Oyster cellular architecture and innate immune programs resolved by single-cell RNA sequencing.

Rotator cuff tear (RCT) is a prevalent age-related condition whose underlying mechanisms remain poorly understood. This study employed spatial transcriptomics and multiplex immunofluorescence (mIF) to investigate gene expression and spatial heterogeneity in rotator cuff tissues from elderly RCT patients compared to age-matched controls, aiming to uncover key molecular pathways. Tendon samples were collected from RCT patients (n = 10) and controls (n = 10). Five from each group underwent spatial transcriptomic sequencing for differential gene expression, functional enrichment, and cell interaction analyzes. Results were validated with mIF on the remaining samples. Compared to controls, the RCT group showed 1261 downregulated and 2789 upregulated genes. Spatial analysis revealed distinct expression gradients: COMP and CHI3L1 were upregulated in the bone region, CHI3L1 and MT1X in the mid-tendon, and MT1X and FMOD in the tendon area-confirmed by mIF. Biological processes also varied regionally: cartilage development and extracellular matrix (ECM) organization were enriched in the bone, while ECM and collagen fibril organization dominated mid-tendon and tendon regions. The PI3K-AKT and ECM-receptor interaction pathways were central to these processes. Tenogenic progenitor-like cells (TPLCs) were significantly reduced in RCT (p < 0.0001), whereas mesenchymal cells increased in bone and mid-tendon areas (p < 0.001, p < 0.01), consistent with structural gradients. These findings suggest that elderly RCT may arise from chronic inflammation, ECM dysregulation, and failed regeneration. Spatial transcriptomics identified repair-related genes (COMP, CHI3L1, MT1X, FMOD) with region-specific expression, providing new insights into pathology and potential therapeutic targets.

Aging is a major risk factor for breast cancer, yet how it shapes tumor development, molecular phenotype, and immune evasion remains incompletely understood. Deciphering how aging influences cancer evolution is critical for improving risk assessment, prevention, and treatment. Here, using a N-nitroso-N-methylurea (NMU)-induced rat mammary tumor model that recapitulates key features of human breast cancer, we integrated bulk and single-cell transcriptomics, whole-exome sequencing, and histopathological analysis to dissect age-associated differences in mammary tumorigenesis. We found that the age at NMU exposure critically influences tumor incidence, mutational burden, molecular subtype, and the tumor immune microenvironment. Tumors arising in aged rats originated from aging luminal progenitor-like cells, exhibited increased genomic instability, reduced immune cell infiltration, and impaired antigen presentation linked to loss of heterozygosity at chromosome (Chr) 20p. The age-associated epithelial and immune changes we identified were conserved in human breast cancers, where the loss of the homologous Chr 6p region correlated with reduced lymphocyte infiltration and shorter relapse-free survival. These findings reveal that aging profoundly affects tumor-initiating cell populations and promotes immune evasion through chromosomal instability-driven defects in antigen presentation. Our work provides a molecular basis for understanding disease onset and progression that may impact efficacy of immunotherapy in older breast cancer patients.

A critical knowledge gap in prostate cancer research is understanding whether castration-tolerant progenitor-like cells that reside in treatment-naïve tumors play a direct role in therapy resistance and tumor progression. Herein, we reveal that the castration tolerance of LSCmed (Lin-, Sca-1+, CD49fmed) progenitor cells, the mouse equivalent of human prostatic Club cells, arises not from intrinsic properties, but from significant transcriptional reprogramming. Utilizing single-cell RNA sequencing of LSCmed cells isolated from prostate-specific Pten-deficient (Ptenpc-/-) mice, we identify the emergence of castration-resistant LSCmed cells enriched in stem-like features, driven by the transcription factor FOSL1/AP-1. We demonstrate that cells exhibiting Ptenpc-/- LSCmed characteristics are prevalent in aggressive double-negative prostate cancer (DNPC) subtypes recently identified in human castration-resistant prostate cancer (CRPC). Furthermore, our findings show that the dual-targeting agents JQ-1 and CX-6258-focused on FOSL1/AP-1 and PIM kinases, respectively-effectively suppress both the progenitor properties and the growth of mouse and human DNPC surrogates in vitro and in vivo. Thus, early eradication of castration-tolerant Club-like cells presents a promising therapeutic strategy to mitigate prostate cancer progression toward CRPC.

Hepatocyte-specific partial cellular reprogramming via selective OSK mRNA lipid nanoparticle attenuates liver fibrosis.

