SOX9 Gene Research Articles

Oligodendrocyte Neurofascin Independently Regulates Both Myelin Targeting and Sheath Growth in the CNS.

SummarySelection of the correct targets for myelination and regulation of myelin sheath growth are essential for central nervous system (CNS) formation and function. Through a genetic screen in zebrafish and complementary analyses in mice, we find that loss of oligodendrocyte Neurofascin leads to mistargeting of myelin to cell bodies, without affecting targeting to axons. In addition, loss of Neurofascin reduces CNS myelination by impairing myelin sheath growth. Time-lapse imaging reveals that the distinct myelinating processes of individual oligodendrocytes can engage in target selection and sheath growth at the same time and that Neurofascin concomitantly regulates targeting and growth. Disruption to Caspr, the neuronal binding partner of oligodendrocyte Neurofascin, also impairs myelin sheath growth, likely reflecting its association in an adhesion complex at the axon-glial interface with Neurofascin. Caspr does not, however, affect myelin targeting, further indicating that Neurofascin independently regulates distinct aspects of CNS myelination by individual oligodendrocytes in vivo.

A Recurrent Rare SOX9 Variant (M469V) is Associated with Congenital Vertebral Malformations.

The genetic variations contributed to a substantial proportion of congenital vertebral malformations (CVM). SOX9 gene, a member of the SOX gene family, has been implicated in CVM. To study the SOX9 mutation in CVM patients is of great significance to explain the pathogenesis of scoliosis (the clinical manifestation of CVM) and to explore the pathogenesis of SOX9-related skeletal deformities. A total of 50 singleton patients with CVM were included in this study. Exome Sequencing (ES) was performed on all the patients. The recurrent candidate variant of SOX9 gene was validated by Sanger sequencing. Luciferase assay was performed to investigate the functional changes of this variant. A recurrent rare heterozygous missense variant in SOX9 gene (NM_000346.3: c.1405A>G, p.M469V) which had not been reported previously was identified in three CVM patients who had the clinical findings of congenital scoliosis without deformities in other systems. This variant was absent from our in-house database and it was predicted to be deleterious (CADD = 24.5). The luciferase assay demonstrated that transactivation capacity of the mutated SOX9 protein was significantly lower than that of the wild-type for the two luciferase reporters (p = 0.0202, p = 0.0082, respectively). This SOX9 mutation (p.M469V) may contribute to CVM without other systematic deformity, which provides important implications and better understanding of phenotypic variability in SOX9-related skeletal deformities.

Relationship of COL9A1 and SOX9 Genes with Genetic Susceptibility of Postmenopausal Osteoporosis

As one of the most common types of osteoporosis, postmenopausal osteoporosis (PMOP) is caused by both genetic and environmental factors. Previous studies have indicated that SOX9 activity is tightly regulated to ensure normal bone mineral density (BMD) in the adult skeleton, and the COL9A1 promoter region can be transactivated by SOX9. In this study, we aimed to investigate the potential association between PMOP and the COL9A1 and SOX9 genes. A total of 10,443 postmenopausal women, including 2288 patients and 3557 controls in the discovery stage and 1566 patients and 3032 controls in the validation stage, were recruited. Forty-three tag SNPs (36 in COL9A1 and 7 in SOX9) were selected for genotyping to evaluate the association of the SOX9 gene with PMOP and BMD. Association and bioinformatics analyses were performed for PMOP. BMD and serum level of SOX9 were also utilized as quantitative phenotypes in further analyses. SNP rs73354570 of SOX9 was significantly associated with PMOP in both discovery stages (OR 1.24 [1.10-1.39], P = 3.56 × 10-4, χ2 = 12.75) and combined samples (OR 1.25 [1.15-1.37], P = 5.25 × 10-7, χ2 = 25.17). Further analyses showed that the SNP was also significantly associated with BMD and serum levels of the SOX9 protein. Our results provide further supportive evidence for the association of the SOX9 gene with PMOP and of the SOX9 gene with the variation of BMD in postmenopausal Han Chinese women. This study supports a role for SOX9 in the etiology of PMOP, adding to the current understanding of the susceptibility of osteoporosis.

