop novel B cell-based therapies to treat a range of human diseases and disorders. Towards this goal, we established a protocol that promotes the efficient development of B lineage cells from definitive hematopoietic progenitors generated from different hPSC lines. Flow cytometric and multi-omic scRNA-seq analyses revealed that B cell development from hPSCs transitions through the well-established pro-B, pre-B and naive B cell stages, accurately recapitulating B lymphopoiesis in the human adult bone marrow. Importantly, the naive B cells generated with this approach could be induced to mature into plasma cells that secrete antibodies and undergo class switching. Analyses of signaling pathways that regulate B lymphopoiesis in these cultures uncovered a potent inhibitory effect of IL-7 on functional IgH rearrangement, resulting in the development of abnormal cells that failed to undergo pre-B cell maturation. Finally, analysis of the different hPSC-derived hematopoietic programs revealed that both definitive and yolk sac progenitors display B cell potential, indicating that there are distinct developmental sources of human B lineage cells. Taken together, these findings demonstrate the efficient generation of B cells from hPSCs and, in doing so, provide a system for further investigating the earliest stages of human B lymphopoiesis and a source of appropriately staged plasma cells for future therapeutic applications.