Molecular Medicine

Type II diabetes mellitus (T2DM) is one of the most prevalent diseases in the United States and is associated with diabetic foot ulcers (DFU) and their impaired, often chronic, wound healing. The T2DM mouse model with dysfunctional leptin receptor (db/db) has been used in basic and translational studies of wound healing due to its systemic phenotypes (hyperphagia, hypometabolism, obesity, T2DM) and its notable delayed skin wound healing. However, a characterization of the temporal cellular dynamics of the db/db wound healing model has not been performed, nor has the model been systematically compared to human DFUs. We performed the first comprehensive single-cell, multi-omic analysis of dermal cells in diabetic (db/db) compared to non-diabetic (ND) mice across three time points ranging from the inflammatory to the delayed proliferative and resolution phases of healing. Single-cell transcriptomics were uniquely linked to their corresponding cells surface protein expressions of cell-specific receptors, including immune cells (CD45) such as neutrophils (CD11b, Ly6G), monocytes/macrophages (CD11b, F4/80, CD11c, Ly6C) and T lymphocytes (CD3, CD4), and dermal cells such as endothelial cells (CD31) and fibroblasts (CD26, CD140a), and showed high concordance between protein cell markers and their gene expressions in major cell types. Differential multi-omic analyses characterized two neutrophil (Tnfaip3+Sod2+Ly6G+, Csf3r+Fos+Ly6G+), three monocyte/macrophage (F4/80highCD11bhigh, Ly6chighCD11bhigh, CD11chighCD11blow) and three fibroblast (Pi16+Dpp4+CD26high, Lrrc15+Tnc+CD140ahigh, Cilp+Mgp+CD26low) subtypes showing dysregulated dynamics across the time course of healing in db/db vs ND mice. Notably, NETotic Tnfaip3+Sod2+Ly6G+ neutrophils and phagocytic F4/80highCD11bhigh macrophage subtypes were drastically up-regulated in diabetic wounds. Differential cell-cell communication analyses revealed striking differences in crosstalk dynamics between fibroblast, macrophage and neutrophil subtypes in the early phase of healing, and ligand-receptor interactome analyses identified CD44 as the hub of dysregulated immune cell interactions in diabetic wounds, implicating cell adhesion, migration and inflammatory pathways, especially those mediated by ICAM1. Inhibition of CD44 using blocking antibodies in primary macrophages from db/db mice and via intradermal injections in db/db mice significantly normalized the early wound immune dysfunction, in part by inhibiting ICAM1 and reversing the excessive neutrophil influx into diabetic wounds. A new integrated dataset of single-cell human chronic wound studies revealed similar CD44-mediated immune cell dysfunctions in diabetic vs non-diabetic foot ulcers, pointing to CD44 as a promising therapeutic target for T2DM-associated chronic wounds.

Structured AbstractO_ST_ABSPurposeC_ST_ABSOrbital fat hyperplasia has a central role in the manifestations of thyroid-associated orbitopathy (TAO). To better understand the pathways involved in adipogenesis in TAO, we have used transcriptomic methods to analyze gene expression in control and TAO patients, as well as in differentiating orbital fibroblasts (OFs). MethodsWe performed bulk RNA sequencing (RNA-Seq) on intraconal orbital fat to compare gene expression in control and TAO patients. We treated cultured OFs derived from TAO patients with media containing dexamethasone, insulin, rosiglitazone, and isobutylmethylxanthine (IBMX) to induce adipogenesis. We used single nuclear RNA-Seq (snRNA-Seq) profiling of treated OFs to compare gene expression over time in order to identify pathways that are involved in orbital adipogenesis in vitro and compared the dynamic patterns of gene expression identify differences in gene expression in control and TAO orbital fat. ResultsOrbital fat from TAO and control patients segregate with principal component analysis (PCA). Numerous signaling pathways are enriched in orbital fat isolated from TAO patients. SnRNA-Seq of orbital fibroblasts undergoing adipogenesis reveals differential expression of adipocyte-specific genes over the developmental time course. Furthermore, genes that are enriched in TAO orbital fat are also upregulated in orbital adipocytes that differentiate in vitro, while genes that are enriched in control orbital fat are enriched in orbital fibroblasts prior to differentiation. ConclusionsDifferentiating orbital fibroblasts serve as a model to study orbital fat hyperplasia seen in TAO. We demonstrate that the insulin-like growth factor-1 receptor (IGF-1R) and Wnt signaling pathways are differentially expressed early in orbital adipogenesis. PrecisTo understand the pathways involved in adipogenesis in TAO, we used transcriptomic methods to analyze gene expression in control and TAO patients, as well as in differentiating OFs. We demonstrate that the IGF-1R and Wnt signaling pathways are differentially expressed during orbital adipogenesis.