LncRNA-mediated regulation of SOX9 expression in basal subtype breast cancer cells.

Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer (BC) subtypes with a poor prognosis and high recurrence rate. Recent studies have identified vital roles played by several lncRNAs (long noncoding RNAs) in BC pathobiology. Cell type-specific expression of lncRNAs and their potential role in regulating the expression of oncogenic and tumor suppressor genes have made them promising cancer drug targets. By performing a transcriptome screen in an isogenic TNBC/basal subtype BC progression cell line model, we recently reported ∼1800 lncRNAs that display aberrant expression during breast cancer progression. Mechanistic studies on one such nuclear-retained lncRNA, linc02095, reveal that it promotes breast cancer proliferation by facilitating the expression of oncogenic transcription factor, SOX9. Both linc02095 and SOX9 display coregulated expression in BC patients as well in basal subtype BC cell lines. Knockdown of linc02095 results in decreased BC cell proliferation, whereas its overexpression promotes cells proliferation. Linc02095-depleted cells display reduced expression of SOX9 concomitant with reduced RNA polymerase II occupancy at the SOX9 gene body as well as defective SOX9 mRNA export, implying that linc02095 positively regulates SOX9 transcription and mRNA export. Finally, we identify a positive feedback loop in BC cells that controls the expression of both linc02095 and SOX9. Thus, our results unearth tumor-promoting activities of a nuclear lncRNA linc02095 by facilitating the expression of key oncogenic transcription factor in BC.

Increased Gene Expression of RUNX2 and SOX9 in Mesenchymal Circulating Progenitors Is Associated with Autophagy during Physical Activity.

Lack of physical exercise is considered an important risk factor for chronic diseases. On the contrary, physical exercise reduces the morbidity rates of obesity, diabetes, bone disease, and hypertension. In order to gain novel molecular and cellular clues, we analyzed the effects of physical exercise on differentiation of mesenchymal circulating progenitor cells (M-CPCs) obtained from runners. We also investigated autophagy and telomerase-related gene expression to evaluate the involvement of specific cellular functions in the differentiation process. We performed cellular and molecular analyses in M-CPCs, obtained by a depletion method, of 22 subjects before (PRE RUN) and after (POST RUN) a half marathon performance. In order to prove our findings, we performed also in vitro analyses by testing the effects of runners' sera on a human bone marrow-derived mesenchymal stem (hBM-MSC) cell line. PCR array analyses of PRE RUN versus POST RUN M-CPC total RNAs put in evidence several genes which appeared to be modulated by physical activity. Our results showed that physical exercise promotes differentiation. Osteogenesis-related genes as RUNX2, MSX1, and SPP1 appeared to be upregulated after the run; data showed also increased levels of BMP2 and BMP6 expressions. SOX9, COL2A1, and COMP gene enhanced expression suggested the induction of chondrocytic differentiation as well. The expression of telomerase-associated genes and of two autophagy-related genes, ATG3 and ULK1, was also affected and correlated positively with MSC differentiation. These data highlight an attractive cellular scenario, outlining the role of autophagic response to physical exercise and suggesting new insights into the benefits of physical exercise in counteracting chronic degenerative conditions.

Effects of combination TGF-B1 transfection and platelet rich plasma (PRP) on three-dimension chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells

ABSTRACT Aim: The aim of this study was to evaluate the effects of standard culture medium and chondrogenic differentiation medium with PRP on chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells (rabbit DPSCs) that are transfected with transforming growth factor-beta 1 (TGF-B1) gene, based on the hypothesis of TGF- B1 and PRP can be effective on the chondrogenesis of stem cells. Materials and Methods: Rabbit DPSCs were characterized by using flow cytometry, immunofluorescent staining, quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and differentiation tests. For the characterization, CD29, CD44 and CD45 mesenchymal cell markers were used. Rabbit DPSCs were transfected with TGF-B1 gene using electroporation technique in group 1; with PRP 10% in group 2; with chondrogenic medium in group 3; with both chondrogenic medium and PRP in group 4. DPSCs were cultured in medium with 10% inactive PRP in group 5, chondrogenic medium in group 6, chondrogenic medium with PRP 10% in group 7. SOX9, MMP13 and Aggrecan gene expression levels were evaluated in 3, 6, 12. and 24. days by qRT-PCR. Results: The expression levels of SOX9, MMP13 and Aggrecan were higher in group 2, 3 and group 7 in 3th day however in 24th day group 7 and group 2 were found higher. The expression levels changed by time-dependent. The extracellular matrix of the cells in experimental groups were positively stained with safranin O and toluidine blue. Conclusion: The combination in culture medium of TGF-B1 gene transfection and 10% PRP accelerates the chondrogenic differentiation of DPSCs.

