To evaluate the feasibility, safety and efficacy of the cutaneous application of Bioengineered Artificial Mesenchymal Sheet (BAMS) in venous leg ulcers (VLUs) versus conventional treatment.

JAK inhibitors (JAKi) are widely used anti-inflammatory drugs. Recent data suggest JAKi have superior effects on pain reduction in rheumatoid arthritis (RA). However, the underlying mechanisms for this observation are not fully understood. We investigated whether JAKi can act directly on human sensory neurons. We analysed RNA sequencing datasets of sensory neurons and found they expressed JAK1 and STAT3. Addition of cell-free RA synovial fluid to human induced pluripotent stem cell (iPSC)-derived sensory neurons led to phosphorylation of STAT3 (pSTAT3), which was completely blocked by the JAKi tofacitinib. Compared to paired serum, RA synovial fluid was enriched for the STAT3 signalling cytokines IL-6, IL-11, LIF, IFN-alpha and IFN-beta, with their requisite receptors present in peripheral nerves post-mortem. Accordingly, these recombinant cytokines induced pSTAT3 in iPSC-derived sensory neurons. Furthermore, IL-6+sIL-6R and LIF upregulated expression of pain-relevant genes with STAT3-binding sites, an effect which was blocked by tofacitinib. LIF also induced neuronal sensitisation, highlighting this molecule as a putative pain mediator. Finally, over time, tofacitinib reduced the firing rate of sensory neurons stimulated with RA synovial fluid. Together, these data indicate that JAKi can act directly on human sensory neurons, providing a potential mechanistic explanation for their suggested superior analgesic properties.

The transforming growth factor β (TGFβ) signaling pathway is critical for survival, proliferation, and cell migration, and is tightly regulated during cardiovascular development. Smads, key effectors of TGFβ signaling, are sequestered by microtubules (MTs) and need to be released for pathway function. Independently, TGFβ signaling also stabilizes MTs. Molecular details and the in vivo relevance of this cross-regulation remain unclear, understanding which is important in complex biological processes such as cardiovascular development. Here, we use rudhira/Breast Carcinoma Amplified Sequence 3 (Bcas3), an MT-associated, endothelium-restricted, and developmentally essential proto-oncogene, as a pivot to decipher cellular mechanisms in bridging TGFβ signaling and MT stability. We show that Rudhira regulates TGFβ signaling in vivo, during mouse cardiovascular development, and in endothelial cells in culture. Rudhira associates with MTs and is essential for the activation and release of Smad2/3 from MTs. Consequently, Rudhira depletion attenuates Smad2/3-dependent TGFβ signaling, thereby impairing cell migration. Interestingly, Rudhira is also a transcriptional target of Smad2/3-dependent TGFβ signaling essential for TGFβ-induced MT stability. Our study identifies an immediate early physical role and a slower, transcription-dependent role for Rudhira in cytoskeleton-TGFβ signaling crosstalk. These two phases of control could facilitate temporally and spatially restricted targeting of the cytoskeleton and/or TGFβ signaling in vascular development and disease.

Zinc finger protein 750 (ZNF750) has been identified as a potential tumor suppressor across multiple malignancies. Nevertheless, the specific involvement of ZNF750 in the regulation of mesenchymal cell differentiation and bone homeostasis has yet to be elucidated. In the current study, we observed a substantial presence of ZNF750 in bone tissue and noted alterations in its expression during osteogenic differentiation of mesenchymal progenitor cells. Functional experiments indicated that ZNF750 promoted osteogenic differentiation while impeding adipogenic differentiation from mesenchymal stem/progenitor cells. Further mechanistic investigations revealed that ZNF750 transcriptionally suppressed the expression of Snail family transcriptional repressor 1 (SNAI1) by binding to the proximal promoter region of Snai1 gene, thereby activating Wnt/β-catenin signaling. SNAI1 exerted opposing effects on cell differentiation towards osteoblasts and adipocytes in comparison to ZNF750. The overexpression of SNAI1 counteracted the dysregulated osteogenic and adipogenic differentiation induced by ZNF750. Furthermore, the transplantation of Znf750-silenced bone marrow stromal cells into the marrow of wild-type mice resulted in a reduction in cancellous and cortical bone mass, alongside a decrease in osteoblasts and an increase in marrow adipocytes, while the number of osteoclasts remained unchanged. This study presents the first demonstration that ZNF750 regulates the differentiation of osteoblasts and adipocytes from mesenchymal stem/progenitor cells by transcriptionally deactivating SNAI1 signaling, thereby contributing to the maintenance of bone homeostasis. It suggests that ZNF750 may represent a promising therapeutic target for metabolic bone disorders such as osteoporosis.

