Cell Proliferation

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

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.

De novo vessel formation (vasculogenesis) in vitro is a key step in tissue engineering to preserve tissue viability for long-term assays and testing therapeutic agents. However, in vitro vasculogenesis is often unreliable due to differences in vascular-supporting cells, including endothelial cells and stromal cells such as smooth muscle cells (SMCs) and fibroblasts. Here, we developed a robust co-culture system of HUVECs and SMCs to generate stable vascular networks capable of maintaining tissue viability over extended periods. Given that SMC plasticity is a major limitation in supporting endothelial network formation, we systematically evaluated the effects of passage number, confluency, and freezing on primary SMC function. To overcome this limitation, we generated immortalized supportive SMCs, which preserved their vasculogenic gene program and functional capacity even at high passage. In addition, we identified and validated key genes associated with endothelial support, including CD248, C3, and FBLN1, all essential for vasculogenesis. Immortalized SMCs consistently maintained expression of these genes and supported robust vessel formation under variable culture conditions. Collectively, this study demonstrates that immortalized SMCs provide a stable, reproducible platform for endothelial-SMC co-cultures, enabling long-term vascularized tumor models suitable for functional studies and therapeutic screening.

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.

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

Bicuspid aortic valve (BAV) is one of the most common congenital heart defects but its genetic basis remains incompletely defined. Extracellular matrix components play key roles in outflow tract (OFT) and valve development, but their contribution to BAV is not fully established. Following the analysis of a cohort of BAV patients, we identified a family harbouring a rare human ELASTIN (ELN) variant (p.Gln691X). To assess its pathogenicity, we generated a zebrafish elna/b double knockout (KO) using an RNAless CRISPR Cas9 strategy to avoid genetic compensation. This mutant exhibited cardiovascular defects including OFT anomalies, reduced stroke volume and dysmorphic aortic valves, highlighting Elastins critical role in cardiac development. We then used this model to test the ELN variant identified in the BAV family. We found that wild-type ELN mRNA was able to restore normal cardiac function and morphology, whereas the variant ELN mRNA failed to do so. This study establishes a robust in vivo model to assess ELN variant pathogenicity and provides evidence linking ELASTIN to BAV, opening new avenues for uncovering the genetic mechanisms underlying BAV.

Human papillomavirus 18 (HPV18) preferentially infects cervical stem cell-like cells and is strongly associated with adenocarcinoma. However, the mechanisms underlying differentiation into cervical adenocarcinoma remain unclear due to the lack of appropriate experimental models. We aimed to establish a model of HPV18-associated cervical adenocarcinoma and elucidate its molecular and cellular differentiation mechanisms. HPV18 E6/E7 were introduced into induced pluripotent stem cell-derived reserve cell-like cells (iRCs) to generate tumor models. Spatial transcriptomics and single-cell multi-omics analyses were performed to integrate histological and molecular data. A distinct component (Gland_A) exhibited morphological and immunohistochemical features of cervical adenocarcinoma and was efficiently induced in iRC-18 tumors. Gland_A showed increased chromatin accessibility and elevated expression of FOXA1, FOXA2, and ALDH1A1. Analysis of clinical samples confirmed enrichment of ALDH1A1 in HPV-associated adenocarcinomas. This model recapitulates key features of HPV18-associated cervical adenocarcinoma and provides insights into its differentiation mechanisms.

Given the critical role of progenitor cells staying within the eye field transcription factor (EFTF) signaling niche for normal eye development, we hypothesized that retinal progenitor cells (RPCs) differentiate within their initial region of inception during eye development. To investigate this, we utilized EosFP, a photoconvertible protein, as a lineage tracer in the model organism Xenopus laevis. By employing confocal laser microscopy for photoconversion, we labeled cells within elongated rectangular regions that encompassed both the eye field and the adjacent tissues. In a separate set of embryos, we identified which portions of these rectangular regions harbored cells destined to become part of the mature eye versus those that would form the surrounding tissues, tracing their development from stage 15 to stage 35. This allowed us to create a fate map of the stage 15 embryo using EosFP to accurately locate and label the eye field to address our hypothesis. With the eye field delineated using our lineage tracer, we further employed EosFP to label RPCs within individual quadrants of the developing eye. Tracking these RPCs from stage 15 to stage 35, we observed the retinal cells organizing into three principal layers of cell bodies, mirroring the layered neuroanatomy characteristic of the mature retina. We observed the red-labeled RPCs proliferated but remained predominantly within their quadrant of inception, with no dispersion into other, unlabeled quadrants of the eye by stage 35. These findings corroborate our hypothesis that RPCs undergo differentiation within their initial locations in the eye field. Our study illuminates the cellular dynamics of eye development in Xenopus laevis and introduces a novel method for lineage tracing of stem cell populations during embryonic development.