Inference of chromosome 3D structures from GAM data by a physics computational approach.

The combination of modelling and experimental advances can provide deep insights for understanding chromatin 3D organization and ultimately its underlying mechanisms. In particular, models of polymer physics can help comprehend the complexity of genomic contact maps, as those emerging from technologies such as Hi-C, GAM or SPRITE. Here we discuss a method to reconstruct 3D structures from Genome Architecture Mapping (GAM) data, based on PRISMR, a computational approach introduced to find the minimal polymer model best describing Hi-C input data from only polymer physics. After recapitulating the PRISMR procedure, we describe how we extended it for treating GAM data. We successfully test the method on a 6 Mb region around the Sox9 gene and, at a lower resolution, on the whole chromosome 7 in mouse embryonic stem cells. The PRISMR derived 3D structures from GAM co-segregation data are finally validated against independent Hi-C contact maps. The method results to be versatile and robust, hinting that it can be similarly applied to different experimental data, such as SPRITE or microscopy distance data.

Improvement of osteogenic differentiation of human mesenchymal stem cells on composite poly l-lactic acid/nano-hydroxyapatite scaffolds for bone defect repair

Tissue engineering offers new approaches to repair bone defects, which cannot be repaired physiologically, developing scaffolds that mimic bone tissue architecture. Furthermore, biomechanical stimulation induced by bioreactor, provides biomechanical cues that regulate a wide range of cellular events especially required for cellular differentiation and function. The improvement of human mesenchymal stem cells (hMSCs) colonization in poly-l-lactic-acid (PLLA)/nano-hydroxyapatite (nHA) composite scaffold was evaluated in terms of cell proliferation (dsDNA content), bone differentiation (gene expression and protein synthesis) and ultrastructural analysis by comparing static (s3D) and dynamic (d3D) 3D culture conditions at 7 and 21 days. The colonization rate of hMSCs and osteogenic differentiation were amplified by d3D when physical stimulation was provided by a perfusion bioreactor. Increase in dsDNA content (p < 0.0005), up-regulation of RUNX2, ALPL, SPP1 (p < 0.0005) and SOX9 (p < 0.005) gene expression, and more calcium nodule formation (p < 0.0005) were observed in d3D cultures in comparison to s3D ones over time. Dynamic 3D culture, mimicking the mechanical signals of bone environment, improved significantly osteogenic differentiation of hMSCs on PLLA/nHA scaffold, without the addition of growth factors, confirming this composite scaffold suitable for bone regeneration.

A critical role of PITX1 in astrocyte differentiation through transcriptional regulation of SOX9 gene

A critical role of PITX1 in astrocyte differentiation through transcriptional regulation of SOX9 gene

Cardiac neural crest contributes to cardiomyocytes in amniotes and heart regeneration in zebrafish.

Cardiac neural crest cells contribute to important portions of the cardiovascular system including the aorticopulmonary septum and cardiac ganglion. Using replication incompetent avian retroviruses for precise high-resolution lineage analysis, we uncover a previously undescribed neural crest contribution to cardiomyocytes of the ventricles in Gallus gallus, supported by Wnt1-Cre lineage analysis in Mus musculus. To test the intriguing possibility that neural crest cells contribute to heart repair, we examined Danio rerio adult heart regeneration in the neural crest transgenic line, Tg(-4.9sox10:eGFP). Whereas the adult heart has few sox10+ cells in the apex, sox10 and other neural crest regulatory network genes are upregulated in the regenerating myocardium after resection. The results suggest that neural crest cells contribute to many cardiovascular structures including cardiomyocytes across vertebrates and to the regenerating heart of teleost fish. Thus, understanding molecular mechanisms that control the normal development of the neural crest into cardiomyocytes and reactivation of the neural crest program upon regeneration may open potential therapeutic approaches to repair heart damage in amniotes.