Knee-Osteoarthritis (Knee-OA) is a prevalent joint disorder lacking FDA-approved cell-therapies to halt its progression. This study uses single-cell RNA sequencing to analyze bone marrow aspirate concentrate (BMAC) and stromal vascular fraction (SVF) samples in a clinical trial of autologous cell therapies. Trial site-specific variability was significant in BMAC, necessitating tailored normalization, whereas SVF was less affected, likely due to uniform subcutaneous fat sampling. Variance partitioning and tensor decomposition identified site effects in BMAC but revealed shared pathways across cell types in both tissues. Differential gene expression (DEG) analysis between responders and non-responders yielded no significant findings, though likelihood ratio testing (LRT) revealed enrichment for DEG patterns linked to disease severity, potentially masked by patient heterogeneity. Key BMAC pathways included oxidative phosphorylation, unfolded protein response, and TNF signaling. Cell-cell communication analysis suggested enhanced HLA signaling in non-responder MSCs, consistent with inflammation, while responders showed more coordinated immune interactions. BMAC-MSCs promoted chondrocyte proliferation, whereas SVF-MSCs emphasized immune regulation. Variability in therapy outcomes reflects patient heterogeneity beyond genomic factors, complicating the immediate use of genomic profiling to guide treatment. Nonetheless, as molecular pathways are better understood, integrating genomic insights into personalized strategies may become feasible.

This study investigates the therapeutic potential of secretomes derived from Adipose-derived Mesenchymal Stem Cells (ADMSC-CM) and Limbal-derived Mesenchymal Stem Cells (LMSC-CM) against oxidative stress-induced damage in Retinal Pigment Epithelium (RPE-1) cells. RPE dysfunction, often triggered by oxidative stress, is a hallmark of various retinal degenerations. Here, we induced RPE-1 injury using H2O2 and evaluated the restorative effects of both MSC-conditioned media (CM). Our results demonstrated that both ADMSC-CM and LMSC-CM significantly enhanced cell viability and successfully reversed H2O2-induced G2/M phase cell cycle arrest. While oxidative stress triggered a pro-inflammatory response characterized by elevated IL-1{beta}, IL-6, and IL-10 expression, MSC-CM treatment, particularly ADMSC-CM, effectively modulated these levels and suppressed the p38 MAPK signaling pathway. Furthermore, MSC-CM reduced the Bax/Bcl-2 ratio, indicating an anti-apoptotic effect, and appeared to stabilize autophagic flux. To investigate the impact of oxidative-stress induced alterations in retinal pigment epithelial cells on angiogenesis, the effects of RPE-derived secreted factors on endothelial cell function were evaluated. Crucially, in terms of safety and secondary complications, neither secretome exhibited pro-angiogenic tendencies; instead, they significantly inhibited HUVEC migration and invasion compared to the H2O2 damaged group. These findings suggest that both ADMSC and LMSC secretomes provide a potent multi-targeted therapeutic effect, making them promising candidates for cell-free therapies in retinal diseases.

BackgroundHepatic stellate cells (HSC) are Vitamin A storing non-parenchymal cells of the liver. During injury and inflammation, HSCs are the major contributors of excessive extracellular matrix (ECM) leading to Liver Fibrosis (LF). Emerging evidence suggests a fibrosis-independent role of these cells as key regulators of liver homeostasis and liver regeneration, emphasising on the dual role of HSCs in liver. HSCs are known to secrete several growth factors through which they largely execute their functions. However, the role of secretome (exosomes) from early activated or undifferentiated HSCs in a fibrotic milieu nor its composition are completely understood. MethodsLX-2 cells were cultured in low to no serum conditions and their isolated exosomes were transplanted into fibrotic severe combined immune deficient (SCID) mice livers, followed by post-transplantation analysis of the liver tissue and compared to the untreated controls. Total proteomic profiling of cell and exosomal cargo was performed using mass spectrometry and the data analysed and compared with the total HSC cell proteome. ResultsSignificant reduction in collagen in the transplanted mice livers compared to untreated fibrotic controls was observed with both the cells and exosomes transplantation. Comparative analysis revealed distinct enrichment of proteins and signaling pathways associated with extracellular matrix regulation, cellular communication, and metabolism in exosomes. Notably, these pathways are prominently represented in the exosomal fraction, suggesting a selective packaging of functional mediators. ConclusionThis study suggests the potential role of HSCs in regulating the complex liver homeostasis via exosomal network of proteins that contribute significantly to liver repair by ECM remodelling and growth factor-mediated signalling to regulate metabolism, fibrosis and liver regeneration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/721862v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@99bbf4org.highwire.dtl.DTLVardef@1029dd0org.highwire.dtl.DTLVardef@c6f578org.highwire.dtl.DTLVardef@1dba81_HPS_FORMAT_FIGEXP M_FIG C_FIG