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.

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.

Inherited retinal diseases (IRDs) are a heterogeneous group of genetic disorders that cause progressive vision loss. A subset of IRDs is associated with ubiquitously expressed genes involved in fundamental cellular processes, often resulting in multisystem disease. Among these is Zellweger spectrum disorder (ZSD), caused by pathogenic variants in PEX genes required for peroxisome biogenesis and function. There are no proven targeted disease-modifying treatments for ZSD, and it is unclear whether localized restoration of peroxisome function is sufficient to mitigate retinal degeneration. We previously demonstrated that HsPEX1 retinal gene augmentation therapy in a mouse model of mild ZSD homozygous for the murine equivalent (PEX1-p.[Gly844Asp]) of the most common deleterious allele in patients (PEX1-c.[2528G>A], PEX1-p.[Gly843Asp]), improved retinal electrophysiological response. Here, we present a comprehensive, dose-range evaluation of a re-designed, clinically relevant AAV8-delivered HsPEX1 subretinal gene therapy, employing expanded outcome measures. We observed a marked improvement in functional vision, retinal response, photoreceptor structure, retinal pigment epithelium integrity, subretinal inflammation, and peroxisomal metabolites, durable to the endpoint of 6 months post single subretinal injection. These studies provide preclinical proof-of-concept that localized retinal gene replacement can mitigate vision loss in peroxisome-mediated IRD.

Xenopus laevis has recently emerged as a vital model for studying functional eye regrowth in pre-metamorphic tadpoles. Following eye removal surgery, tailbud embryos have been shown to regenerate a functionally complete eye within a 3-5 day period. While current studies have primarily focused on the signaling mechanisms required for this rapid regeneration, less is known about the specific stem cell populations and modes of regeneration employed by the embryo. In both the adult and tadpole, eye tissue regeneration can be facilitated through a combination of a pre-existing stem cell niche and the transdifferentiation of cells surrounding retinal or lens injuries, depending on the extent of the tissue removal. Notably, in the Xenopus eye regrowth assay, surgeries typically leave behind approximately 15% of the ocular tissue, indicating a post-surgical stem cell niche with potential for regeneration. In this study, we explored the hypothesis that a residual retinal progenitor cell (RPC) niche is critical for the rapid eye regrowth observed in Xenopus tadpoles. By utilizing a photoconvertible protein, EosFP, which changes permanently from green to red fluorescence, we selectively marked retinal progenitor cells (RPCs) in the presumptive eye area with red fluorescence. We then carefully preserved a small population of these red-labeled RPCs within the post-surgical wound. This progenitor cell niche, comprising not only the red-labeled RPCs but also the surrounding cells, creates a unique signaling environment. This specialized microenvironment is crucial, as it may provide specific signals that dictate the developmental outcomes of the RPCs, effectively controlling their fate. Observations made throughout the regrowth process revealed that the eye predominantly regrew from this red-labeled RPC niche within three days, with all retinal layers comprising red-labeled cells. The regrown lens was observed to be composed of a mix of both cells outside the RPC lineage and RPC progeny. Of interest, we observed cells of the closing optic fissure and ventral retina incorporate progeny from cells outside the labeled RPC lineage. These findings support the notion that the primary mode of regeneration in pre-metamorphic Xenopus eye regrowth involves the use of a pre-existing stem cell niche, and may also involve transdifferentiation, thus providing new insights into the mechanisms of embryonic eye regrowth in Xenopus laevis.

Liver sinusoidal endothelial cells (LSEC) rapidly dedifferentiate in 2D-monoculture, losing their high endocytic activity and characteristic morphology, limiting their use in mechanistic studies. We established and validated culture conditions that preserve LSEC endocytic capacity for at least 10 days, enabling efficient in vitro siRNA-mediated gene silencing. Mouse LSEC were cultured in 5% oxygen, growth media partially exchanged daily and assessed for cell viability, endocytic capacity, morphology and ultrastructure. Despite typical culture-induced defenestration, the cells showed high viability and efficient endocytosis via scavenger-receptors. This allowed for siRNA-mediated mannose receptor knockdown exemplified by 96% and 76% reduction in Mrc1 mRNA and protein expression at 72h (validated by qPCR and Western blot), with functional assays confirming decreased mannose-receptor-mediated endocytosis. Extended maintenance of LSEC viability and functions, previously restricted to complex co-culture systems, provide a practical platform for investigating LSEC-specific molecular mechanisms and hepatic sinusoid physiology.