Exostosin-1 enhances canonical Wnt signaling activity during chondrogenic differentiation

Exostosin-1 (Ext1) encodes a glycosyltransferase required for heparan sulfate (HS) chain elongation in HS-proteoglycan biosynthesis. HS chains serve as binding partners for signaling proteins, affecting their distribution and activity. The Wnt/β-catenin pathway emerged as critical regulator of chondrogenesis. Yet, how EXT1 and HS affect Wnt/β-catenin signaling during chondrogenesis remains unexplored. Ext1 was stably knocked-down or overexpressed in ATDC5 chondrogenic cells cultured as micromasses. HS content was determined using ELISA. Chondrogenic markers Sox9, Col2a1, Aggrecan, and Wnt direct target gene Axin2 were measured by RT-qPCR. Proteoglycan content was evaluated by Alcian blue and DMMB assay, canonical Wnt signaling activation by β-catenin Western blot and TOP/FOP assay. ATDC5 cells and human articular chondrocytes were treated with Wnt activators CHIR99021 and recombinant WNT3A. Ext1 knock-down reduced HS, and increased chondrogenic markers and proteoglycan accumulation. Ext1 knock-down reduced active Wnt/β-catenin signaling. Conversely, Ext1 overexpressing cells, with higher HS content, showed decreased chondrogenic differentiation and enhanced Wnt/β-catenin signaling. Wnt/β-catenin signaling activation led to a down-regulation of Ext1 expression in ATDC5 cells and in human articular chondrocytes. EXT1 affects chondrogenic differentiation of precursor cells, in part via changes in the activity of Wnt/β-catenin signaling. Wnt/β-catenin signaling controls Ext1 expression, suggesting a regulatory loop between EXT1 and Wnt/β-catenin signaling during chondrogenesis.

Enhanced chondrogenesis from human embryonic stem cells

Human embryonic stem cells (hESCs) have great potential for the repair of damaged articular cartilage. We developed a serum-free 14-day protocol for hESC differentiation into chondrocyte progenitors, which surprisingly lacked strong cartilage matrix production in in vitro tests. In order to direct these progenitors to a more mature phenotype, we investigated substituting different members of the TGFβ family in the protocol. Initially, we supplemented, or substituted GDF5 (day 11–14), with combinations of BMP7 and TGFβ-1, or −3, but these modifications yielded no improvement in matrix gene expression. However, replacing BMP4 with BMP2 (days 3–10 of the protocol) resulted in a more rapid increase in SOX9 gene expression and increased expression of chondrogenic genes SOX5, ACAN and COL2A1. The replacement of BMP4 with BMP2 also enhanced the formation of chondrogenic cell aggregates, with greater deposition of type II collagen. This change was not accompanied by hypertrophic chondrocyte marker COL10A1 expression. The results demonstrate that BMP2 has greater specificity for the generation of chondrogenic cells from hESCs than BMP4 and this was consistent in two hESC lines (HUES1 and MAN7). hESC-chondrogenic cells derived with either BMP2 or BMP4 were tested in vivo by implanting them in fibrin into osteochondral defects in the femur of RNU rats. Repaired cartilage tissue, positive for Safranin O and type II collagen was detected at 6 and 12 weeks with both cell sources, but the BMP2 cells scored higher for tissue quality (Pineda score). Therefore, BMP2 is more effective at driving chondrogenic differentiation from human pluripotent stem cells than BMP4 and the effect on the resulting chondroprogenitors is sustained in an in vivo setting.

Specific miRNA-G Protein-Coupled Receptor Networks Regulate Sox9a/Sox9b Activities to Promote Gonadal Rejuvenation in Zebrafish.