Renal artery stenosis (RAS) is the leading cause of secondary hypertension worldwide. However, current medical and surgical treatment modalities provide minimal benefits for kidney injury. Recent preclinical RAS models have demonstrated promising potential of human mesenchymal stem cells (MSC) and their daughter extracellular vesicles (EV) in improving murine renal function and attenuating inflammation. However, the extent and mechanisms underlying immune rejection of xenogeneic MSCs or EVs are yet undetermined. Therefore, adipose tissue was harvested from adult healthy patients. Adipose-derived MSCs were extracted and cultured, and EVs were isolated from their supernatants via ultra-centrifugation. Then, mice randomly assigned to RAS or sham surgery were divided into 6 groups: sham surgery, RAS, sham + MSC, RAS + MSC, sham + EV, and RAS + EV. Two weeks after intra-aortic injection of MSCs (5 × 105) or EVs (20 µg protein), we compared the intrarenal T-cell and macrophage accumulation, splenic B-cell numbers, circulating cytokines and anti-human antibodies levels among the groups. MSCs and EVs did not influence intrarenal immune cell infiltrations. However, MSCs significantly increased circulating anti-human antibodies. In the spleen, RAS + EV mice showed higher memory IgM+ B-cells but reduced CD19+ B-cells compared to RAS + MSC. In vitro T-cell recall assay showed that both MSCs and EVs exhibited reduced IFN-γ release upon re-stimulation, indicating an immunosuppressive effect. Therefore, xenogeneic MSCs induced a greater humoral response in mice, while EVs triggered a splenic cellular response, but neither elicits discernible kidney rejection. Our results provide key insights into the immunomodulatory mechanisms of MSCs and EVs and immune mechanisms underlying xenograft rejection.

In this single-center, double-blinded, randomized controlled trial, we investigated whether human umbilical cord-derived mesenchymal stromal cells loaded collagen scaffolds (hUC-MSC/CS) could improve the cumulative live-birth rate (cLBR) in infertile women with refractory thin endometrium (RTE). We randomly assigned 25 subfertile women with RTE, in a 1:1 ratio, to receive hysteroscopic adhesiolysis and plowing plus either hUC-MSC/CS or saline/CS (control) for intrauterine implantation. Uterine fluid was collected on the embryo transfer day for RNA-sequencing to explore the potential mechanisms by which hUC-MSCs exert their effects. The primary outcome was the cLBR. Live births occurred in 3 out of 11 women in the hUC-MSC/CS group and in 1 out of 13 women in the control group (27.3% vs 7.7%; relative risk [RR], 3.55; 95% confidence interval [CI], 0.43 to 29.42; P = .30). The cumulative frequencies of clinical pregnancy were 5/11 and 1/13 in the hUC-MSC/CS group and control group, respectively (45.5% vs. 7.7%; RR, 5.91; 95% CI, 0.81-43.28; P = .06). Two of 11 participants developed urticaria in the hUC-MSC/CS group. Enrichment analysis showed that T-cell activation had the largest proportion in the biological process category. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that most genes were related to cytokine-cytokine receptor interaction. In conclusion, there was a non-significant trend toward a higher cLBR with hUC-MSC/CS compared to controls, potentially through the cytokine-cytokine receptor interaction pathway. hUC-MSCs appeared to be relatively safe in a 1-year follow-up. Therefore, this novel therapy can be proposed to patients with RTE.

