Molecular Medicine

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.

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

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.

Background Genome-wide association studies have identified >200 loci associated with IBD, yet the causal gene for most remains unknown. As multiple epithelial functions have been linked with susceptibility to IBD, there is a need to prioritize candidate causal genes for functional studies in this cellular context. Methods Using a standardized definition of regions implicated by index SNPs from three GWAS studies, we categorized regions as containing: (1) a known casual gene, (2) a single gene or (3) multiple genes. We then developed an IBD Priority Score to rank genes based on genetic, genomic and functional data. We next developed and applied an Epithelial Priority Score, based on expression patterns and quantitative traits, to prioritize genes for functional validation in epithelial models. Two candidate genes identified through this approach were tested for their impact on viral response pathways in HT-29 cells. Results The IBD Priority Score prioritized a single gene in 71 of the 104 regions containing multiple genes. The Epithelial Priority Score identified 31 epithelial candidates. Functional studies demonstrated that IRF6 enhanced, whereas IRF8 suppressed, antiviral responses in intestinal epithelial cells stimulated with Poly(I:C). Conclusions Combining multiple genetic, genomic, and functional data is a useful approach for prioritizing the most likely causal gene within IBD GWAS loci, and for prioritizing functional validation studies in epithelial cells and tissues. Moreover, we provide functional evidence for two IBD genes playing a role in the regulation of anti-viral responses in intestinal epithelial cells.

BackgroundKnee osteoarthritis (KOA) is a prevalent degenerative joint disease in which synovial inflammation and fibrosis are closely linked to pain, stiffness, and functional limitation. Growing evidence suggests that metabolic dysregulation, particularly in lipid metabolism, is involved in KOA pathogenesis, but the underlying mechanisms remain incompletely defined. MethodsSprague Dawley rats underwent bilateral anterior cruciate ligament transection to establish a KOA model; sham-operated rats served as controls. RNA sequencing of synovial tissues was performed to identify differentially expressed genes (DEGs) and enriched pathways, followed by GO/KEGG and GSEA analyses. In vivo, adeno-associated virus vectors were used to overexpress or knock down PPAR-{gamma} and phosphoenolpyruvate carboxykinase 1 (PCK1) via intra-articular injection. Ex vivo, primary rat fibroblast-like synoviocytes (FLSs) were stimulated with IL-1{beta} and transfected with PPAR-{gamma} or PCK1 siRNA/overexpression plasmids. synovitis and fibrosis were evaluated by HE, Masson, and Sirius Red staining, immunofluorescence, ELISA, RT-qPCR, and Western blotting. ResultsRNA-seq revealed 621 up-regulated and 228 down-regulated genes in KOA synovium versus sham, with DEGs significantly enriched in PPAR signaling, adipocytokine, and AMPK pathways. Metabolism-related genes including Fabp5, Plin1, Adipoq, Lep, and Pck1 were up-regulated. GSEA indicated downregulation of PPAR-{gamma} signaling in KOA synovium. In vivo and ex vivo, PPAR-{gamma} expression was reduced in KOA, whereas PCK1, FABP5, and ADIPOQ were increased. PPAR-{gamma} overexpression alleviated synovial inflammation, collagen I deposition, and fibrosis, and suppressed FABP5, ADIPOQ, and PCK1 expression; PPAR-{gamma} knockdown produced the opposite effects. Functional studies showed that PCK1 overexpression aggravated synovial inflammatory cell infiltration and fibrosis, elevated IL-1{beta}, IL-18, and TGF-{beta}, and decreased TIMP1 levels in serum, synovial tissue, and FLSs supernatants, whereas PCK1 silencing reversed these changes. ConclusionsThe PPAR-{gamma}/PCK1 metabolic axis modulates synovitis and fibrosis in KOA. Downregulation of PPAR-{gamma} and consequent upregulation of PCK1 promote synovitis and fibrotic remodeling. These findings identify the PPAR-{gamma}/PCK1 pathway as a potential therapeutic target for KOA.

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.

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.

Inflammatory bowel disease (IBD) often involves disrupted intestinal epithelial barrier, but therapies specifically targeting this barrier are limited. We found that downregulated AUF1 (HNRNPD) contributes to defective barrier integrity in ulcerative colitis (UC). Compared to controls, its expression level was decreased and inversely correlated with clinical severity. Knocking down AUF1 in human and mouse colonic organoids led to impaired barrier function, with reduced Occludin and upregulated Claudin-2, mimicking characteristic IBD-associated mucosal alterations. Distinct RNA-binding activity of AUF1 protein isoforms contributed to these changes: p37 stabilized Occludin mRNA and blocked microRNA-122/Ago2-mediated repression, whereas p40 promoted Claudin-2 mRNA degradation via ubiquitin-proteasome pathway. Restoring AUF1 expression in organoids enhanced epithelial properties and, when transplanted into mice with established colitis, accelerated mucosal healing and epithelial regeneration in recipient mice and decreased fibrosis. Our study unravelled a post-transcriptional mechanism important for intestinal homeostasis and demonstrated a concept of using engineered organoids for treating IBD.