Fertility and endocrine function rely on a tightly regulated synchronicity within the hypothalamic-pituitary-gonadal axis, for which the sex gonad serves as the primary source of sex steroid hormones and germ cells. To maintain hormonal stasis and fertility throughout the lifespan, inducing gonadal stem cell renewal is an attractive strategy. The follicle-stimulating hormone/cAMP/MAPK/Sox9 signaling axis and its regulated specific miRNAs are thought to regulate vertebrate gonadal development and sex differentiation, yet the regulatory networks are largely unknown. By genome-wide transcriptome mining and gonadal microinjections, we identify two G protein-coupled receptor (GPCR)-regulatory circuits: miR430a-Sox9a in the testis and miR218a-Sox9b in the ovary. Coinjection of a Sox9a-miR430a mixture promotes spermatogenesis, whereas Sox9b-miR218a mixture increases primordial ovarian follicles. Coimmunoprecipitation and mass spectrometry indicate that the two mixtures differentially modulate Sox9a/Sox9b multiple covalent modifications. We further reveal that miR430a and Sox9a synergistically activate testicular protein kinase C (PKC)/Akt signaling, whereas the miR218a and Sox9b mixture constrains ovary PKC/Akt signaling. pMIR-GFP reporter assay demonstrate that miR430a and miR218a target the 3' untranslated region (UTR) of four GPCR targets (lgr4, grk5l, grk4, and grp157). Knockdown of these GPCR genes or two Sox9 genes alters miR430a and miR218a regulation in the above gonad-specific PKC and Akt signaling pathways. These results establish two specific miRNA-GPCR-Sox9 networks and provide mechanistic insight into gonadal differentiation and rejuvenation. Stem Cells 2019;37:1189-1199.

Campomelic dysplasia: an overview of a rare genetic disorder

Campomelic dysplasia is a rare and severe genetic condition that is characterized by shortening and bowing of the long bones, abnormal face, multiple congenital anomalies, and ambiguous genitalia. Having conducted a review of the existing literature on this rare genetic disorder, we herein present the most pertinent and essential data on the condition viewed from the clinical perspective. In the majority of cases when the neonate survives the condition, since the underlying cause is more often than not a de novo mutation of the SOX9 gene, there is no increased risk of recurrence. Diagnosis is tentatively made based on skeletal findings during routine prenatal ultrasound; it may subsequently be confirmed via either prenatal or postnatal molecular genetic testing or else radiologic evaluation. In general, the condition is considered to be lethal in the neonatal period, there is no prenatal treatment and pregnancy termination is an option.

Mechanical stimulation promotes the proliferation and the cartilage phenotype of mesenchymal stem cells and chondrocytes co-cultured in vitro

Mesenchymal stem cells and chondrocytes are an important source of the cells for cartilage tissue engineering. Therefore, the culture and expansion methods of these cells need to be improved to overcome the aging of chondrocytes and induced chondrogenic differentiation of mesenchymal stem cells. The aim of this study was to expand the cells for cartilage tissue engineering by combining the advantages of growing cells in co-culture and under a mechanically-stimulated environment. Rabbit chondrocytes and co-cultured cells (bone mesenchymal stem cells and chondrocytes) were subjected to cyclic sinusoidal dynamic tensile mechanical stimulationusing the FX-4000 tension system. Chondrocyte proliferation was assayed by flow cytometry and CFSE labeling. The cell cartilage phenotype was determined by detecting GAG, collagen II and TGF-β1 protein expression by ELISA and the Col2α1, TGF-β1 and Sox9 gene expression by RT-PCR. The results show that the co-culture improved both the proliferation ability of chondrocytes and the cartilage phenotype of co-cultured cells. A proper cyclic sinusoidal dynamic tensile mechanical stimulation improved the proliferation ability and cartilage phenotype of chondrocytes and co-cultured cells. These results suggest that the co-culture of mesenchymal stem cells with chondrocytes and proper mechanical stimulation may be an appropriate way to rapidly expand the cells that have an improved cartilage phenotype for cartilage tissue engineering.

Association between polymorphisms in the SOX9 region and canine disorder of sex development (78,XX; SRY-negative) revisited in a multibreed case-control study.