In both Drosophila and mammals, the brain contains the most diverse population of cell types of any tissue. It is generally accepted that transcriptional diversity is an early step in generating neuronal and glial diversity, followed by the establishment of a unique gene expression profile that determines morphology, connectivity, and function. In Drosophila, there are two types of neural stem cells, called Type 1 (T1) and Type 2 (T2) neuroblasts. The diversity of T2-derived neurons contributes a large portion of the central complex (CX), a conserved brain region that plays a role in sensorimotor integration. Recent work has revealed much of the connectome of the CX, but how this connectome is assembled remains unclear. Mapping the transcriptional diversity of T2-derived neurons is a necessary step in linking transcriptional profile to the assembly of the adult brain. Here we perform single nuclei RNA sequencing of T2 neuroblast-derived adult neurons and glia. We identify clusters containing all known classes of glia, clusters that are male/female enriched, and 161 neuron-specific clusters. We map neurotransmitter and neuropeptide expression and identify unique transcription factor combinatorial codes for each cluster. This is a necessary step that directs functional studies to determine whether each transcription factor combinatorial code specifies a distinct neuron type within the CX. We map several columnar neuron subtypes to distinct clusters and identify two neuronal classes (NPF+ and AstA+) that both map to two closely related clusters. Our data support the hypothesis that each transcriptional cluster represents one or a few closely related neuron subtypes.

Non-obstructive azoospermia (NOA) belongs to male infertility due to spermatogenesis failure. However, evidence for cell type-specific abnormalities of spermatogenesis disorders in NOA remains lacking. We performed single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on testicular tissues from patients with obstructive azoospermia (OA) and NOA. HE staining confirmed the structural abnormalities of the seminiferous tubules in NOA patients. We identified 12 germ cell subtypes (spermatogonial stem cell-0 [SSC0], SSC1, SSC2, diffing-spermatogonia [Diffing-SPG], diffed-spermatogonia [Diffed-SPG], pre-leptotene [Pre-Lep], leptotene-zygotene [L-Z], pachytene [Pa], diplotene [Di], spermatids-1 [SPT1], SPT2, and SPT3) and 8 Sertoli cell subtypes (SC1-SC8). Among them, three novel Sertoli cell subtype phenotypes were identified, namely SC4/immature, SC7/mature, and SC8/further mature Sertoli cells. For each germ or Sertoli cell subtype, we identified unique new markers, among which immunofluorescence confirmed co-localization of ST3GAL4, A2M, ASB9, and TEX19 and DDX4 (classical marker of germ cell). PRAP1, BST2, and CCDC62 were co-expressed with SOX9 (classical marker of Sertoli cell) in testes tissues also confirmed by immunofluorescence. The interaction between germ cell subtypes and Sertoli cell subtypes exhibits stage-specific-matching pattern, as evidenced by SC1/2/5/7 involving in SSC0-2 development, SC3 participating in the whole process of spermiogenesis, SC4/6 participating in Diffing and Diffed-SPG development, and SC8 involving in the final stage of SPT3. This pattern of specific interactions between subtypes of germ cell and Sertoli cell was confirmed by immunofluorescence of novel markers in testes tissues. The interaction was mainly regulated by the Notch1/2/3 signaling. Our study profiled the single-cell transcriptome of human spermatogenesis and provided many potential molecular markers for developing testicular puncture-specific marker kits for NOA patients.

Mechanisms by which mucosal regeneration is abrogated in inflammatory bowel disease (IBD) are still under investigation, and a role for an intestinal stem cell (ISC) defect is now emerging. Herein, we report an abnormal ISC death that occurs in Crohn's disease, which exacerbates colitis, limits ISC-dependent mucosal repair, and is controlled through the death factor Transmembrane protein 219 (TMEM219). Large alterations in TMEM219 expression were observed in patients with Crohn's disease, particularly in those with active disease and/or those who were nonresponders to conventional therapy, confirming that TMEM219 signaling is abnormally activated and leads to failure of the mucosal regenerative response. Mechanistic studies revealed a proapoptotic TMEM219-mediated molecular signature in Crohn's disease, which associates with Caspase-8 activation and ISC death. Pharmacological blockade of the IGFBP3/TMEM219 binding/signal with the recombinant protein ecto-TMEM219 restored the self-renewal abilities of miniguts generated from patients with Crohn's disease in vitro and ameliorated DSS-induced and T cell-mediated colitis in vivo, ultimately leading to mucosal healing. Genetic tissue-specific deletion of TMEM219 in ISCs in newly generated TMEM219fl/flLGR5cre mice revived their mucosal regenerative abilities both in vitro and in vivo. Our findings demonstrate that a TMEM219-dependent ISC death exacerbates colitis and that TMEM219 blockade reestablishes intestinal self-renewal properties in IBD.