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes with no disease modifying treatments. Despite the prevalence, the molecular mechanisms of DPN are not fully characterized. Among the various molecular regulators, microRNAs (miRNAs) control protein synthesis and are essential for normal development and homeostasis, with dysregulation implicated in cancer and neurodegenerative diseases. In this study, we performed small RNA-sequencing to profile the miRNA landscape of human sural nerves from individuals with and without DPN. Our analysis revealed that nearly 10% of all miRNAs detected are dysregulated and among those 74% are significantly downregulated in DPN. Target gene enrichment analysis of the differentially expressed miRNAs yielded pathways significantly associated with nerve regeneration, metabolic dysfunction, and immune cell activity. In particular, miR-21-5p is significantly upregulated in DPN, showed a positive association with axonal loss severity, and localizes to Schwann cells, consistent with its broader role as an injury- and inflammation-responsive miRNA that shifts from early pro-regenerative functions to maladaptive, inflammation-amplifying effects that impair Schwann cell mediated nerve repair. These results suggest that miRNAs may contribute to peripheral nerve degeneration by promoting inflammation, apoptosis, oxidative stress, and impaired nerve regeneration, while also opening potential avenues for biomarker discovery and therapeutic intervention. Article highlightsO_LIWe undertook this study to address the limited understanding of molecular changes contributing to diabetic peripheral neuropathy (DPN) in humans. C_LIO_LIWe sought to profile microRNAs (miRNAs), key post-transcriptional regulators of gene expression, in human sural nerves and developed a dedicated computational pipeline for robust miRNA quantification, differential expression, and target enrichment analysis. C_LIO_LIOur analyses revealed widespread miRNA dysregulation in DPN, with most altered miRNAs downregulated and miR-21-5p significantly upregulated in DPN, highly correlated with axonal loss severity and localized to Schwann cells. C_LIO_LIThese findings suggest that miRNA imbalance, including elevated Schwann cell miR-21-5p, may contribute to nerve dysfunction in DPN and provide new opportunities for biomarker development and therapeutic targeting. C_LI

BackgroundInflammation-induced pancreatic islet-cell death and dysfunction are key aspects of both type 1 and type 2 diabetes. Stem cell-derived islets (SC-islets) are an emerging tool in diabetes research, however, our understanding of how inflammation affects SC-islet function is incomplete. We therefore aimed to thoroughly characterize how SC-islets respond to pro-inflammatory cytokines at the functional and transcriptomic levels in comparison with human primary islets and EndoC-{beta}H5 cells. MethodA 7-stage differentiation protocol was used to generate SC-islets with insulin-, glucagon-, and somatostatin-positive cells. SC-islets, primary human islets and EndoC-{beta}H5 cells were exposed to different doses of pro-inflammatory cytokines (IL-1{beta} + IFN{gamma} + TNF) including a high dose for up to 48 h and a low dose up to 144 h to mimic the intense islet inflammation in T1D and chronic low-grade inflammation in T2D, respectively. Differential gene expression (RNA-seq), cell death, activation of key signalling proteins, hormones, and chemokine secretion were determined. ResultsBasal expression of key islet-cell identity genes in SC-islets correlated well with that of primary islets and EndoC-{beta}H5 cells. In SC islets, cytokines dose-dependently induced activation of key proximal signalling pathways (NF{kappa}B, STAT1, and JNK), upregulation of major histocompatibility complex (MHC) class I, and increased cell death (cytotoxicity and caspase 3/7 activity). In head-to-head experiments, SC-islets displayed similar cytokine responses particularly as primary islets regarding induction of cell death, chemokine secretion, differential gene expression, and protein levels of cell death executioners (gasdermin D and caspase-7). Cytokines increased insulin release in SC-islets and primary islets, while diminishing insulin secretion in EndoC-{beta}H5 cells. Cytokines reduced glucagon release in SC-islets, which was partially restored by treatment with the incretin hormone glucose-dependent insulinotropic peptide (GIP) with or without a glucagon-like peptide 1 (GLP-1) receptor agonist (liraglutide). ConclusionSC-islets are highly responsive to inflammation with a high degree of similarity to primary islets. Our results support the use of SC-islets as a valid tool in inflammation and diabetes research.

