SOX9 Gene Research Articles

Allografts for partial meniscus repair: an in vitro and ex vivo meniscus culture study.

The purpose of this study was to evaluate the treatment potential of a human-derived demineralized scaffold, Spongioflex® (SPX), in partial meniscal lesions by employing in vitro models. In the first step, the differentiation potential of human meniscal cells (MCs) was investigated. In the next step, the ability of SPX to accommodate and support the adherence and/or growth of MCs while maintaining their fibroblastic/chondrocytic properties was studied. Control scaffolds, including bovine collagen meniscus implant (CMI) and human meniscus allograft (M-Allo), were used for comparison purposes. In addition, the migration tendency of MCs from fresh donor meniscal tissue into SPX was investigated in an ex vivo model. The results showed that MCs cultured in osteogenic medium did not differentiate into osteogenic cells or form significant calcium phosphate deposits, although AP activity was relatively increased in these cells. Culturing cells on the scaffolds revealed increased viability on SPX compared to the other scaffold materials. Collagen I synthesis, assessed by ELISA, was similar in cells cultured in 2D and on SPX. MCs on micro-porous SPX (weight >0.5g/cm3) exhibited increased osteogenic differentiation indicated by upregulated expression of ALP and RUNX2, while also showing upregulated expression of the chondrogen-specific SOX9 and ACAN genes. Ingrowth of cells on SPX was observed after 28days of cultivation. Overall, the results suggest that SPX could be a promising biocompatible scaffold for meniscal regeneration.

Decreased expression of GEM in osteoarthritis cartilage regulates chondrogenic differentiation via Wnt/β-catenin signaling

BackgroundGEM (GTP-binding protein overexpressed in skeletal muscle) is one of the atypical small GTPase subfamily members recently identified as a regulator of cell differentiation. Abnormal chondrogenesis coupled with an imbalance in the turnover of cartilaginous matrix formation is highly relevant to the onset and progression of osteoarthritis (OA). However, how GEM regulates chondrogenic differentiation remains unexplored.MethodsCartilage tissues were obtained from OA patients and graded according to the ORASI and ICRS grading systems. The expression alteration of GEM was detected in the Grade 4 cartilage compared to Grade 0 and verified in OA mimic culture systems. Next, to investigate the specific function of GEM during these processes, we generated a Gem knockdown (Gem-Kd) system by transfecting siRNA targeting Gem into ATDC5 cells. Acan, Col2a1, Sox9, and Wnt target genes of Gem-Kd ATDC5 cells were detected during induction. The transcriptomic sequencing analysis was performed to investigate the mechanism of GEM regulation. Wnt signaling pathways were verified by real-time PCR and immunoblot analysis. Finally, a rescue model generated by treating Gem-KD ATDC5 cells with a Wnt signaling agonist was established to validate the mechanism identified by RNA sequencing analysis.ResultsA decreased expression of GEM in OA patients’ cartilage tissues and OA mimic chondrocytes was observed. While during chondrogenesis differentiation and cartilage matrix formation, the expression of GEM was increased. Gem silencing suppressed chondrogenic differentiation and the expressions of Acan, Col2a1, and Sox9. RNA sequencing analysis revealed that Wnt signaling was downregulated in Gem-Kd cells. Decreased expression of Wnt signaling associated genes and the total β-CATENIN in the nucleus and cytoplasm were observed. The exogenous Wnt activation exhibited reversed effect on Gem loss-of-function cells.ConclusionThese findings collectively validated that GEM functions as a novel regulator mediating chondrogenic differentiation and cartilage matrix formation through Wnt/β-catenin signaling.

Thy-1 knockdown promotes the osteogenic differentiation of GMSCs via the Wnt/β-catenin pathway.

Gingival mesenchymal stem cells (GMSCs) are newly developed seed cells for tissue engineering owing to their easy isolation, abundance and high growth rates. Thy-1 is an important regulatory molecule in the differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the function of Thy-1 in the osteogenic differentiation of GMSCs by reducing the expression of Thy-1 using a lentivirus. The results demonstrated that Thy-1 knockdown promoted the osteogenic differentiation of GMSCs in vitro. Validation by RNA-seq revealed an obvious decrease in Vcam1 and Sox9 gene expression with Thy-1 knockdown. Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the differentially expressed genes were enriched in the Wnt signalling pathway. We further demonstrated that Thy-1 knockdown promoted osteogenic differentiation of GMSCs by activating the Wnt/β-catenin signalling pathway. Therefore, Thy-1 has a key regulatory role in the differentiation of GMSCs and maybe a core molecule connecting transcription factors related to the differentiation of MSCs. Our study also highlighted the potential of Thy-1 to modify MSCs, which may help improve their use in tissue engineering.

