Increase In Sox9 Research Articles

Changes in equine intestinal stem/progenitor cell number at resection margins in cases of small intestinal strangulation.

Intestinal epithelial stem cells (ISC) are responsible for epithelial regeneration and are critical to the intestine's ability to regain barrier function following injury. Evaluating ISC biomarker expression in cases of small intestinal strangulation (SIS) may provide insight into clinical progression. Intestinal resection margins from cases of SIS were evaluated to determine if (1) evidence of injury could be identified using histomorphometry, (2) ISC biomarker expression was decreased in the proximal resection margin compared to control and distal resection margin, and (3) the ISC biomarker expression was associated with the number of preoperative risk factors negatively related to outcome, post-operative complications, or case outcome. Retrospective cohort study. Intestinal samples were obtained intraoperatively from resection margins of adult horses with SIS and horses euthanised for reasons unrelated to colic. Preoperative risk factors negatively related to outcome, post-operative complications, and case outcome were obtained from medical records. Horses were grouped as euthanised intraoperatively, postoperatively, or survived to discharge. Histomorphometry and immunofluorescence were performed to evaluate tissue architecture and ISC and progenitor cell number. Groups were compared using one-way ANOVA. Associations between biomarker expression and the number of preoperative risk factors and post-operative complications negatively related to outcome were determined using linear regression modelling. Thirty-six cases of SIS were evaluated. Ki67+ cell counts were decreased in the proximal (mean=15.45 cells; 95% CI= 10.27-20.63; SD=4.17; p= 0.02) and distal resection margins (mean=15.05; 95% CI=8.46-21.64; SD=4.141; p= 0.03) in horses euthanised postoperatively compared to control (mean=23.62 cells; 95% CI=19.42-27.83; SD=5.883). In the distal resection margin, an increase in SOX9+ Ki67+ cells were associated with a decrease in the total number of preoperative risk factors negatively related to outcome (95% CI = 0.236-1.123; p= 0.008, SE=0.1393). Small population size. Proliferating cell and ISC numbers may be associated with case outcome.

Increased Expression of Flightless I in Cutaneous Squamous Cell Carcinoma Affects Wnt/β-Catenin Signaling Pathway.

Cutaneous squamous cell carcinoma (cSCC) accounts for 25% of cutaneous malignancies diagnosed in Caucasian populations. Surgical removal in combination with radiation and chemotherapy are effective treatments for cSCC. Nevertheless, the aggressive metastatic forms of cSCC still have a relatively poor patient outcome. Studies have linked actin cytoskeletal dynamics and the Wnt/β-catenin signaling pathway as important modulators of cSCC pathogenesis. Previous studies have also shown that the actin-remodeling protein Flightless (Flii) is a negative regulator of cSCC. The aim of this study was to investigate if the functional effects of Flii on cSCC involve the Wnt/β-catenin signaling pathway. Flii knockdown was performed using siRNA in a human late stage aggressive metastatic cSCC cell line (MET-1) alongside analysis of Flii genetic murine models of 3-methylcholanthrene induced cSCC. Flii was increased in a MET-1 cSCC cell line and reducing Flii expression led to fewer PCNA positive cells and a concomitant reduction in cellular proliferation and symmetrical division. Knockdown of Flii led to decreased β-catenin and a decrease in the expression of the downstream effector of β-catenin signaling protein SOX9. 3-Methylcholanthrene (MCA)-induced cSCC in Flii overexpressing mice showed increased markers of cancer metastasis including talin and keratin-14 and a significant increase in SOX9 alongside a reduction in Flii associated protein (Flap-1). Taken together, this study demonstrates a role for Flii in regulating proteins involved in cSCC proliferation and tumor progression and suggests a potential role for Flii in aggressive metastatic cSCC.

Improved Mesenchymal Stem Cell Proliferation, Differentiation, Epithelial Transition, and Restrained Senescence on Hierarchically Patterned Porous Honey Silk Fibroin Scaffolds.

We report a significant improvement of adipose-derived mesenchymal stem cells' (ADMSCs) biocompatibility and proliferation on hierarchically patterned porous honey-incorporated silk fibroin scaffolds fabricated using a combination of soft lithography and freeze-drying techniques. Parametric variations show enhanced surface roughness, swelling, and degradation rate with good pore interconnectivity, porosity, and mechanical strength for soft-lithographically fabricated biomimetic microdome arrays on the 2% honey silk fibroin scaffold (PHSF2) as compared to its other variants, which eventually made PHSF2 more comparable to the native environment required for stem cell adhesion and proliferation. PHSF2 also exhibits sustained honey release with remarkable antibacterial efficacy against methicillin-resistant Staphylococcus aureus (MRSA). Honey incorporation (biochemical cue) influences microdome structural features, that is, biophysical cues (height, width, and periodicity), which further allows ADMSCs pseudopods (filopodia) to grasp the microdomes for efficient cell-cell communication and cell-matrix interaction and regulates ADMSCs behavior by altering their cytoskeletal rearrangement and thereby increases the cellular spreading area and cell sheet formation. The synergistic effect of biochemical (honey) and biophysical (patterns) cues on ADMSCs studied by the nitro blue tetrazolium assay and DCFDA fluorescence spectroscopy reveals limited free radical generation within cells. Molecular expression studies show a decrease in p53 and p21 expressions validating ADMSCs senescence inhibition, which is further correlated with a decrease in cellular senescence-associated β galactosidase activity. We also show that an increase in CDH1 and CK19 molecular expressions along with an increase in SOX9, RUNX2, and PPARγ molecular expressions supported by PHSF2 justify the substrate's efficacy of underpinning mesenchymal to epithelial transition and multilineage trans-differentiation. This work highlights the fabrication of a naturally healing nutraceutical (honey)-embedded patterned porous stand-alone tool with the potential to be used as smart stem cells delivering regenerative healing implant.