Uterine leiomyomas or fibroids are highly prevalent benign tumors of the female reproductive tract, often causing significant symptoms and requiring surgical intervention, leading to substantial healthcare costs worldwide. Their molecular pathogenesis remains incompletely understood, but evidence suggests that somatic stem cells play a pivotal role in myometrial growth, whereas a genetic alteration, particularly mutations in MED12, may transform a myometrial stem cell into a tumor-initiating cell, promoting fibroid growth. In organ cultures of fibroids and myometrium, most differentiated cells degenerated by day 7, whereas stem cells remained quiescent and viable within their native niches. Notably, between days 15 and 29, hypoxia-induced activation triggered stem cell proliferation and differentiation within the ex vivo slices. Transcriptomic profiling revealed statistically significant upregulation of stemness-associated genes, including HMGA, ITG, KLF, HOX, and SOX family members, in long-term cultured slices compared with baseline tissue, and between normal and tumor cultures. Reactome pathway enrichment analysis further identified distinct metabolic, extracellular matrix remodeling, immune surveillance, angiogenic, and cell death- related programs distinguishing myometrial from leiomyoma cultures. Furthermore, previously reported gene and pathway differences between healthy and fibroid tissues were robustly confirmed, validating the culture model. In conclusion, our findings establish long-term organ culture as a powerful, physiologically relevant platform for investigating stem cell dynamics in myometrium and uterine leiomyoma. They also provide proof of concept for extending this approach to other tissue types, enabling the discovery of mechanisms underlying stem cell activation, differentiation, and death, with broad translational potential in regenerative medicine and cancer biology.

1.BACKGROUND & AIMSUlcerative colitis (UC) is an inflammatory bowel disorder with unknown etiology. Given its complex nature, in vivo studies to investigate its pathophysiology is vital. Animal models play an important role in molecular profiling necessary to pinpoint mechanisms that contribute to human disease. Thus, we aim to identify common conserved gene expression signatures and differentially regulated pathways between human UC and a mouse model hereof, which can be used to identify UC patients from healthy individuals and to suggest novel treatment targets and biomarker candidates. METHODSTherefore, we performed high-throughput total and small RNA sequencing to comprehensively characterize the transcriptome landscape of the most widely used UC mouse model, the dextran sodium sulfate (DSS) model. We used this data in conjunction with publicly available human UC transcriptome data to compare gene expression profiles and pathways. RESULTSWe identified differentially regulated protein-coding genes, long non-coding RNAs and microRNAs from colon and blood of UC mice and further characterized the involved pathways and biological processes through which these genes may contribute to disease development and progression. By integrating human and mouse UC datasets, we suggest a set of 51 differentially regulated genes in UC colon and blood that may improve molecular phenotyping, aid in treatment decisions, drug discovery and the design of clinical trials. CONCLUSIONGlobal transcriptome analysis of the DSS-UC mouse model supports its use as an efficient high-throughput tool to discover new targets for therapeutic and diagnostic applications in human UC through identifying relationships between gene expression and disease phenotype.