Genetic Variants Associated With Hidradenitis Suppurativa

Hidradenitis suppurativa (HS) is a common and severely morbid chronic inflammatory skin disease that is reported to be highly heritable. However, the genetic understanding of HS is insufficient, and limited genome-wide association studies (GWASs) have been performed for HS, which have not identified significant risk loci. To identify genetic variants associated with HS and to shed light on the underlying genes and genetic mechanisms. This genetic association study recruited 753 patients with HS in the HS Program for Research and Care Excellence (HS ProCARE) at the University of North Carolina Department of Dermatology from August 2018 to July 2021. A GWAS was performed for 720 patients (after quality control) with controls from the Add Health study and then meta-analyzed with 2 large biobanks, UK Biobank (247 cases) and FinnGen (673 cases). Variants at 3 loci were tested for replication in the BioVU biobank (290 cases). Data analysis was performed from September 2021 to December 2022. Main outcome measures are loci identified, with association of P < 1 × 10-8 considered significant. A total of 753 patients were recruited, with 720 included in the analysis. Mean (SD) age at symptom onset was 20.3 (10.57) years and at enrollment was 35.3 (13.52) years; 360 (50.0%) patients were Black, and 575 (79.7%) were female. In a meta-analysis of the 4 studies, 2 HS-associated loci were identified and replicated, with lead variants rs10512572 (P = 2.3 × 10-11) and rs17090189 (P = 2.1 × 10-8) near the SOX9 and KLF5 genes, respectively. Variants at these loci are located in enhancer regulatory elements detected in skin tissue. In this genetic association study, common variants associated with HS located near the SOX9 and KLF5 genes were associated with risk of HS. These or other nearby genes may be associated with genetic risk of disease and the development of clinical features, such as cysts, comedones, and inflammatory tunnels, that are unique to HS. New insights into disease pathogenesis related to these genes may help predict disease progression and novel treatment approaches in the future.

Gene signature reveals decreased SOX10-dependent transcripts in malignant cells from immune checkpoint inhibitor-resistant cutaneous melanomas

Evidence is mounting for cross-resistance between immune checkpoint and targeted kinase inhibitor therapies in cutaneous melanoma patients. Since the loss of the transcription factor, SOX10, causes tolerance to MAPK pathway inhibitors, we used bioinformatic techniques to determine if reduced SOX10 expression/activity is associated with immune checkpoint inhibitor resistance. We integrated SOX10 ChIP-seq, knockout RNA-seq, and knockdown ATAC-seq data from melanoma cell models to develop a robust SOX10 gene signature. We used computational methods to validate this signature as a measure of SOX10-dependent activity in independent single-cell and bulk RNA-seq SOX10 knockdown, cell line panel, and MAPK inhibitor drug-resistant datasets. Evaluation of patient single-cell RNA-seq data revealed lower levels of SOX10-dependent transcripts in immune checkpoint inhibitor-resistant tumors. Our results suggest that SOX10-deficient melanoma cells are associated with cross-resistance between targeted and immune checkpoint inhibitors and highlight the need to identify therapeutic strategies that target this subpopulation.

SOX9 and IL1A as the Potential Gene Biomarkers of the Oral Cancer.