Microtia is a common congenital craniofacial malformation characterized by the partial or complete absence of the external ear structure. Despite its relatively high incidence, the pathogenesis of microtia remain poorly understood. In this study, we analyzed both single-cell and bulk RNA sequencing data from microtia cases and identified a population of COL1+HES1+ mesenchymal stem cell in perichondrium with significantly higher expression of the CRABP2 gene, a gene that encodes a nuclear transporter of retinoic acid. Gene expression analysis further confirmed that the RA signaling intensity and stemness are both higher in COL1+HES1+ perichondral stem cells from microtia patients, possibly due to elevated CRABP2 levels. Through histological verification we further confirmed the presence of this cell population with high CRABP2 expression in the perichondrium. Mechanistically, the elevated CRABP2 expression in perichondral stem cells seen in microtia patients may cause dysregulated RA signaling and disrupt the regulation of stem cell differentiation during auricular development. Histological analysis further revealed higher KLF2 expression as well as cartilage hypoplasia in microtia samples. Our study identified that the CRABP2-induced RA dysregulation in COL1+HES1+ perichondral stem cells may contribute to microtia. These findings offer new insights into the etiology of microtia and provide potential directions for prenatal prevention and tissue engineering treatments.

IntroductionSkeletal muscle wasting is a common complication of chronic kidney disease which leads to a loss of muscle function. The pathogenesis of skeletal muscle wasting is incompletely understood, which is preventing the development of targeted therapeutics. Recent evidence implicates miRNAs in the of skeletal muscle wasting. Our aim was to firstly examine miRNA profiles of CKD human skeletal muscle for the identification of aberrant expression patterns compared to a healthy control (HC) cohort, and secondly, investigate the role these miRNAs may play in inducing or promoting skeletal muscle atrophy using a novel human primary skeletal muscle cell model of CKD skeletal muscle. MethodsFor the comparison between CKD and HC populations, skeletal muscle biopsies were collected from the vastus lateralis of n=15 non-dialysis dependent CKD patients stage 3b-5 CKD patients, and n=15 healthy controls matched for age, gender and physical activity. n=5 biopsies from each group underwent next generation sequencing to obtain complete microRNA profiles in CKD vs HC cohorts, which were then validated in a separate cohort by PCR (N=10 in each group). A causative role in muscle wasting was determined by transfection of key microRNAs into a primary culture model of CKD skeletal muscle and changes in protein degradation determined by L-[3H]- phenylalanine release into the media. ResultsNext Generation Sequencing identified differential expression of 16 miRNAs in skeletal muscle of CKD patients versus controls, and PCR validation confirmed miRNA-148a-3p expression was significantly decreased in CKD patients. The reduced miRNA-148a-3p expression was also maintained in the primary culture model. Upon overexpression of miRNA-148a-3p in CKD myotubes, protein degradation rates were decreased non-significantly (p=0.28) by 16.3% compared to un-transfected CKD cells. ConclusionCKD was associated with a significant reduction in miRNA-148a-3p expression in skeletal muscle compared to non-CKD controls which was retained in our in vitro model. Overexpression of miRNA-148a-3p in primary skeletal myotubes non-significantly decreased muscle protein degradation by 16.3%. In order to determine the importance of miRNA-148a-regulation of protein degradation, a deeper understanding of miRNA-148a-3p targets and their associated pathways with respect to those dysregulated in skeletal muscle wasting is required.

MicroRNAs (miRNAs) are evolutionarily conserved post-transcriptional regulators that play critical roles in cellular development and differentiation across species. Although the importance of miRNAs in oligodendrocyte lineage cell (OLLC) differentiation has been extensively studied in rodent models, their roles in human OL development remain less understood. To address this gap, we used a human embryonic stem cell (hESC) reporter system designed to study human OLs and OL progenitor cells (OPCs). Using an optimized differentiation protocol, we used the reporter hESCs to generate and isolate well-characterized OLLCs at specific developmental stages and performed next-generation sequencing-based miRNA profiling to identify stage-specific miRNAs enriched during OL lineage specification and maturation. In addition to canonical miRNAs known to be enriched at various stages of OL development, our study identified several lesser-known miRNAs with distinct stage-specific enrichment patterns that may serve as useful molecular markers for classifying human CNS cell types in future studies. Target analysis of OPC-and OL-enriched miRNAs revealed key genes, including transcription factors ZNF488 and DLX1, cytoskeletal regulator CSNK2B, and potassium channel gene KCNJ1, along with key signaling pathways such as AKT, SMAD2/3, estrogen receptor, and insulin signaling, which regulate OPC and OL lineage function. These findings advance our understanding of the OLLC-specific miRNAs, and miRNA-mediated regulatory networks governing human OL differentiation and maturation and provide promising therapeutic targets for future studies aimed at restoring myelin integrity and improving outcomes in demyelinating diseases.