ObjectiveThis retrospective cohort investigated the role of leptins promoter methylation and microRNA targeting profile in developing adiposity and inflammation in neonates, using umbilical cord blood from preterm (n=67) and term (n=71) mothers. MethodsGlobal DNA methylation and leptin promoter methylation were performed. ELISA determined leptin and IGF1 levels. Real-time PCR measured mRNA levels. MicroRNA target prediction on the human leptin gene (LEP) was done in silico using network analysis. ResultsPreterm cord blood significantly reduced genome-wide (p<0.001) and LEP promoter methylation (p=0.001), increased LEP & LEPR expression (p=0.04), and circulatory leptin (p=0.41). Neonatal birth weight positively correlated with leptin and IGF1 levels in preterm (r=0.47, p=0.04) but not in the term. IL6 expression showed a positive correlation with circulatory leptin (r= 0.687, p=0.008), LEP (r= 0.763, p=0.009), and an inverse association with LEP promoter methylation (r= -0.636, p=0.04) in preterm. The obtained LEP targeting miRNAs showed their affinities for critical genes associated with body fat distribution, fat cell differentiation, and energy regulation, implicating a close association in the LEP-miRNA-obesity axis. ConclusionsThe strong correlation between LEP methylation and pro-inflammatory cytokine influences each other in developing chronic inflammation in preterm neonates, which might predispose them to obesity in later life. Study importanceWhat is already known? O_LILeptin communicates about the bodys fat deposits to the brain and aids in maintaining energy homeostasis and stable body weight. C_LIO_LIPreterm exhibit lower body weight and fat mass at birth than term neonates, who often show rapid compensatory catch-up growth. C_LI What does this study add? O_LILeptin gene (LEP) promoter methylation was reduced in preterm cord blood compared to term. C_LIO_LIHigher interleukin-6 (IL6) and tumour necrosis factor-alpha (TNF) expression in preterm but not in term. IL6 correlated positively with circulatory leptin and LEP expression while inversely associated with LEP-specific promoter methylation, indicating that a dysregulated epigenetic control can promote low-grade inflammation in preterm neonates. C_LIO_LILEP-targeting micro-RNAs showed affinities for critical genes associated with fat cell differentiation, energy regulation, and other processes. C_LI How might these results change the direction of research or the focus of clinical practice? O_LISince others observed dysregulated LEP methylation in the adipose tissue of obese subjects, these data imply that leptin could mediate the risk for obesity during preterm birth. C_LIO_LIWhile short-term outcomes of preterm birth are well addressed, its effect on long-term metabolic health is of concern as it might elevate the risk of obesity. C_LI Graphical AbstractMaternal factors leading to preterm birth and cord blood leptin dysregulation in predicting obesity. Elevated blood pressure, infection, and lower haemoglobin in preterm disrupted epigenetic control of leptin and activated inflammation that might induce leptin resistance. The latter is known to reduce satiety and increase body mass, elevating the risk of obesity. Solid arrows depict present data, and dotted lines indicate possible pathways. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/24319077v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1f3604corg.highwire.dtl.DTLVardef@13723b5org.highwire.dtl.DTLVardef@1094bfborg.highwire.dtl.DTLVardef@15b5ebc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background/ObjectivesPancreatic cancer (PC) is an aggressive malignancy with rising incidence and poor survival rates. While Hispanic/Latino (H/L) patients have a lower overall incidence compared to Non-Hispanic White (NHW) patients, they are diagnosed at younger ages, often present with more advanced disease, and experience worse survival outcomes. The molecular drivers underlying these disparities remain poorly understood. Key oncogenic pathways, including TP53, WNT, PI3K, TGF-Beta, and RTK/RAS, play crucial roles in tumor progression, therapy resistance, and response to targeted treatments. However, their ethnicity-specific alterations and prognostic implications in PC remain largely unexplored. This study aims to characterize pathway-specific mutations in PC among H/L and NHW patients, assess tumor mutation burden, and identify ethnicity-specific oncogenic drivers using publicly available datasets. The findings may provide critical insights to optimize precision medicine strategies and enhance targeted therapies for underrepresented populations. MethodsA bioinformatics analysis was performed using publicly available PC datasets to evaluate mutation frequencies in genes associated with the TGF-Beta, RTK/RAS, WNT, PI3K, and TP53 pathways. The study included 4,248 patients, with 407 identified as H/L and 3,841 as NHW. Patients were stratified by ethnicity to assess differences in mutation prevalence. Chi-squared tests were conducted to compare mutation rates between groups, while Kaplan-Meier survival analysis was performed to evaluate overall survival differences based on pathway-specific alterations. ResultsSignificant differences were observed in the TGF-Beta pathway between H/L and NHW patients. TGF-Beta mutations were less prevalent in H/L patients (18.4% vs. 24.4%, p = 8.6e-3). Additionally, genes related to the TGF-Beta pathway showed significant alterations, with SMAD2 (1.5% vs. 0.4%, p = 6.3e-3) and SMAD4 (15% vs. 19.9%, p = 0.02) exhibiting notable differences. Although RTK/RAS, WNT, PI3K, and TP53 pathway mutations were not statistically significant overall, borderline significance was observed in genes associated with these pathways, including ERBB4 (3.4% vs. 1.8%, p = 0.03), ALK (2.7% vs. 1.1%, p = 0.01), HRAS (1.2% vs. 0.1%, p = 1.3e-4), and RIT1 (0.7% vs. 0.1%, p = 0.03) in the RTK/RAS pathway, as well as CTNNB1 (2.9% vs. 1.3%, p = 0.01) in the WNT pathway. Survival analysis revealed no significant differences in overall survival among H/L patients. However, NHW patients with TP53 pathway alterations exhibited borderline significant differences in survival outcomes.