Inflammatory Bowel Diseases (IBD), including Crohn's disease and ulcerative colitis, pose challenges due to their complex pathophysiology and high prevalence. Despite advances in immune-targeted therapies, a substantial number of patients fail to achieve mucosal healing, highlighting the need for alternative therapeutic strategies. In this issue of the JCI, D'Addio et al. identified another mechanism underlying impaired epithelial regeneration in Crohn's disease. They found that abnormal cell death in intestinal epithelial stem cells, mediated by altered TMEM219 signaling, led to impaired mucosal healing. Targeting TMEM219 with ecto-TMEM219, which blocks its activation, restored stem cell function and promoted mucosal healing in vitro and in vivo. These findings suggest a promising therapeutic avenue focusing on epithelial repair. Additionally, patient-derived organoids (PDOs) emerge as a valuable tool for personalized treatment strategies and for advancing the field of IBD research. This study underscores the importance of epithelial cell biology in developing innovative IBD therapies.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. Clemastine fumarate, the over-the-counter antihistamine and muscarinic receptor blocker, has remyelinating potential in MS. A clemastine arm was added to an ongoing platform clinical trial, targeting residual activity by precision, biomarker-guided combination therapies of multiple sclerosis (TRAP-MS) (ClinicalTrials.gov NCT03109288), to identify a cerebrospinal fluid (CSF) remyelination signature and to collect safety data on clemastine in patients progressing independently of relapse activity (PIRA). The clemastine arm was stopped per protocol-defined criteria when 3 of 9 patients triggered individual safety stopping criteria. Clemastine-treated patients had significantly higher treatment-induced disability progression slopes compared with the remaining TRAP-MS participants. Quantification of approximately 7,000 proteins in CSF samples collected before and after clemastine treatment showed significant increases in purinergic signaling and pyroptosis. Mechanistic studies showed that clemastine with sublytic doses of extracellular adenosine triphosphate (ATP) activates inflammasome and induces pyroptotic cell death in macrophages. Clemastine with ATP also caused pyroptosis of induced pluripotent stem cell-derived human oligodendrocytes. Antagonist of the purinergic channel P2RX7, which is strongly expressed in oligodendrocytes and myeloid cells, blocked these toxic effects of clemastine. Finally, reanalysis of published single-nucleus RNA-Seq (snRNA-Seq) studies revealed increased P2RX7 expression and pyroptosis transcriptional signature in microglia and oligodendrocytes in the MS brain, especially in chronic active lesions. The CSF proteomic pyroptosis score was increased in untreated MS patients, was higher in patients with progressive than relapsing-remitting disease, and correlated significantly with the rates of MS progression. Collectively, this identifies pyroptosis as a likely mechanism of CNS injury underlying PIRA even outside of clemastine toxicity.

Cancer, next to cardiovascular diseases, remains the primary concern of modern medicine in developed countries. Despite the unprecedented progress in targeted therapies and personalised medicine, including immunotherapy and gene therapy, we are still unable to efficiently treat many malignancies. One of the major obstacles to treating cancer is its ability to metastasise. Hence, a better understanding of cancer biology with emphasis on the metastasis formation may hold the key to further ameliorating cancer treatment. Nowadays, there is a growing body of evidence for the common denominator of neoplasia, which seems to be universal - cancer stem cells which are being found in a growing number of cancers.

The interaction between CD47 expressed on cancer cells and signal regulatory protein-α located on macrophages blocks the phagocytosis of tumor cells by macrophages. Our data reveal that human endometrial cancer cells (hECCs) upregulate the CD47 level on their surface and that there is a high density of tumor-associated macrophages within the microenvironment of human endometrial cancer. In vitro functional assay shows that an anti-CD47 monoclonal antibody (mAb) promotes the phagocytosis of hECCs by macrophages. Systemic and in situ treatments with an anti-CD47 mAb effectively reduce tumor burden in vivo in a genetically engineered mouse model of endometrial cancer. Thus, this study provides preclinical evidence that CD47 blockade using an anti-CD47 mAb to augment macrophage phagocytosis is a potential therapeutic strategy for endometrial cancer.

Liver fibrosis has been proposed as the most important predictive indicator affecting prognosis of patients with chronic liver disease. It is defined by an abnormal accumulation of extracellular matrix components that results from necrotic and inflammatory processes and eventually impairs organ function. With no approved therapy, comprehensive cellular models directly derived from patient's cells are necessary to understand the mechanisms behind fibrosis and the response to anti-fibrotic therapies. Primary human cells, human hepatic cell lines and human stem cells-derived hepatic stellate-like cells have been widely used for studying fibrosis pathogenesis. In this paper, we depict the cellular crosstalk and the role of extracellular matrix during fibrosis pathogenesis and summarize different in vitro models from simple monolayers to multicellular 3D cultures used to gain deeper mechanistic understanding of the disease and the therapeutic response, discussing their major advantages and disadvantages for liver fibrosis modelling.