Wnt-dependent modulation of alveolar epithelial phenotypes and barrier function in human progenitor-like cells.

Sex-based disparities remain a major challenge in musculoskeletal medicine. Women and men experience different anterior cruciate ligament (ACL) injury rates and severity, but the causes remain unclear. We hypothesized that cellular differences in human progenitor cells contribute to the higher ACL tear risk observed in females. ACL samples were collected from 4 male and 5 female patients undergoing ACL reconstruction surgery. Live cells were collected through flow cytometry and sent for single-cell RNA sequencing. Significantly greater expression in either sex relative to the other was defined as a >25% increase in expression level (log2 fold change > 0.32) and p < 0.05). Subpopulation characterization was performed with immunofluorescence on tissue sections. We discovered sex-based differences in all of the native cell types within the ACL. In particular, fibroblast progenitor-like (TPPP3+) cells from female patients expressed genes associated with dysregulation and degradation of collagen more highly than progenitor cells from male patients. These results highlight a ligament progenitor population with a sex-dependent gene expression profile. This work suggests that sex-based differences in stem cell populations may drive differential injury rates and outcomes between male and female patients with musculoskeletal injuries. The differential gene expression among TPPP3+ progenitor-like cells provides a possible target population for studying ligamentous injury and regeneration. Differential expression of collagen and extracellular matrix-related genes provides evidence of specific genes that could be therapeutically targeted to strengthen the ACL and reduce the risk of rupture, particularly in female athletes.

Abstract Introduction ZFTA-RELA (ZR) is the most recurrent genetic alteration seen in pediatric supratentorial ependymoma (EPN) and is sufficient to initiate tumors in mice. Despite the oncogenic potential of ZR, it is observed nearly exclusively in childhood EPN, with tumors located distinctly in the supratentorial region of the central nervous system (CNS). We hypothesized that specific chromatin modules accessible during brain development would render distinct cell lineage programs at direct risk of transformation by ZR. Methods To this end, we performed combined single nucleus ATAC and RNA (snMultiome) sequencing of the developing mouse forebrain, as compared to ZR-driven mouse and human EPN. Results We demonstrate that specific developmental lineage programs present in transient progenitor cells and regulated by PLAG/L family transcription factors (TF) are at risk of neoplastic transformation. Binding of this chromatin network by ZR or other PLAG/L family motif targeting fusion oncoproteins lead to persistent chromatin accessibility at oncogenic loci and oncogene expression. Cross-species analysis of mouse and human ZR EPN reveals significant cell type heterogeneity mirroring incomplete neurogenic and gliogenic differentiation, with a small percentage of cycling progenitor-like or radial glial-like cells that establish a putative tumor cell hierarchy. In vivo lineage tracing studies reveal neoplastic clones that aggressively dominate tumor growth and establish the entire EPN cellular hierarchy. Conclusions These findings unravel developmental epigenomic states critical for fusion oncoprotein-driven transformation and elucidate how these states continue to shape tumor progression. Citation Format: Alisha Kardian, Hua Sun, Stephen Mack. Dominant Malignant Clones Leverage Lineage Restricted Epigenomic Programs to Drive Ependymoma Development [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr LT004.

Glioblastoma is characterized by heterogeneous and plastic cellular populations that adopt transcriptional programs shaped by genetic alterations and microenvironmental cues. Recent studies have identified at least four partially inconvertible cell states-astrocytic-like, neural progenitor-like, oligodendrocyte progenitor-like, and mesenchymal-like-that represent aberrant developmental programs. Expanded analysis further reveals hybrid and intermediate states that form continuous transcriptional and metabolic gradients. These states exhibit spatial organization, assembling into three distinct microanatomical niches: a perivascular niche enriched with mesenchymal-like and oligodendrocyte progenitor-like cells, a hypoxic niche harboring quiescent and stressed cells of all states, and an invasive niche containing astrocyte-like or proneural populations. Niches continuously remodel as cell states transition, migrate, and re-establish new programming in response to angiogenesis, hypoxia, immune infiltration, and neuronal activity. This interplay between states and the microenvironment generates a self-renewing spatial architecture, maintaining expansion at the edge and protection within the core. This review integrates single-cell, single-nucleus, and spatial studies to describe a microenvironmental-driven model of cell state organization. Understanding how these multiscale drives converge to generate a continuum of cell state identities may reveal strategies to disrupt the spatial architecture underlying glioblastoma plasticity and recurrence.