Gastric bypass surgery results in long-term weight loss due to re-routing of the gastro-intestinal anatomy and dietary intake alterations. Studies have examined protein change during rapid weight loss (up to 1 year post-surgery), but whether protein changes are maintained long-term after weight stabilization is unknown. To identify proteins and pathways involved with the long-term beneficial effects of weight loss, abundances of 1297 blood-circulating proteins were measured at baseline, 2 and 12 years after Roux-en-Y gastric bypass surgery. Protein changes were compared between 234 surgery and 144 non-surgery subjects with severe obesity, with discovery and replication subgroups. Seventy-one protein changes were associated with 12-year BMI changes and 58 (7 unique) with surgical status. Protein changes, including ApoM, were most strongly associated with long-term changes in lipids (HDL-C and triglycerides). Inflammation, adipogenesis, cellular signaling, and complement pathways were implicated. Short-term improvements in protein levels were maintained long-term, even after some weight regain.

New approach methodologies (NAMs), including induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) and retinal pigment epithelium (iRPE), are increasingly applied to study retinal disease mechanisms and therapeutic strategies. However, these models often remain relatively immature. Given the high lipid content and complex metabolism of the retina, it is unclear to what extent iPSC-derived systems recapitulate the human retinal lipidome. Here, we compared the lipidomic profiles of ROs and iRPE, collected at several differentiation stages, with those of post-mortem adult human macular, non-macular and RPE plus choroid (pmRPE). The lipidome of iRPE differed markedly from pmRPE, whereas prolonged differentiation of ROs resulted in a lipidomic profile increasingly resembling that of the post-mortem retina. Moreover, ROs showed similarities to both macular and non-macular lipidome. These findings show that iPSC-derived models can become valuable NAMs to study lipid-related retinal disorders and provide a framework to optimize differentiation protocols.

BackgroundCKD affects approximately 850 million people worldwide and is a leading cause of mortality. Podocytes, cells in the kidney are terminally differentiated and incapable of division in vivo making the establishment of primary cultures particularly challenging. The ability of cells to proliferate and avoid senescence is closely linked to telomere length. When telomere length becomes critically reduced, it results in cellular senescence. MethodsWe present the successful rejuvenation of a human SIX2-positive renal progenitor cell line derived from the urine of a 30-year-old West African male (UM30-OSN). To achieve partial reprogramming, plasmids expressing the Yamanaka factors OCT4, SOX2, NANOG, c-Myc, and KLF4 were employed. ResultsUM30-OSN expresses the pluripotency-associated marker SSEA4, renal stem cell markers such as SIX2, CD133 and CD24, determined by immunofluorescence, FACS and qPCR. Expression analysis revealed downregulation of senescence markers p21and p53 and upregulation of proliferation-associated genes PCNA, KI67 and TERT, confirming rejuvenation. Upon podocyte differentiation, UM30-OSN cells expressed podocyte-specific markers NPHS1, NPHS2, SYNPO and CD2AP. Comparative transcriptome analyses revealed a correlation co-efficiency (R2 = 0.88) with the immortal podocyte line AB 8/13. To demonstrate the usefulness of UM30-OSN to model APOL1-mediated kidney disease, we investigated the effects of Interferon-{gamma} (IFN-{gamma}) on UM30-OSN derived podocytes and evaluated the potential of the JAK1/JAK2 inhibitor Baricitinib to mitigate IFN-{gamma}-induced cellular responses. IFN-{gamma} stimulation resulting in increased phosphorylation of STAT1, activation of APOL1, upregulation of pro-inflammatory and fibrotic markers such as, IL-6, TGF-{beta}, Vimentin, Fibronectin, and morphological changes indicative of cell stress. Pre-treatment with Baricitinib effectively inhibited STAT1 phosphorylation, reduced expression of pro-inflammatory and fibrosis-associated genes, and preserved podocyte morphology. ConclusionGiven their robust proliferation capacity, UM30-OSN cells represent a valuable additional model for investigating kidney-associated diseases such the contribution of APOL1 high-risk variants to kidney injury and fibrosis.