Oral cancer is one of the most common malignant tumors in the head and neck. It is easy to relapse, and the prognosis is poor. However, the molecular mechanism in the development of oral cancer is still unclear. A total of 30 normal individuals and 30 patients with head and neck cancer who underwent surgery were recruited in the Fourth Hospital of Hebei Medical University between February 2019 and November 2021. Furthermore, Human Protein Atlas (HPA) analysis, RT-qPCR, and immunofluorescence were used to verify the expression of SOX9 and IL1A. The GSE69002 dataset was downloaded from the Gene Expression Omnibus (GEO) database. GEO2R was used to identify the differentially expressed genes (DEGs). The Protein-Protein Interaction (PPI) network was constructed by using the STRING, and Cytoscape software was performed for visualization. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for enrichment analysis were made via the DAVID, Metascape, Gene Set Enrichment Analysis (GSEA), and Bin Gene Ontology (BINGO) analysis. Gene Expression Profiling Interactive Analysis (GEPIA) analysis was used to analyze the expression level of hub genes and pathological stage. The cBioPortal can be used for mutation analysis and pathway prediction of hub genes. Kaplan Meier Plotter was used for survival analysis of hub genes. The relative expression level of SOX9 (P=0.021, t=4.332) and IL1A (P=0.011, t= -4.213) in oral cancer was significantly higher than that in the standard group (P<0.05). The DEGs are mainly enriched in cell division, inflammation, interleukin-12 beta-subunit binding, and interleukin- 10 receptor binding. All the differentially expressed gene pathways eventually converge in cell growth and apoptosis. No relationship between the pathologic stage and the expression of hub genes. The poor overall survival of patients with the high expression of SOX9 (Hazard Ratio (HR) = 1.46, P = 0.009) and IL1A (HR = 1.49, P = 0.008). There were strong correlations between the hub genes and the head and neck neoplasms via the Comparative Toxicogenomics Database (CTD). The immunofluorescence and PCR results showed that the level of SOX9 (P<0.001, t = -23.368) in the cancer group was significantly higher than that in the normal group; The level of IL1A in the cancer group was significantly higher than that in the normal group (P<0.001, t = -11.960). SOX9 and IL1A genes are highly expressed in oral cancer and might be potential therapeutic targets for oral cancer. The poor overall survival of patients with the high expression of SOX9 and IL1A.

Decellularized Lung Extracellular Matrix Scaffold Promotes Human Embryonic Stem Cell Differentiation towards Alveolar Progenitors.

Efficient production of functional and mature alveolar epithelial is a major challenge for developing any cell replacement therapy for lung degenerative diseases. The extracellular matrix (ECM) pro-vides a dynamic environment and mediates cellular responses during development and maintenance of tissue functions. The decellularized ECM (dECM) which retains its native-like structure and bio-chemical composition can provide the induction of embryonic stem cell (ESC) differentiation toward the tissue-specific lineages during in vitro culture. Therefore, the aim of this study was to evaluate the effect of sheep lung dECM-derived scaffold on differentiation and further maturation of ESC-derived lung progenitor cells. This study was an experimental study. In the first step, a sheep lung was decellularized to achieve dECM scaffolds and hydrogels. Afterwards, the obtained dECM scaffold was evaluated for collagen and glycosaminoglycan contents, DNA quantification, and its ultrastructure. Next, the three experimental groups: i. Sheep lung dECM-derived scaffold, ii. Sheep lung dECM-derived hydrogel, and iii. Fibronectin-coated plates were compared in their abilities to induce further differentiation of human embryonic stem cells (hESCs)-derived definitive endoderm (DE) into lung progenitor cells. The comparison was evaluated by immuno-staining and real-time polymerase chain reaction (PCR) assessments. We found that the dECM-derived scaffold preserved its composition and native porous structures while lacking nuclei and intact cells. All experimental groups displayed lung progenitor cell differen-tiation as revealed by the RNA and protein expression of NKX2.1, P63 and CK5. DE cells differenti-ated on dECM-derived scaffold and dECMderived hydrogel showed significant upregulation of SOX9 gene expression, a marker of the distal airway epithelium. DE cells differentiated on the dECM-derived scaffold compared to the two other groups, showed enhanced expression of SFTPC (type 2 alveolar epithelial [AT2] cell marker), FOXJ1 (ciliated cell marker), and MUC5A (secretory cell marker) genes. Overall, our results suggest that dECM-derived scaffold improves the differentiation of DE cells towards lung alveolar progenitor cells in comparison with dECM-derived hydrogel and fibronectin-coated plates.