Adult pancreatic tissue contains cell populations with latent regenerative potential, but the processes governing their expansion and differentiation into endocrine lineages remain unclear. Adult human pancreatic cells obtained from donor tissue were isolated and expanded and analyzed for lineage potential using single-cell RNA sequencing, flow cytometry, and functional assays. A CD9+ PROCR+ RGS16+ subpopulation, termed islet progenitor-like cells (IPCs), was evaluated for proliferative capacity and differentiation potential. IPCs exhibited robust proliferative capacity and, upon differentiation, formed insulin- and glucagon-secreting organoids. Treatment of IPCs with the small molecule ISX9 induced expression of key transcription factors RFX6 and NEUROD1 through calcium-dependent chromatin remodeling mediated by NFAT recruitment of p300 and displacement of histone deacetylases (HDAC1-3). Pharmacologic inhibition of HDACs further enhanced IPC maturation and glucose-stimulated insulin secretion. These findings define the molecular and epigenetic mechanisms driving the expansion and differentiation of adult IPCs into functional islet-like organoids, providing a foundation for future regenerative approaches using adult pancreatic tissue as a renewable source for endocrine cell replacement.

The Stathmin 1-lineage Contributes to Acinar Regeneration but not to Neoplasia Upon Oncogenic Kras Expression.

MAP2K4 encodes a kinase that activates the c-Jun N-terminal kinase (JNK) pathway, which is essential for human neurodevelopment. While somatic MAP2K4 loss has been observed in cancer, germline variants have not previously been linked to human disease. We describe ten individuals with de novo or presumed de novo MAP2K4 variants who present with a novel syndromic neurodevelopmental disorder. Shared features include developmental delay or intellectual disability, epilepsy, and variable congenital malformations, most commonly affecting the genitourinary system. To define the mechanism, we generated CRISPR-edited iPSC-derived neurons with MAP2K4 deficiency. These neurons showed reduced JNK pathway activation and abnormal differentiation, characterized by persistence of progenitor-like cells and disrupted neurite morphology. Our findings establish MAP2K4 as a Mendelian neurodevelopmental disorder gene and identify impaired JNK signaling as the underlying mechanism. More broadly, this work expands the spectrum of JNK-pathway disorders and underscores the critical role of JNK signaling in human brain development.

The impact of circadian rhythm disruption on immune system function has been widely studied; however, the precise mechanisms by which it modulates immune system structure and function at the single-cell level remain poorly understood. This study was conducted from April to November 2024 in the Department of Pediatrics, the Third Xiangya Hospital, Central South University. In this study, a zebrafish model of circadian rhythm disruption was established by exposing larvae to constant light (24 h LL) for three days, while control groups were maintained under a standard 14-hour light/10-hour dark (14∶10 h LD) cycle. Single-cell RNA sequencing was performed to comprehensively profile the immune cells from both groups. Immune cell subpopulations, their proportional shifts, and developmental trajectories were characterized through UMAP-based clustering, marker gene annotation, and Monocle pseudotime trajectory analysis. A total of seven immune-related cell subtypes were identified, including hematopoietic progenitors, T cell progenitor-like cells, activated T cell-like cells, macrophages, neutrophils, NK cells, and migratory/repair-associated cells. Notably, the proportion of activated T cells was increased in the circadian rhythm-disrupted group (18.11%) compared with the control group (7.61%), accompanied by enhanced expression of immune activation markers such as cd40lg, il2rb,tnfrsf9b (log2FC=7.49, 5.58, 3.76, adjusted P<0.01). Pseudotime analysis revealed bifurcated differentiation paths along myeloid and lymphoid lineages. Although the core developmental trajectories remained intact under circadian rhythm disturbance, alterations were observed in the distribution and maturation pace of terminal lymphoid cells. Overall, the findings demonstrate that circadian rhythm disruption induces structural and functional remodeling of the zebrafish immune system, characterized by enhanced activation of T and NK cells and a shift in immune response status. These results provide single-cell-level insights into the immunological mechanisms underlying sleep-related disorders.