Liver sinusoidal endothelial cells promote the differentiation and survival of mouse vascularised hepatobiliary organoids

The structural and physiological complexity of currently available liver organoids is limited, thereby reducing their relevance for drug studies, disease modelling, and regenerative therapy. In this study we combined mouse liver progenitor cells (LPCs) with mouse liver sinusoidal endothelial cells (LSECs) to generate hepatobiliary organoids with liver-specific vasculature. Organoids consisting of 5x103 cells were created from either LPCs, or a 1:1 combination of LPC/LSECs. LPC organoids demonstrated mild hepatobiliary differentiation in vitro with minimal morphological change; in contrast LPC/LSEC organoids developed clusters of polygonal hepatocyte-like cells and biliary ducts over a 7 day period. Hepatic (albumin, CPS1, CYP3A11) and biliary (GGT1) genes were significantly upregulated in LPC/LSEC organoids compared to LPC organoids over 7 days, as was albumin secretion. LPC/LSEC organoids also had significantly higher in vitro viability compared to LPC organoids. LPC and LPC/LSEC organoids were transplanted into vascularised chambers created in Fah−/−/Rag2−/−/Il2rg−/− mice (50 LPC organoids, containing 2.5x105 LPCs, and 100 LPC/LSEC organoids, containing 2.5x105 LPCs). At 2 weeks, minimal LPCs survived in chambers with LPC organoids, but robust hepatobiliary ductular tissue was present in LPC/LSEC organoids. Morphometric analysis demonstrated a 115-fold increase in HNF4α+ cells in LPC/LSEC organoid chambers (17.26 ± 4.34 cells/mm2 vs 0.15 ± 0.15 cells/mm2, p = 0.018), and 42-fold increase in Sox9+ cells in LPC/LSEC organoid chambers (28.29 ± 6.05 cells/mm2 vs 0.67 ± 0.67 cells/mm2, p = 0.011). This study presents a novel method to develop vascularised hepatobiliary organoids, with both in vitro and in vivo results confirming that incorporating LSECs with LPCs into organoids significantly increases the differentiation of hepatobiliary tissue within organoids and their survival post-transplantation.

623 Changing endothelial cell fate in wound healing through modulation of Sox9 to reduce scarring

Endothelial cells that line the vascular endothelium undergo a variety of cellular transitions, with one such transition known as endothelial to mesenchymal transition (EndMT). We identified transcription factor Sox9 to be highly expressed in a progenitor population in the endothelium. We here aimed to investigate the potential molecular regulation of EndMT by the transcription factor Sox9 as well as its interaction with Notch signaling. A vascular-specific Sox9 knock-out (Sox9fl/flCdh5CreERt2Rosa-YFP (Sox9eKO)) and a vascular specific rbpj knock-out mouse model were used to assess EndMT. Wild-type mice were used for Sox9 siRNA knock-down. Loss of rbpj resulted in an increase in Sox9 expression in the vasculature. Full thickness excisional large wounds were used to assess fibrotic scar area following Sox9eKO or Sox9 siRNA knock-down. Full thickness small wounds were used to assess the changes in cellular phenotype in EndMT following Sox9eKO or Sox9 siRNA knock-down via immunofluorescence staining and flow cytometry. A significant reduction in fibrotic scar area was seen following Sox9eKO (**P<0.01) and siRNA knock-down (*P<0.05). Immunofluorescence quantification showed a significant reduction in the number of YFP labelled endothelial cells, vessels stained for endothelial (CD31) and mesenchymal/EndMT (α-SMA, FSP-1 and SLUG) markers across time-points assessed (D3 to D7), in both Sox9eKO and Sox9 siRNA knock-down. Flow cytometry analysis showed a significant reduction in the intensity of the pro-fibrotic marker CD26 in both Sox9eKO and Sox9 siRNA knock-down (***P<0.001). Loss of rbpj resulted in an increase in Sox9, EndMT and fibrosis. In conclusion, the dramatic decrease in fibrotic scar area following both Sox9eKO and Sox9 siRNA knock-down highlights Sox9s role in scar formation through EndMT. This highlights Sox9 as a novel regulator for EndMT and contributes to both an understanding into the fundamentals of cardiovascular biology and identification of a potential therapeutic target using siRNA in reducing scar area.