Testicular or ovotesticular disorders of sex development (DSD) in individuals with female karyotype (XX) lacking the SRY gene has been observed in several mammalian species, including dogs. A genetic background for this abnormality has been extensively sought, and the region harboring the SOX9 gene has often been considered key in canine DSD. Three types of polymorphism have been studied in this region to date: a) copy number variation (CNV) in a region about 400 kb upstream of SOX9, named CNVR1; b) duplication of SOX9; and c) insertion of a single G-nucleotide (rs852549625) approximately 2.2 Mb upstream of SOX9. The aim of this study was thus to comprehensively analyze these polymorphisms in a large multibreed case-control cohort containing 45 XX DSD dogs, representing 23 breeds. The control set contained 57 fertile females. Droplet digital PCR (ddPCR) was used to study CNVR1 and the duplication of SOX9. Fluorescent in situ hybridization (FISH) was used to visualize copy numbers on a cellular level. The Sanger sequencing approach was performed to analyze the region harboring the G-insertion. We confirmed that CNVR1 is highly polymorphic and that copy numbers varied between 0 and 7 in the case and control cohorts. Interestingly, the number of copies was significantly higher (P = 0.038) in XX DSD dogs (mean = 2.7) than in the control females (mean = 2.0) but not in all studied breeds. Duplication of the SOX9 gene was noted only in a single XX DSD dog (an American Bully), which had three copies of SOX9. Distribution of the G-nucleotide insertion was similar in the XX DSD (frequency 0.20) and control (frequency 0.14) cohorts. Concluding, our study showed that CNVR1, located upstream of SOX9, is associated with the XX DSD phenotype, though in a breed-specific manner. Duplication of the SOX9 gene is a rare cause of this disorder in dogs. Moreover, we did not observe any association of G-insertion with the DSD phenotype. We assume that the genetic background of XX DSD can be different in certain breeds.

Proper mechanical stimulation improve the chondrogenic differentiation of mesenchymal stem cells: Improve the viscoelasticity and chondrogenic phenotype

Mesenchymal stem cells are ideal seed cell alternatives for articular cartilage tissue engineering, and the methods of the expansion of seed cells need to be improved. The mechanical factors play a significant role in the process of articular cartilage development and regeneration. The aim of this study was to improve the chondrogenic differentiation of mesenchymal stem cells and to expand seed cells for articular cartilage tissue engineering based on mechanical factors. Rabbit bone mesenchymal stem cells were subjected to cyclic dynamic square wave tensile mechanical loading using the FX-4000 tension system. The viscoelasticity of cells was investigated using the micropipette aspiration technique combined with the Kelvin standard linear viscoelastic solid model. The cell chondrogenic phenotype was assessed by detecting characteristic chondrocyte biomarkers; the expression of the GAG and TGF-β1 was analyzed by ELISA, and the expression of the Col2α1 and Sox9 gene was analyzed by RT-PCR. The results show that proper tensile mechanical stimulation improves the viscoelasticity and chondrogenic phenotype of mesenchymal stem cells such that it is similar to that of chondrocytes. These results suggest that viscoelasticity is a specific marker of chondrogenic differentiation and that proper mechanical stimulation culture methods can be used to expand seed cells and improve the chondrogenic phenotype for articular cartilage tissue engineering.

Effect of Matrix Metalloproteinase 3 Gene Silencing and SRY-Related High Mobility Group-Box Gene 9 Gene Overexpression on Human Degeneration Nucleus Pulposus Cells In Vitro

This study aims to detect the biological effects of matrix metalloproteinase 3 (MMP3) and SRY-related high mobility group-box gene 9 (SOX9) gene regulation in the human intervertebral disc degeneration of nucleus pulposus cells mediated by lentiviral vectors in vitro. The culture and development of human degenerated intervertebral disc nucleus pulposus cells was performed. The cell morphology and structure were observed, as identified by hematoxylin and eosin (H&amp;E) staining. The experiment was divided into five groups: blank control group, SOX9 gene overexpression group, MMP3 gene silencing group, SOX9 gene overexpression+MMP3 gene silencing group, and blank vector group. RT-PCR and western blot were performed to detect the influence of MMP3 gene silencing and SOX9 gene overexpression on degenerated human intervertebral disc nucleus pulposus cells, include the secretion of polysaccharide and expression of type II collagen, at the mRNA and protein level. The originally generated degenerated human intervertebral disc nucleus pulposus cells had the same cellular morphology. The MTT assay revealed that the blank group and blank vector group had no statistical significance in cell proliferation. The MMP3 gene silencing and SOX9 gene overexpression could promote the proliferation of degenerated human intervertebral disc nucleus pulposus cells, when compared with the blank control group and blank vector group, and this more obvious in the MMP3 gene silencing group and SOX9 gene overexpression group. RT-PCR and western blot revealed that MMP3 gene silencing and SOX9 gene overexpression can promote cells to secrete polysaccharide and type-II collagen (P &lt; 0.05). MMP3 gene silencing and SOX9 gene overexpression can promote the proliferation of degenerated human intervertebral disc nucleus pulposus cells, and their ability to secrete polysaccharide and type-II collagen.