Abstract H3K27M-mutant gliomas are lethal pediatric brain tumors driven by progenitor-like cells that subvert normal differentiation but retain some developmental plasticity, as evidenced by the presence of partially differentiated, non-tumorigenic H3K27M-positive cells within the tumor mass. This progenitor-to differentiated fate transition is also induced by the common cell culture supplement, serum. However, the identity of the pro-differentiation serum component is a key unanswered question. Through integrated lipidomic, chromatin, and single-cell transcriptomic analyses, we identify n-3 polyunsaturated fatty acids (n3-PUFAs) as the key serum and dietary components necessary and sufficient to induce differentiation of H3K27M glial progenitors. Remarkably, dietary enrichment of n3-PUFAs triggers this developmental shift in H3K27M-mutant glioma orthotopic models, reduces tumor burden, and increases survival. These findings uncover a previously unrecognized role for n3-PUFAs in governing glioma cell fate with promising therapeutic implications. Ongoing studies are focused on elucidating the molecular mechanisms whereby n3-PUFAs drive this differentiation program.

Abstract Pediatric cancers are often driven by unique alterations suggesting mechanistic ties to underlying developmental programs. Ependymoma (EPN) is an example of a brain cancer subtype driven by gene fusions involving ZFTA-RELA (ZR) that are exclusive to this disease. This leads to our hypothesis that specific chromatin states in developmental lineage programs are at risk of oncogenic transformation, and that these chromatin signatures persist during tumor development and govern tumor cell heterogeneity. To address this question, we constructed an integrated single-nucleus Multiome (snRNA-seq and snATAC-seq) atlas of the developing mouse forebrain and compared it with snMultiome analysis of ZR-driven mouse and human EPN. We identified specific developmental lineage programs present in glial progenitor cells enriched with the Plagl family transcription factors; the precise DNA binding sites of ZFTA fusion oncoproteins. In both mouse and human EPN, ZR expression maintains accessibility of developmental epigenomic programs leading to persistent cell proliferation and tumorigenicity. Cross-species analysis of mouse and human EPN revealed significant cell type heterogeneity mirroring incomplete neurogenic and gliogenic differentiation, with a small percentage of cycling intermediate progenitor-like cells that established a putative tumor cell hierarchy. These findings were expanded by applying in vivo lineage barcode tracing methods in mouse EPN, which revealed dominant cell lineages that aggressively expanded and recapitulated the entire tumor cell diversity. Our findings unravel the intersection between developmental and oncogenic epigenomic states critical for ZR driven brain cancer. These findings shape our understanding of the earliest stages of epigenetic programs in brain cancer, and how cancer drivers such as ZR intersect with developmental programs to establish tumor cell heterogeneity.

Liver transplantation remains constrained by the scarcity of donor organs and the risks inherent in the procedure, underscoring the urgent need for novel cirrhosis therapies. We developed a protocol to convert human primary hepatocytes into expandable hepatocyte-derived liver progenitor-like cells (HepLPCs), which secrete high levels of matrix metalloproteinases and hepatocyte growth factor. In a thioacetamide-induced rat model of cirrhosis, human HepLPCs demonstrated potent anti-fibrotic properties and promoted liver regeneration. Biodistribution studies revealed that most xenogenic HepLPCs were cleared from the body within one week, suggesting that their therapeutic benefits likely arise from paracrine signaling rather than long-term engraftment. We initiated a first-in-human clinical trial involving nine patients with cirrhosis to evaluate the feasibility and safety of HepLPCs. Preclinical toxicity assessments in 36 crab-eating macaques confirmed the safety of HepLPC treatment. In the clinical trial, nine patients (mean age: 53 years), primarily with HBV-related cirrhosis, received HepLPCs via trans-hepatic arterial infusion without immunosuppressants. No serious adverse event was observed, and the minor adverse events were consistent with those commonly seen in cirrhosis patients. The treatment was well tolerated, with no transfusion reactions or dose-limiting toxicities. While significant changes in Child-Pugh and MELD scores were not observed, some patients showed improvements in liver biochemical parameters, coagulation profiles, and portal hypertension indicators during the six-month follow-up. These findings indicate that HepLPC therapy is safe and feasible, offering a promising new strategy for treating cirrhosis. Further clinical trials are needed to assess its efficacy in patients with decompensated cirrhosis and acute-on-chronic liver failure.

Matrix stiffness regulates profibrotic fibroblast differentiation and fibrotic niche activation in systemic sclerosis.