A novel variant of the SOX10 gene associated with Waardenburg syndrome type IV

BackgroundWaardenburg syndrome (WS) is a rare genetic disorder characterized by varying degrees of sensorineural hearing loss and accumulated pigmentation in the skin, hair and iris. The syndrome is classified into four types (WS1, WS2, WS3, and WS4), each with different clinical phenotypes and underlying genetic causes. The aim of this study was to identify the pathogenic variant in a Chinese family with Waardenburg syndrome type IV.MethodsThe patient and his parents underwent a thorough medical examination. We applied whole exome sequencing to identify the causal variant on the patient and other family members.ResultsThe patient presented with iris pigmentary abnormality, congenital megacolon and sensorineural hearing loss. The clinical diagnosis of the patient was WS4. The whole exome sequencing (WES) revealed a novel variant (c.452_456dup) in the SOX10 gene, which could be responsible for the observed pathogenic of WS4 in this patient. Our analysis suggests that this variant produces a truncated protein that contributes to the development of the disease. The genetic test confirmed the diagnosis of WS4 in the patient from the studied pedigree.ConclusionsThis present study demonstrated that genetic test based on WES, an effective alternative to regular clinical examinations, helps diagnose WS4. The newly identified SOX10 gene variant can expand the understanding of WS4.

Molecular functions of Mrhl lncRNA in mouse spermatogenesis.

Mrhl lncRNA regulates the Wnt signaling pathway in mouse spermatogonial cells, resulting in the commitment of B-type spermatogonia to meiotic entry through Mrhl lncRNA-mediated regulation of Sox8. Mrhl lncRNA regulates chromatin dynamics of the Sox8 promoter/enhancer interaction through chromatin looping. Proliferation and meiotic division of spermatogonial stem cells are highly regulated biological processes that occur during spermatogenesis. In addition to protein-coding genes, the mammalian genome encodes thousands of lncRNAs which are spatio-temporally expressed and play key role(s) in cellular differentiation and development. Mrhl lncRNA is one such mono-exonic polyadenylated non-coding RNA encoded within the 15th intron of the mouse Phkb gene. Mrhl lncRNA is expressed in mouse testis amongst other tissues. The RNA is nuclear localized and predominantly bound to chromatin in GC-1 spg cells (derived from B-type spermatogonia). It regulates multiple genes belonging to different biological processes including Wnt signaling. Wnt activation of GC-1 spg cells downregulates Mrhl lncRNA expression, in turn activating the expression of meiotic marker genes and downregulating stem cell markers. We have mapped the genomic loci bound by Mrhl lncRNA and identified 37 genes to be regulated by its physical association. Sox8 gene, one among these, is regulated through its interaction at its promoter through RNA:DNA:DNA triplex structure and chromatin looping mediated by the architectural proteins CTCF and YY1. Here, we summarize our major findings on this novel lncRNA starting from its discovery to biological function(s), particularly during meiotic commitment/initiation in mouse spermatogenesis.

Transplanting Human Neural Stem Cells with ≈50% Reduction of SOX9 Gene Dosage Promotes Tissue Repair and Functional Recovery from Severe Spinal Cord Injury.

Neural stem cells (NSCs) derived from human pluripotent stem cells (hPSCs) are considered a major cell source for reconstructing damaged neural circuitry and enabling axonal regeneration. However, the microenvironment at the site of spinal cord injury (SCI) and inadequate intrinsic factors limit the therapeutic potential of transplanted NSCs. Here, it is shown that half dose of SOX9 in hPSCs-derived NSCs (hNSCs) results in robust neuronal differentiation bias toward motor neuron lineage. The enhanced neurogenic potency is partly attributed to the reduction of glycolysis. These neurogenic and metabolic properties retain after transplantation of hNSCs with reduced SOX9 expression in a contusive SCI rat model without the need for growth factor-enriched matrices. Importantly, the grafts exhibit excellent integration properties, predominantly differentiate into motor neurons, reduce glial scar matrix accumulation to facilitate long-distance axon growth and neuronal connectivity with the host as well as dramatically improve locomotor and somatosensory function in recipient animals. These results demonstrate that hNSCs with half SOX9 gene dosage can overcome extrinsic and intrinsic barriers, representing a powerful therapeutic potential for transplantation treatments for SCI.