Qualifying mscs for clinical applications -differential gene expression analyses of pluripotent, differentiation and surface marker genes of early and late passage mscs, in hypoxic and normal oxygen level in culture conditions.

Background & Aim With the increasing focus on the large scale expansion of MSCs required for the clinical application, the optimisation of in-vitro expansion of MSCs requires careful consideration to maintain the native MSCs characteristics. Physiological parameters such as oxygen concentration, media constituents and passage numbers have much influence on the properties of MSCs and may have major impact on the therapeutic potential of MSCs. Cells grown under hypoxic (low oxygen) conditions have been widely documented in clinical use however culturing MSCS in large scale requires bioreactor and it is challenging to maintain the low oxygen and other physiological parameters over several passages in big vessels of bioreactors. Similarly, the culture conditions in the bioreactor including pH, dissolved oxygen and the sheer force exerted on cells may change the cellular characteristics including gene expression Methods, Results & Conclusion These culture conditions may have different potency attributes clinically. For these reasons, our study investigated differential expression of 11 key genes of MSCs for pluripotent genes POU5F1, NANOG and KLF4, differentiation genes RUNX2, COL1A1, SOX9, COL2A1, PPARG and CD marker genes THY1, ENG and NT5E from adipose derived MSCs, grown under both the normal oxygen or hypoxic conditions were analysed. Two different cells age early (P5) and late passage (P9) cells. The result demonstrated marked differences in gene expression pattern of the early and late passage cells grown under normoxic (normal oxygen) when compared with the hypoxic conditions. Pluripotent markers KLF4 in early passage cells was expressed more than two-fold in the normoxic cells, and PPARG was expressed more in the late passage cells under normoxic condition. Differentiation marker for RUNX2 and NT5E were highly expressed in early passage cells. These differences in gene expression may have critical impact on the potency of the cells grown in large scale in bioreactor. Additionally, the effects of prolonged culture of MSCs in-vitro will need be qualified prior to use when manufactured in large scale bioreactor for the clinical dose to ensure the therapeutic efficacy.

Sox9 deletion in the intestinal epithelium increases house dust mite induced allergic asthma symptoms in mice

Abstract Paneth cells regulate many key aspects of gastrointestinal health through the antimicrobial products and cytokines they produce. Loss or defective Paneth cell functions leads to dysbiosis and is one of the causes of Inflammatory Bowel Disease (IBD). Mice with Sox9 gene deletion within the intestinal epithelium (Sox9 ΔIEC mice) lack Paneth cells making these mice a great model for the study or IBD and other consequences of dysbiosis. Our separate studies have shown that dysbiosis of the gut microbiota could enhance allergic sensitization in the gut and subsequent allergic responses to nasal allergen challenge. We investigated whether dysbiosis caused by a Sox9 deletion in the intestinal epithelium of mice could affect allergic sensitization in the airway and development of asthma responses. For this purpose, control wild-type and Sox9 ΔIEC mice were sensitized tohouse dust mite (HDM) by daily intranasal administration over two weeks period. Upon nasal HDM challenge, Sox9 ΔIEC mice showed more severe signs of allergic asthma than wild-type C57 BL/6 mice as indicated by their higher hypothermia. We also established that the increased allergic symptoms in Sox9 ΔIEC mice correlated with higherIgE levels in serum and bronchoalveolar lavage fluid, and higher frequency of HDM-specific Th2 cells but no difference in Th1 or Th17 responses. These results further illustrate that the gut immune homeostasis plays an important role in allergic responses at distant mucosal sites.