Mixed-lineage leukemia translocated to 3 (MLLT3) is vital for maintaining the stemness of hematopoietic stem cells. Loss of MLLT3 in megakaryocyte (MK)-erythrocyte progenitor (MEP) cells leads to its differentiation into MKs. Despite its significance in stemness, the regulatory mechanism of MLLT3 during differentiation remains elusive. In this study, we investigate the regulatory role of histone deacetylase 6 (HDAC6) in modulating MLLT3 levels via heat shock protein 90 (Hsp90) activation during myeloid lineage differentiation into MKs, monocytes, and macrophages. We found that HDAC6 activates Hsp90 through deacetylation, enabling Hsp90 to retain MLLT3 in the cytoplasm where protein kinase C (PKC) phosphorylates MLLT3 at serine residues; leading to loss of MLLT3 during MK and macrophage differentiation but not during monocyte differentiation. This research provides valuable insights into the regulatory mechanisms underlying myeloid lineage commitment and opens new avenues for future investigations into stem cell biology and therapeutic applications.

Background and PurposeCardiotoxicity is a major cause for drug failure throughout the drug development process, with particular concern for action potential prolongation and arrhythmia. Hence, such liabilities are heavily considered during the early phases of drug design to pre vent dangerous compounds from progressing. New approach methodologies (NAMs) that efficiently examine this risk early in the discovery pipeline should help streamline drug development programs. We developed a cardiac NAM, a 384-well open bath platform consisting of cardiac tissue derived from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, enabling high-throughput drug screening while maintaining the structural and functional complexity of 3D cardiac micromuscles. MethodsWe dramatically increased throughput without compromising physiological relevance provided by the 3D micromuscle structure. Our 384-well open bath high-throughput platform allowed evaluation of multiple compounds at a time, enabling us to study the CiPA (comprehensive in vitro proarrhythmia assay) drug panel for proarrhythmia screening. We obtained phenotypic fingerprints of all 28 compounds (9 low, 11 intermediate, and 8 high arrhythmia risk; https://cipaproject.org) in dose-escalation studies around their respective clinical concentrations. The analysis was augmented with an in silico pipeline that used phenotypic biomarkers to invert data into a mathematical model of cellular currents to infer which ion channels were affected upon drug exposure, and then trained a ML model to predict channel block. Results and ConclusionsWe found accurate detection of arrhythmic potential for most of the compounds, and the in silico model inversions were consistent with published values of compound channel block. All the high risk compounds showed action potential duration (APD) prolongation coupled with either action potential abnormalities, early afterdepolarizations (EADs), or beat cessation. For the intermediate risk group, 9 out of 11 compounds caused APD prolongation alone or in combination with EADs while 2 others showed either beat cessation or beat rate change. Augmentation of APD analysis with detailed biophysical modeling and ML tools provided meaningful insight into the mechanisms involved in APD changes. Overall, our cardiac NAM allowed for fast and relevant screening for mechanistic understanding of APD prolongation and proarrhythmic activity, at massively increased throughput compared to other 3D micromuscle models. SummaryCardiotoxicity testing is critical in drug development to prevent arrhythmogenic side effects. Current stringent regulations have greatly reduced market withdrawals; however, these strict evaluations often lead to costly late-stage failures and loss of promising candidates as false positives. We developed a cardiac new approach methodology (NAM), a 384-well open bath cardiac micromuscle platform created from hiPSC-derived cardiomyocytes, enabling high-throughput drug screening while maintaining the structural and functional complexity of 3D cardiac micromuscles. Using the comprehensive in vitro proarrhythmia assay (CiPA) drug panel, we validated the system to accurately detect proarrhythmic potential. Our assay provided phenotypic fingerprints based on mechanical and electrophysiological biomarkers. Integration with computational modeling offered insights into multi-ion channel effects (MICE). Particularly, we identified sodium channel block contributions as a significant factor for poor risk prediction based on traditional parameters. The combined experimental and computational platform can enhance early drug screening, thereby reducing late-stage failures and promoting the progression of low-risk compounds with complex electrophysiological profiles.