The transcription factors Creb1 and CEBPB regulate Sox9 promoter activity in TM4 Sertoli cells

In Sertoli cells, the Sox9 gene is essential for testicular development and normal spermatogenesis. SOX9 is critical for postnatal Sertoli cells differentiation and proliferation in the testis. However, the molecular mechanisms that specifically regulate its expression are not entirely understood. Sox9 expression is regulated by CREB1 and CEBPB in other biological contexts such as during chondrogenesis and in rat thyroid follicular cells. We hypothesized that Sox9 promoter activity is regulated by CREB1 and CEBPB in Sertoli cells. Our results show that Sox9 expression is dependent on the activation of these transcription factors by the cAMP/PKA signaling pathway in TM4 Sertoli cells. Chromatin immunoprecipitation and promoter/reporter luciferase assays with 5′ promoter deletions and site-directed mutagenesis demonstrated that CREB1 is being recruited to a DNA regulatory element at -141 bp of the Sox9 promoter region. Such regulation is dependent on the cAMP/PKA signaling pathway, resulting in phosphorylation of CREB1. Activation of Sox9 expression by CEBPB may involve its recruitment to the proximal promoter region by protein–protein interaction with CREB1. Thus, we have shown that the Sox9 promoter is being regulated by the transcription factors CREB1 and CEBPB in TM4 Sertoli cells and involve their recruitment to the proximal promoter region.

The role of three-dimensional scaffolds based on polyglycerol sebacate/ polycaprolactone/ gelatin in the presence of Nanohydroxyapatite in promoting chondrogenic differentiation of human adipose-derived mesenchymal stem cells

BackgroundTissue engineering for cartilage regeneration has made great advances in recent years, although there are still challenges to overcome. This study aimed to evaluate the chondrogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) on three-dimensional scaffolds based on polyglycerol sebacate (PGS) / polycaprolactone (PCL) / gelatin(Gel) in the presence of Nanohydroxyapatite (nHA).Materials and methodsIn this study, a series of nHA-nanocomposite scaffolds were fabricated using 100:0:0, 60:40:0, and 60:20:20 weight ratios of PGS to PCL: Gel copolymers through salt leaching method. The morphology and porosity of prepared samples was characterized by SEM and EDX mapping analysis. Also, the dynamic contact angle and PBS adsorption tests are used to identify the effect of copolymerization and nanoparticles on scaffolds' hydrophilicity. The hydrolytic degradation properties were also analyzed. Furthermore, cell viability and proliferation as well as cell adhesion are evaluated to find out the biocompatibility. To determine the potential ability of nHA-nanocomposite scaffolds in chondrogenic differentiation, RT-PCR assay was performed to monitor the expression of collagen II, aggrecan, and Sox9 genes as markers of cartilage differentiation.ResultsThe nanocomposites had an elastic modulus within a range of 0.71–1.30 MPa and 0.65–0.43 MPa, in dry and wet states, respectively. The PGS/PCL sample showed a water contact angle of 72.44 ± 2.2°, while the hydrophilicity significantly improved by adding HA nanoparticles. It was found from the hydrolytic degradation study that HA incorporation can accelerate the degradation rate compared with PGS and PGS/PCL samples. Furthermore, the in vitro biocompatibility tests showed significant cell attachment, proliferation, and viability of adipose-derived mesenchymal stem cells (ADMSCs). RT-PCR also indicated a significant increase in collagen II, aggrecan and Sox9 mRNA levels.ConclusionsOur findings demonstrated that these nanocomposite scaffolds promote the differentiation of hADSCs into chondrocytes possibly by the increase in mRNA levels of collagen II, aggrecan, and Sox9 as markers of chondrogenic differentiation. In conclusion, the addition of PCL, Gelatin, and HA into PGS is a practical approach to adjust the general features of PGS to prepare a promising scaffold for cartilage tissue engineering.Graphical

Mesenchymal stem cell-derived secretome enhances nucleus pulposus cell metabolism and modulates extracellular matrix gene expression in vitro.