BRCA1/2 genes are considered tumor suppressor genes and help repair damaged DNA. Pathogenic germline mutations of BRCA1/2 genes are the most common hereditary cause of breast cancer and ovarian cancer. It has been established that BRCA1 mutations increase the risk of brain metastasis compared to the BRCA1 wildtype, and once metastasis occurs to the brain the disease is considered uncurable. The blood-brain barrier (BBB) is essential for maintaining and regulating homeostasis of the central nervous system and is composed of highly specialized brain endothelial cells. Using a human induced pluripotent stem cell (hiPSC) based model, we characterized an hiPSC line from an invasive cancer patient harboring a BRCA1 mutation. This patient-derived hiPSC line can be utilized to study BBB properties as after differentiation into brain-like endothelial cells (BECs), BECs derived from this line express BBB markers such as tight junction proteins, and functional efflux transporters. Future application of patient-derived stem cell models could provide a platform to discover genetic predispositions to BBB disruption in individuals with BRCA1 mutations, as well as the potential molecular mechanisms contributing to brain metastasis.

CRISPR/Cas9-edited induced pluripotent stem cells (iPSCs) are valuable research models for mechanistic studies. However, gene conversion between a gene-pseudogene pair that share high sequence identity and form direct repeats in proximity on the same chromosome can interfere with the precision of gene editing. Mutations in the human beta-glucocerebrosidase gene (GBA1) are associated with Gaucher disease, Parkinson's disease, and Lewy body dementia. During the creation of a GBA1 KO iPSC line, we detected about 70% gene conversion from its pseudogene GBAP1. These events maintained the reading frame and resulted from GBA1-specific cleavage by CRISPR/Cas9, without disrupting the GBA1 gene.

Restoring fertility in male cancer individuals through testicular tissue transplantation faces challenges due to hypoxia-induced loss of spermatogonial stem cells (SSCs). Hydrogel encapsulation was explored to minimize hypoxic damage in testicular tissue transplantation. For this purpose, human amnion membrane (hAM)-derived hydrogel could be an alternative.

Radiation-induced thrombocytopenia (RIT) is a serious complication of cancer radiotherapy, for which therapeutic options are limited. This study investigates wedelolactone (WED), a metabolite of a botanical drug, as a potential treatment for RIT.

Cancer remains a major global health challenge, necessitating innovative prevention and treatment approaches. Certain plants, adapted to specific environments, may exhibit bioactive properties with potential anticancer applications.

Liposarcoma (LPS) is the most common soft-tissue sarcoma in adults, and well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are the most frequent subtypes. These LPSs are considered to develop due to disturbances in the adipogenic differentiation of mesenchymal stem cells. However, the molecular mechanisms underlying the disturbances remain unclear. Here, we aimed to identify the mechanism and explore its therapeutic advantages focusing upon their epigenetic alterations, known to be important in differentiation. First, we conducted a genome-wide DNA methylation analysis using 15 LPSs (6 WDLPSs and 9 DDLPSs) and 6 normal adipose tissues. Unsupervised hierarchical cluster analysis using DNA methylation profiles at enhancers classified the samples into the three histological types, whereas analysis using promoters did not. Principal component analysis revealed that normal adipose tissues and WDLPSs were grouped closely, whereas DDLPSs were scattered. Genomic regions hypermethylated in DDLPSs were enriched for enhancers, especially super-enhancers (13.5% of hypermethylated regions and 7.0% of the whole genome), which were located in the genes involved in adipogenesis, such as PPARG2 and its target genes (FABP4 and PLIN1). In addition, marked decreases in PPARG2 and FABP4 expression were confirmed in DDLPSs. Then, treatment of PPARG2-expressing DDLPS cell lines with 5-aza-2'-deoxycytidine, a DNA demethylating agent, and rosiglitazone, a PPARγ agonist, was shown to induce differentiation with enhanced expression of FABP4. These findings indicate that aberrant DNA methylation of adipogenic gene enhancers plays a crucial role in the development of DDLPS and can be a therapeutic target.