PurposeTo identify the transcriptomic changes induced by dexamethasone (DEX) in trabecular meshwork (TM) and Schlemms canal endothelial (SCE) cells with RNA-sequencing (RNA-seq). MethodsHuman TM (n=10) and SCE cell strains (n=5) were isolated from healthy donor eyes and exposed to DEX 100nM and vehicle (control). Three DEX exposure times were evaluated: 1-hour, 6-hours, and 2 days. RNA-seq was performed on Illuminas TruSeq platform and gene expression was quantified using featureCount. DESeq2 paired (treated and untreated) sample test was applied to identify genes transcriptionally responsive to DEX (DEGs) at false discovery rate <0.05. Gene-set enrichment analyses were performed on DEGs. DEGs were tested for association with glaucoma (POAG) and intraocular pressure (IOP). ResultsNine TM and 4 SCE strains passed quality control. After 2-day DEX exposure, there were 857 and 2,086 DEGs in TM and SCE, respectively. Of these, 411 genes were differentially expressed in both TM and SCE, including FKBP5 (17.3-fold-change, p=6.9x10-53) and FAM107A (25.1-fold-change, p=4.0x10-240), the most significant DEG after 2-day DEX exposure in TM and SCE, respectively. The 2-day DEX DEGs in TM and SCE were enriched in cell adhesion, extracellular matrix, and response to stimulus in Gene Ontologies (p<3.7x10-6). Early response DEGs were enriched in immune-related processes. Thirteen DEGs in TM were significant at all three time points, including PER1. LTBP2 is a TM-only DEG and FAM105A a SCE-only DEG associated with IOP and POAG risk. ConclusionsThis study identified candidate genes and pathways for glucocorticoid-induced ocular hypertension which can be further explored in human genetic analyses.

Retinal degenerative diseases (RDD) such as retinitis pigmentosa (RP) have no treatment. Stem cell-based therapies could provide promising opportunities to repair the damaged retina and restore vision. We investigated a novel approach in which human retinal progenitor cells (RPCs) derived from primitive mesenchymal stem cells (pMSCs) were examined to treat retinal degeneration in an rd12 mouse model of RP. Intravitreally transplanted cells improved retinal function and significantly increased retinal thickness. Transplanted cells homed, survived, and integrated to various retinal layers. They also induced anti-inflammatory and neuroprotective responses and upregulated neurogenesis genes. We found that RPCs were more efficacious than pMSCs in improving the retinal structure and function. RNA analyses suggest that RPCs promote neuroprotection and neuronal differentiation by activating JAK/STAT and MAPK, and inhibiting BMP signaling pathways. These promising results provide the basis for clinical studies to treat RDD using RPCs derived from pMSCs.

Porcine organotypic retinal explant cultures are widely used to study retinal neurodegeneration under controlled conditions, but the biological process that occurs in the retinal explant over time due to preparation-induced injury and culture are not well understood. Here, we generated a time-resolved transcriptomic reference for porcine neural retinal explants-maintained ex vivo for 10 days. Global expression profiles are strongly separated by culture time, with Day 0 clearly distinct from cultured samples and at Day 7 and Day 10 showing the highest similarity, indicating a transition toward a later stabilized state. Across the time course, 3,187 genes were differentially expressed relative to Day 0, with the largest shifts occurring at an early stage of culture (Day 1-Day 3). Pathway-level analyses revealed coordinated remodeling involving inflammatory signaling, and metabolic/bioenergetic changes, including reduced mitochondrial and oxidative phosphorylation-related programs at later time points. Here, we provide a time-resolved transcriptomics reference dataset for cultured porcine retinal explants. These data can build a foundation to interpret data generated in this model, differentiate changes inherent to the explant culture from treatment-specific effects and to select appropriate experimental windows for mechanistic studies of retinal degeneration.