Introduction: Intradiscal mesenchymal stromal cell (MSC) therapies for intervertebral disc degeneration (IDD) have been gaining increasing interest due to their capacity to ameliorate intervertebral disc metabolism and relieve low back pain (LBP). Recently, novel investigations have demonstrated that most of MSC anabolic effects are exerted by secreted growth factors, cytokines, and extracellular vesicles, collectively defined as their secretome. In this study, we aimed to evaluate the effect of bone-marrow-MSCs (BM-MSCs) and adipose-derived stromal cells (ADSCs) secretomes on human nucleus pulposus cells (hNPCs) in vitro. Methods: BM-MSCs and ADSCs were characterized according to surface marker expression by flow cytometry and multilineage differentiation by Alizarin red, Red Oil O and Alcian blue staining. After isolation, hNPCs were treated with either BM-MSC secretome, ADSC secretome, interleukin (IL)-1β followed by BM-MSC secretome or IL-1β followed by ADSC secretome. Cell metabolic activity (MTT assay), cell viability (LIVE/DEAD assay), cell content, glycosaminoglycan production (1,9-dimethylmethylene blue assay), extracellular matrix and catabolic marker gene expression (qPCR) were assessed. Results: 20% BM-MSC and ADSC secretomes (diluted to normal media) showed to exert the highest effect towards cell metabolism and were then used in further experiments. Both BM-MSC and ADSC secretomes improved hNPC viability, increased cell content and enhanced glycosaminoglycan production in basal conditions as well as after IL-1β pretreatment. BM-MSC secretome significantly increased ACAN and SOX9 gene expression, while reducing the levels of IL6, MMP13 and ADAMTS5 both in basal conditions and after in vitro inflammation with IL-1β. Interestingly, under IL-1β stimulation, ADSC secretome showed a catabolic effect with decreased extracellular matrix markers and increased levels of pro-inflammatory mediators. Discussion: Collectively, our results provide new insights on the biological effect of MSC-derived secretomes on hNPCs, with intriguing implications on the development of cell-free approaches to treat IDD.

Evaluation of BDE-47-induced neurodevelopmental toxicity in zebrafish embryos.

There are growing concerns about the neurodevelopmental toxicity of polybrominated diphenyl ethers (PBDEs), but the toxicological phenotypes and mechanisms are not well elucidated. Here, zebrafish (Danio rerio) were exposed to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) from 4 to 72 h post-fertilization (hpf). The results showed that BDE-47 stimulated the production of dopamine and 5-hydroxytryptamine, but inhibited expression of Nestin, GFAP, Gap43, and PSD95 in 24 hpf embryos. Importantly, we unraveled the inhibitory effects of BDE-47 on neural crest-derived melanocyte differentiation and melanin syntheses process, evidenced by disrupted expression of wnt1, wnt3, sox10, mitfa, tyrp1a, tyrp1b, tryp2, and oca2 gene in 72 hpf embryos and decreased tyrosinase activities in embryos at 48 and 72 hpf. The transcriptional activities of myosin VAa, kif5ba, rab27a, mlpha, and cdc42 genes, which are associated with intracellular transport process, were also disturbed during zebrafish development. Ultimately, these alterations led to fast spontaneous movement and melanin accumulation deficit in zebrafish embryos upon BDE-47 exposure. Our results provide an important extension for understanding the neurodevelopmental effects of PBDEs and facilitate the comprehensive evaluation of neurotoxicity in embryos.

IL-6, IL-1β and TNF-α regulation of the chondrocyte phenotype: a possible mechanism of haemophilic cartilage destruction

ABSTRACTObjective:Proinflammatory cytokines are considered to be one of the key causes of haemophilic cartilage destruction by inducing chondrocyte apoptosis and extracellular matrix degradation. However, few studies have focused on how proinflammatory cytokines regulate the phenotypic changes of chondrocytes, which may be an important factor in haemophilic cartilage degradation pathogenesis. More understanding is needed about the effect of proinflammatory cytokines on phenotypic changes of the chondrocyte. The objective of this study was to examine how IL-6, TNF-α and IL-1β regulate the chondrocyte phenotype, which may be an important factor in haemophilic cartilage degradation pathogenesis.Methods:HUM-iCell-s018 chondrocytes were treated with increasing concentrations of TNF-α, IL-6 or IL-1β (0, 1, 5, 10 ng/ml) for 24 h, then FGF23 and SOX9 expression was determined by qRT-PCR and WB, respectively.Results:We found that TNF-α, IL-6 and IL-1β induced FGF23 and suppressed SOX9 expression in chondrocytes in a dose-dependent manner. IL-1β had a stronger regulatory effect on FGF23, while TNF-α and IL-6 had stronger regulatory effects on SOX9.Conclusions:These findings suggest that IL-6, IL-1β and TNF-α may be involved in haemophilic cartilage destruction pathogenesis by altering the chondrocyte phenotype through modulation of FGF23 and SOX9 gene expression.