The endometrium is a hormonally responsive tissue that undergoes cyclic remodeling. Although endometrial organoids have been established in several species, detailed characterization remains limited. Here, we assessed the structural and molecular fidelity of equine endometrial organoids across reproductive cycle stages and during extended culture. Organoids were generated from biopsies collected during estrus and diestrus and analyzed using histology, immunohistochemistry, electron microscopy, and bulk- and single-cell RNA sequencing. Organoids formed polarized cystic structures composed of columnar cells with microvilli, tight junctions, and secretory vesicles. Compared to native tissue, organoids showed higher expression of genes involved in proliferation and metabolism, and lower expression of genes related to differentiation, angiogenesis, and immune responses. Single-cell analysis identified diverse epithelial and stromal populations in both tissue and organoids. While most cell types were preserved, organoids were enriched in progenitor-like cells but underrepresented in ciliated, proliferative glandular, endothelial, smooth muscle, and antigen-presenting cells. Cycle-specific differences were observed in morphology, hormone receptor expression, and gene expression profiles. Estrus-derived organoids showed increased proliferation and metabolic activity. Although organoids retained transcriptional signatures reflective of the hormonal cycle stage of the source tissue, these signatures faded with prolonged culture, despite overall transcriptomic stability. In summary, equine endometrial organoids replicate key features of the native tissue, retain reproductive cycle characteristics, and maintain transcriptomic stability over time. Endometrial organoids provide a robust platform to study the equine endometrium, though native tissue differences should be considered in the experimental design and data interpretation.

Adipose tissue heterogeneity has emerged as a central factor in regulating adipose tissue function in physiology and pathophysiology, yet tools to model and study this diversity in vitro remain limited. Here, we performed single-cell RNA sequencing on cultured primary white and brown preadipocytes to assess how in vitro conditions impact progenitor identity. We identified two major subpopulations in both depots: committed adipogenic precursors (CAPs) and fibro-adipogenic progenitor-like cells (FAPLs). Remarkably, FAPLs were also present in brown adipose tissue, expanding the known landscape of progenitor populations in this depot. Trajectory and regulon analyses revealed that both white and brown FAPLs exhibit similar pro-fibrotic, stress-responsive signatures and diverge early from proliferating progenitor states. Integration of datasets showed that FAPLs from both depots cluster together, emphasizing their conserved identity, while CAPs remain depot-specific. Comparison to previously published in vivo single-cell datasets revealed that these in vitro populations, including brown adipose FAPLs, correspond to adipose-resident progenitor subtypes, validating the physiological relevance of this model for studying adipose tissue heterogeneity and development.

Abstract BACKGROUND Gangliogliomas (GGs) are rare glio-neuronal tumors frequently associated with pharmacoresistant focal epilepsy. Although patients often achieve favorable seizure outcomes following surgery, the cellular and molecular basis of epileptogenesis in GGs remains poorly understood. In this study, we focus on the role of extracellular matrix (ECM) remodeling and immature neuronal components in shaping the tumor microenvironment and driving epileptogenicity. MATERIAL AND METHODS We analyzed spatial transcriptomic profiles of eight GG samples (BRAF wild-type, n = 3; BRAF-mutant, n = 5) using the 10X Visium platform. Data were processed with SPATA2. Cell type deconvolution (cell2location) and Weighted Gene Correlation Network Analysis (WGCNA) were applied to characterize transcriptional programs within the tumor microenvironment. RESULTS WGCNA identified eight transcriptional modules reflecting distinct cellular programs. These included signatures of astrogliosis and oligodendroglial reactivity with active myelination (GFAP, S100A1, MBP, PLP1), as well as neuron-rich clusters enriched for synaptic signaling (SNAP25, SYP; p &amp;lt; 0.001) and plasticity-associated genes (ENC1, CHGA, CHGB; p &amp;lt; 0.001). Immature neuronal populations showed enrichment for progenitor-like genes (FABP7, HOPX), indicative of impaired neuronal maturation. Additional clusters reflected reactive gliosis (THY1, PDGFRA, SERPINA3; p &amp;lt; 0.001) and immune infiltration involving microglia and macrophages (CD63, FGF12, S100A10; p &amp;lt; 0.001), suggesting immune-tumor interactions. ECM-related transcriptional programs were upregulated in regions with histological evidence of matrix remodeling and pial boundary disruption, implying increased infiltrative potential—features previously linked to higher seizure risk and recurrence. CONCLUSION GGs are composed of both glial and neuronal elements, including a population of progenitor-like neuronal cells and infiltrating immune components. Our findings highlight inflammatory ECM remodeling as a central feature that shapes the tumor microenvironment and might interact with immature and dysfunctional neuronal populations to promote epileptogenicity.