22.1 BackgroundAdipogenesis is a highly organised series of events that facilitates the healthy expansion of adipose tissue, beginning during embryogenesis and continuing throughout life. White adipogenesis protects against lipotoxicity, influencing insulin resistance and obesity-related comorbidities. Brown adipogenesis enhances energy expenditure, thereby counteracting weight gain, lipotoxicity and insulin resistance. Recently, there has been a significant increase in interest regarding adipocyte differentiation, mainly focusing on the interplay between microRNAs (miRNAs) and the transcriptional cascade that governs adipogenesis and metabolic dysfunction. This study aimed to identify miRNAs regulating white and brown adipocyte differentiation and define miRNA action in a stem cell model of adipogenesis. 2.2 MethodsSmall RNAseq analysis of primary mouse brown and white adipocytes (WAs) identified miR-10b to be upregulated in mature brown adipocytes (BAs). We generated two model systems: 1) immortalized brown pre-adipocytes treated with miRNA inhibitors and 2) CRISPR/Cas9 KO of miR-10b in E14 mouse embryonic stem cells (mESCs). Both cell models were differentiated into mature adipocytes. To unravel the pathways that are affected by miR-10b depletion, a transcriptomic analysis was performed at key time points. 2.3 ResultsBoth cell models showed that miR-10b-5p depletion severely impaired differentiation into mature adipocytes, as indicated by a lack of lipid droplet formation and reduced adipogenic gene expression. Gene expression analysis supports that miR-10b-5p directs embryonic stem (ES) cells towards the mesoderm lineage, promoting commitment to pre-adipocytes by downregulating Gata6 and its downstream target Bmp2. This mechanism appears to be unaffected in BAs. Our study demonstrated that miR-10b-5p regulates the later stages of adipogenesis, at least in part, by downregulating Tub, a direct target of miR-10b-5p. We also confirmed that miR-10b-5p alleviated the halted differentiation phenotypes of adipocytes by supressing the G Protein Signalling pathway mediated by Tubby. 2.4 ConclusionsThese results evidence that miR-10b inhibition plays a dynamic role in adipocyte biology, as its inhibitory effects manifest differently during the stem cell preadipocyte proliferation state and during the maturation phase of adipocytes. Collectively, our study demonstrated that miR-10b-5p may represent a new potential therapeutic target for lipodystrophy and obesity.

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.

BackgroundChronic kidney disease (CKD) is a growing public health emergency with a global prevalence of approximately 14%. Sarcopenia is a common complication of CKD contributing to functional decline and poor outcomes. However, the molecular mechanisms driving muscle wasting in CKD remain incompletely understood. This study aimed to characterise the transcriptomic profile in individuals with CKD compared to healthy control counterparts, to identify key pathways implicated in muscle dysfunction. MethodsVastus lateralis muscle biopsy samples were obtained from n=10 people with CKD and n=9 healthy controls matched for age, sex, ethnicity and physical activity. Bulk RNA sequencing was performed on all samples. Differential gene expression was assessed using DESeq2 and pathway enrichments analyses were conducted using Gene Ontology (GO) and KEGG databases. ResultsA total of 76 genes were differentially expressed in CKD muscle (FDR < 0.05, |log2FC| [&ge;] 1), with 62 downregulated and 14 upregulated. Transcriptomic analysis revealed suppression of immune-related pathways, including leukocyte chemotaxis and macrophage-associated signalling (e.g., CD163, CXCL14, MPEG1). GO and KEGG analyses further supported downregulation of immune surveillance and inflammatory pathways. Several genes implicated in muscle regeneration (e.g., MEGF10, PODN, SOX4) were also differentially expressed, suggesting impaired regenerative signalling. Classical markers of myogenesis and protein degradation were unchanged, indicating a blunted rather than overtly inflammatory or catabolic muscle environment. ConclusionsSkeletal muscle in CKD exhibits a distinct transcriptional profile marked by suppression of immune and regenerative processes. These findings refine our understanding of CKD-associated sarcopenia and may inform the development of targeted therapeutic strategies beyond conventional exercise-based interventions.