Whole-exome sequencing reveals genetic variants in low-risk and high-risk neuroblastoma

This study aimed to investigate the genetic aberrations in neuroblastoma (NB) by comparing high and low-risk NB patients by whole-exome sequencing (WES) and to reveal the heterogeneity and association between somatic variants and clinical features. Seven NB patients with available clinical data were included in the study (4 in the low-risk group and 3 in the high-risk group). WES was performed and somatic variants associated with NB genes in the COSMIC database were selected through bioinformatics pipeline analysis. Variants were determined using the Integrative Genomics Viewer (IGV). Some gene variations were found in both groups, including variations in oncogene and tumor suppressor genes. In general, candidate gene variations were associated with chromatin remodeling complexes, the RAS pathway, cell proliferation, and DNA repair mechanism. Some variations in CSF1R, MSH6, PTPN11, SOX9, RET, TSC1, and DNMT1 genes were detected only in high-risk patients, while EP300, TET2, MYCN, PRDM1, and ARID2 gene variations were detected only in low-risk patients. When high-risk gene variants were compared with the cBioportal cancer genomic database, two common gene variants (ARID1A and NCOR2) were identified. However, when low-risk gene variants were compared with the cBioportal cancer genomic database, no common genes were found. GO/KEGG enrichment analysis was performed to find relevant biological processes and molecular pathways related to gene variants, which will help to decipher the molecular mechanisms of NB tumorigenesis and the phenotypic differences between high-risk and low-risk patients.

Pregnenolone enhances the proliferation of mouse neural stem cells and promotes oligodendrogenesis, together with Sox10, and neurogenesis, along with Notch1 and Pax6.

Pregnenolone is a precursor of various steroid hormones involved in osteoblast proliferation, microtubules polymerization and cell survival protection. Previous reports focused on the effects of pregnenolone metabolites on stem cell proliferation and differentiation; however, the effects of pregnenolone itself has not been well explored. The present study aimed to investigate the role of pregnenolone on NSC proliferation and to determine the doses required for NSC differentiation as well as the various genes involved in its mechanism of action. NSCs were isolated from the embryonic cortex of E14 mice, incubated for 5 days, and then treated with pregnenolone doses of 2, 5, 10, 15 and 20μM for another 5 days. The number of neurospheres and neurosphere derived cells were then counted. Flow cytometry was used to evaluate the differentiation of NSCs into oligodendrocytes, astrocytes, and neurons. The expression level of Notch1, Pax6 and Sox10 genes were also measured by Real Time PCR after 5 days of treatment. Our data suggest that treatment with 10μM pregnenolone is optimal for NSC proliferation. In fact, this concentration caused the highest increase in the number of neurospheres and neurosphere derived cells, compared to the control group. In addition, treatment with low doses of pregnenolone (5 and 10μM) caused a significant increase in NSC differentiation towards immature (Olig2+) and mature (MBP+) oligodendrocyte cell populations, compared to controls. However, NSC differentiation into neurons (beta III tubulin+cells) increased in all treatment groups, with the highest and most significant increase obtained at 15μM concentration. It is worth noting that pregnenolone at the highest concentration of 15μM decreased the number of astrocytes (GFAP+). Furthermore, there was an increase of Sox10 expression with low pregnenolone doses, leading to oligodendrogenesis, whereas Notch1 and Pax6 gene expression increased in pregnenolone groups with more neurogenesis. Pregnenolone regulates NSCs proliferation in vitro. Treatment with low doses of pregnenolone caused an increase in the differentiation of NSCs into mature oligodendrocytes while higher doses increased the differentiation of NSCs into neurons. Oligodendrogenesis was accompanied by Sox10 while neurogenesis occurred together with Notch1 and Pax6 expression.