Inflammatory bowel disease (IBD) frequently co-occurs with immune-mediated and metabolic disorders, but whether these associations reflect shared genetics or causal effects remains unclear. We performed two-sample Mendelian randomization (MR) using large-scale genome-wide association study (GWAS) summary statistics to investigate potential causal effects of immune-mediated diseases and lifestyle traits on IBD, Crohns disease (CD), and ulcerative colitis (UC). SNP-based heritability and genetic correlations were estimated to contextualize findings. Following false discovery rate correction, genetically predicted psoriasis was positively associated with IBD (OR 1.15), CD (OR 1.23), and UC (OR 1.10), with the strongest effect observed for CD. Genetically predicted type 2 diabetes mellitus (T2DM) showed a modest inverse association with UC (OR 0.88). No lifestyle-related traits remained significant after correction. Sensitivity analyses indicated heterogeneity across instruments and evidence of directional pleiotropy in selected models, whereas no pleiotropy was detected for the T2DM-UC association. These findings support a role of psoriasis-related immune pathways in IBD susceptibility and suggest a potential inverse association between genetic liability to T2DM and UC.

BackgroundNeuroblastoma, the most common extracranial solid tumor in children, exhibits considerable clinical heterogeneity influenced by genetic predisposition. While genome-wide association studies (GWAS) in European populations have identified eight susceptibility loci, the genetic basis of neuroblastoma in East Asian populations remains poorly understood. MethodsWe conducted the first GWAS in a Chinese cohort comprising 235 neuroblastoma patients and 3,100 controls, followed by multi-omics analyses of gene expression. The novel risk loci were further validated in an independent East Asian cohort (76 cases/269 controls). Functional characterization of a novel locus was carried out in neuroblastoma cell lines using CRISPR/Cas9-mediated deletion and overexpression assays to evaluate its regulatory effects on candidate genes. FindingsWe replicated six of eight known loci including genome-wide significant associations at CASC15 (6p22.3; P = 1.55 x 10-) and BARD1 (2q35; P = 3.44 x 10-), and identified 11 novel risk loci. These novel associations implicate genes involved in DNA repair (MUTYH at 1p34.1), neurodevelopment (BASP1 at 4q13.2 and SLC22A4/SLC22A5 at 5q31.1), and immune regulation (HLA at 6p21 and IDO1/IDO2 at 5q31.1). Multi-omics integration revealed that lead variants modulate gene expression (cis-eQTLs) and DNA methylation (mQTLs) in neural crest-derived tissues and immune cells. Two loci (rs2631372 at 5q31.1: P= 0.045; rs2956095 at 11p13: P= 0.027) showed consistent associations in the replication cohort. Functional studies demonstrated that deletion of the 5q31.1 risk interval reduced expression of SLC22A4, SLC22A5, and LOC553103, while their overexpression promoted neuroblastoma cell proliferation. InterpretationThese findings highlight both shared and population-specific genetic contributions to neuroblastoma susceptibility, underscoring the importance of diversifying GWAS efforts to advance ancestry-informed risk assessment and therapeutic strategies.

The mammalian retina lacks meaningful regenerative capacity, and degeneration usually leads to irreversible vision loss. Although lower vertebrates regenerate retinal neurons through Muller glia, this capacity has generally been considered absent in humans. Using long-term organotypic retinal cultures from 39 adult donors, we show that defined humoral cues alone are sufficient to unlock a latent neurogenic program in human Muller cells. FGF-2 treatment and GSK-3 inhibition induced robust proliferation across both peripheral and central retina, with 79.09 {+/-} 6.32% of dividing cells identified as Muller glia, some completing multiple cell cycles. Single-cell transcriptomics revealed activation of progenitor-like and neuronal differentiation pathways, whereas immunohistochemistry demonstrated expression of early and late neuronal markers spanning all major retinal lineages. Newly generated cells expressed markers of cone, rod, bipolar, horizontal, amacrine, and ganglion cell identities, together with evidence of early synaptogenesis. These findings reveal an intrinsic regenerative potential in adult human Muller glia, with implications for future vision-restoration strategies in degenerative retinal disease. SummaryAdult human Muller glia retain an intrinsic capacity for proliferation and neural lineage commitment independent of donor age or gender. In long-term organotypic cultures of human donor retina, defined humoral cues, without genetic manipulation, induce Muller glia proliferation and the onset of neuronal differentiation. These findings reveal intrinsic neurogenic potential in human Muller glia and provide a human-relevant platform for retinal regeneration studies.