Precision medicine aims to advance our ability from a "one-size-fits-all" approach to personalized and predictive healthcare across diverse populations. It promotes integration of multi-omics and phenotypic data to understand disease mechanisms and discover novel biomarkers and risk factors, which could be used to predict and prevent critical diseases in individual patients across diverse populations. The potential implications of precision medicine approach can accelerate our ability to classify patients at higher risk of developing critical diseases, improve diagnostic capabilities, develop deeper understanding of individual risk, investigate racial differences and demographic characteristics, and find relationships between genetic variants, expressions, and diseases. This study focuses on implementing an innovative and data driven framework of translational bioinformatics and Machine Learning (ML) techniques to analyze multi-omics, including RNA-seq and Whole-Genome Sequencing (WGS) data, generated using blood samples of randomly consented patients. First, we utilized bioinformatics pipelines to identify differentially expressed genes and their pathogenic and likely pathogenic variants for the downstream data analysis, annotation, and visualization. Then, applied a nexus of ML models for multi-omics biomarker discovery, disease prediction, density-based clustering, single-patient profiling, and pathogenicity classification. WGS data analysis supported the exploration of genetic variation and diversity among patients to identify known and novel biomarkers, whereas RNA-seq data analysis improved our understanding of functional and biological pathways that underlying disease states. We classified and clustered pathogenic variants and expressions across various genes and discovered numerous diseases leading risk factors. Our results include gene-disease associations and captured common pathways across the broader population, demonstrating a level of sensitivity and accuracy that has broad clinical implications. We validated our results through clinical records, and state of the science literature. This study delves into the strengths of multi-omics data integration and capabilities of ML application in genetically diverse and complex patient cohorts. Our approach has the potential to elucidate complex gene-disease interactions for genetically diverse populations, which can support earlier diagnoses for patients in many disease realms.

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.

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) deficiency is a rare genetic cause of neurodevelopmental disorders (NDDs) lacking targeted therapies. Here, we developed a transcriptomic-guided compound prioritization pipeline using Connectivity Map (CMap) analysis on multi-model transcriptomic signatures from HNRNPU-deficient human cells and mouse models. Ten compounds were selected through manual curation and functionally screened in patient-derived HNRNPU-deficient neuroepithelial stem (NES) cells with earlier observed cellular phenotypes. Two of the compounds, AS601245 and Lenalidomide, significantly reduced the elevated neural progenitor population during differentiation, and their combination further decreased primary cilia incidence, indicating partial rescue of the patient-specific cellular phenotypes. To understand the mechanisms underlying the partial rescue, we employed proteome integral solubility alteration (PISA) and expression proteomics. PISA assay identified TMEM150C and GSK3A as proximal targets of combined treatment. Additionally, we observed reversal of multiple biological pathways including downregulation of Wnt signalling and upregulation of mitochondrial pathways and transmembrane proteins. Altogether, we established a computational-experimental pipeline for transcriptomic-guided drug repurposing for a monogenic NDD, and demonstrated that the network-level modulation partially rescues the delayed neural differentiation in HNRNPU-deficient neural cells.

I.Understanding the mechanisms governing neuronal differentiation is essential for elucidating neurodevelopmental processes and identifying therapeutic targets for neurological disorders. In this study, we optimized serum-dependent induction conditions and benchmarked multiple RNA-seq pipelines to establish a robust in-vitro model of neurogenesis using P19 embryonal carcinoma cells. Retinoic acid (RA, 0.5 {micro}M) was used to induce neuronal differentiation under varying concentrations (1%, 2%, and 5%) of fetal bovine serum (FBS) obtained from three suppliers. Morphological observation and marker gene analysis (MAP2, OCT4) revealed that serum concentration strongly influenced aggregation, survival, and neuronal commitment, with 2-5% FBS yielding optimal neurogenic differentiation. Total RNA extracted on day 10 of differentiation was subjected to RNA sequencing, and the resulting datasets were analyzed using four independent bioinformatics workflows: a Linux-based R pipeline (HISAT2 + featureCounts + DESeq2), nf-core, Galaxy, and BGIs Dr. Tom platform. Differential gene expression analysis identified 9,943 differentially expressed genes (DEGs) (FDR < 0.05, |log2FC| > 1), enriched in synaptic assembly and axon development among upregulated genes, and in ribosome biogenesis and RNA processing among downregulated genes. Comparison across all pipelines revealed 62 consistently upregulated and 63 downregulated genes, representing a robust core signature of P19 neurogenesis. Together, these findings establish an optimized and reproducible framework for in-vitro neuronal differentiation and transcriptomic analysis, providing a foundation for mechanistic and disease-modeling studies in neurodevelopmental biology.

UT-018, a stem cell chemoattractant formulation, demonstrated significant regenerative activity across independent murine wound-healing and hair-regeneration studies. Topical treatment accelerated wound closure, enhanced granulation tissue formation, improved collagen organization, increased fibroblast proliferation, and enhanced dermal vascularization. Separate hair-growth studies demonstrated increased follicular density, deeper follicular penetration, enhanced dermal vascularization, and induction of anagen-phase transition by UT-018. Mechanistic studies demonstrated strong intracellular cAMP generation and activation-associated {beta}-catenin phosphorylation consistent with GPCR-mediated regenerative signaling.