Additional evidence for the role of chromosomal imbalances and SOX8, ZNRF3 and HHAT gene variants in early human testis development

BackgroundForty-six ,XY Differences/Disorders of Sex Development (DSD) are characterized by a broad phenotypic spectrum ranging from typical female to male with undervirilized external genitalia, or more rarely testicular regression with a typical male phenotype. Despite progress in the genetic diagnosis of DSD, most 46,XY DSD cases remain idiopathic.MethodsTo determine the genetic causes of 46,XY DSD, we studied 165 patients of Tunisian ancestry, who presented a wide range of DSD phenotypes. Karyotyping, candidate gene sequencing, and whole-exome sequencing (WES) were performed.ResultsCytogenetic abnormalities, including a high frequency of sex chromosomal anomalies (85.4%), explained the phenotype in 30.9% (51/165) of the cohort. Sanger sequencing of candidate genes identified a novel pathogenic variant in the SRY gene in a patient with 46,XY gonadal dysgenesis. An exome screen of a sub-group of 44 patients with 46,XY DSD revealed pathogenic or likely pathogenic variants in 38.6% (17/44) of patients.ConclusionRare or novel pathogenic variants were identified in the AR, SRD5A2, ZNRF3, SOX8, SOX9 and HHAT genes. Overall our data indicate a genetic diagnosis rate of 41.2% (68/165) in the group of 46,XY DSD.

Induced Chondrogenic Differentiation of hESCs by hESC-Derived MSCs Conditioned Medium and Sequential 3D-2D Culture System

Background and Aims: It has been proven that human mesenchymal stem cells (MSCs) conditioned medium (hMSCs-CM) can influence human embryonic stem cells (hESCs) chondrogenic differentiation. In this study, we hypothesized that conditioned medium (CM) from hESCs-derived MSCs in a sequential 3D-2D culture system could facilitate the induction of chondrogenesis in hESCs.&#x0D; Materials and Methods: CM was collected from Yazd2 (hESCs; 46, XY) derived MSCs confluent cultures and stored at -20 °C. Yazd4 hESC line (46, XX) was induced for differentiation using EB formation as 3D culture into SD (spontaneously differentiation) and CM groups (differentiation using conditioned medium) for four days. Cell culture continued in a 2D (monolayer) culture system for both groups till day 14. Ultimately, chondrogenic differentiation was assessed by Alcian blue and masson′s trichrome staining at 4 and 14 days of differentiation, and quantitative real-time polymerase chain reaction (PCR) for NANOG, MEOX1, SOX5, SOX6, SOX9, ACAN, COL2A1 and RUNX2 genes for SD and CM groups on days 0 and 4.&#x0D; Results: The gene expression profile for chondrogenic genes in the CM group was significantly more than the SD group (p&lt; 0.05). Furthermore, chemical staining assessment illustrated a significant GAG and collagen II difference between the CM and SD groups at days 4 and 14 (p&lt; 0.05).&#x0D; Conclusions: Our findings would pave the way for creating an in vitro human chondrogenesis model for further studies in the developmental biology of articular cartilage tissue, which lend itself to cell-based therapy to cure joint diseases such as osteoarthritis.

Modeling the Differentiation of Embryonic Limb Chondroprogenitors by Cell Death and Cell Senescence in High Density Micromass Cultures and Their Regulation by FGF Signaling

Considering the importance of programmed cell death in the formation of the skeleton during embryonic development, the aim of the present study was to analyze whether regulated cell degeneration also accompanies the differentiation of embryonic limb skeletal progenitors in high-density tridimensional cultures (micromass cultures). Our results show that the formation of primary cartilage nodules in the micromass culture assay involves a patterned process of cell death and cell senescence, complementary to the pattern of chondrogenesis. As occurs in vivo, the degenerative events were preceded by DNA damage detectable by γH2AX immunolabeling and proceeded via apoptosis and cell senescence. Combined treatments of the cultures with growth factors active during limb skeletogenesis, including FGF, BMP, and WNT revealed that FGF signaling modulates the response of progenitors to signaling pathways implicated in cell death. Transcriptional changes induced by FGF treatments suggested that this function is mediated by the positive regulation of the genetic machinery responsible for apoptosis and cell senescence together with hypomethylation of the Sox9 gene promoter. We propose that FGF signaling exerts a primordial function in the embryonic limb conferring chondroprogenitors with their biological properties.