Transcription factor Sox10 regulates oligodendroglial Sox9 levels via microRNAs.

During development of myelin-forming oligodendrocytes in the central nervous system the two closely related transcription factors Sox9 and Sox10 play essential roles that are partly shared and partly unique. Whereas Sox9 primarily functions during oligodendroglial specification, Sox10 is uniquely required to induce terminal differentiation and myelination. During this process, Sox10 protein levels rise substantially. As this coincides with a reciprocal decrease in Sox9, we postulated that Sox10 influences Sox9 amounts in differentiating oligodendrocytes. Here we show that Sox9 levels are indeed inversely coupled to Sox10 levels such that Sox10 deletion in oligodendroglial cells evokes a reciprocal increase in Sox9. We furthermore provide evidence that this coupling involves upregulation of microRNAs miR335 and miR338 as direct transcriptional targets of Sox10. The two microRNAs in turn recognize the 3'-UTR of Sox9 mRNA and may thereby reduce Sox9 protein levels posttranscriptionally in oligodendroglial cells. Such a mechanism may enable oligodendroglial cells to adapt the ratio of both related Sox proteins in a manner required for successful lineage progression and differentiation. Mathematical modeling furthermore shows that the identified regulatory circuit has the potential to convert a transient stimulus into an irreversible switch of cellular properties and may thus contribute to terminal differentiation of oligodendrocytes.

Rapamycin-Induced Hypoxia Inducible Factor 2A Is Essential for Chondrogenic Differentiation of Amniotic Fluid Stem Cells

Amniotic fluid stem (AFS) cells represent a major source of donor cells for cartilage repair. Recently, it became clear that mammalian target of rapamycin (mTOR) inhibition has beneficial effects on cartilage homeostasis, but the effect of mTOR on chondrogenic differentiation is still elusive. Therefore, the objectives of this study were to investigate the effects of mammalian target of rapamycin complex 1 (mTORC1) modulation on the expression of SOX9 and on its downstream targets during chondrogenic differentiation of AFS cells. We performed three-dimensional pellet culturing of AFS cells and of in vitro-expanded, human-derived chondrocytes in the presence of chondrogenic factors. Inhibition of mTORC1 by rapamycin or by small interfering RNA-mediated targeting of raptor (gene name, RPTOR) led to increased AKT activation, upregulation of hypoxia inducible factor (HIF) 2A, and an increase in SOX9, COL2A1, and ACAN abundance. Here we show that HIF2A expression is essential for chondrogenic differentiation and that AKT activity regulates HIF2A amounts. Importantly, engraftment of AFS cells in cell pellets composed of human chondrocytes revealed an advantage of raptor knockdown cells compared with control cells in their ability to express SOX9. Our results demonstrate that mTORC1 inhibition leads to AKT activation and an increase in HIF2A expression. Therefore, we suggest that mTORC1 inhibition is a powerful tool for enhancing chondrogenic differentiation of AFS cells and also of in vitro-expanded adult chondrocytes before transplantation. Repair of cartilage defects is still an unresolved issue in regenerative medicine. Results of this study showed that inhibition of the mammalian target of rapamycin complex 1 (mTORC1) pathway, by rapamycin or by small interfering RNA-mediated targeting of raptor (gene name, RPTOR), enhanced amniotic fluid stem cell differentiation toward a chondrocytic phenotype and increased their engrafting efficiency into cartilaginous structures. Moreover, freshly isolated and in vitro passaged human chondrocytes also showed redifferentiation upon mTORC1 inhibition during culturing. Therefore, this study revealed that rapamycin could enable a more efficient clinical use of cell-based therapy approaches to treat articular cartilage defects.

Human Migratory Meniscus Progenitor Cells Are Controlled via the TGF-β Pathway

SummaryDegeneration of the knee joint during osteoarthritis often begins with meniscal lesions. Meniscectomy, previously performed extensively after meniscal injury, is now obsolete because of the inevitable osteoarthritis that occurs following this procedure. Clinically, meniscus self-renewal is well documented as long as the outer, vascularized meniscal ring remains intact. In contrast, regeneration of the inner, avascular meniscus does not occur. Here, we show that cartilage tissue harvested from the avascular inner human meniscus during the late stages of osteoarthritis harbors a unique progenitor cell population. These meniscus progenitor cells (MPCs) are clonogenic and multipotent and exhibit migratory activity. We also determined that MPCs are likely to be controlled by canonical transforming growth factor β (TGF-β) signaling that leads to an increase in SOX9 and a decrease in RUNX2, thereby enhancing the chondrogenic potential of MPC. Therefore, our work is relevant for the development of novel cell biological, regenerative therapies for meniscus repair.

Regenerative potential of TGFβ3 + Dex and notochordal cell conditioned media on degenerated human intervertebral disc cells

Injection of soluble cell signaling factors into degenerated intervertebral discs (IVDs) offers a minimally invasive treatment that could limit the processes of degeneration by stimulating native matrix repair. This study evaluated the regenerative capacity of degenerated nucleus pulposus (NP) cells obtained from patients undergoing anterior interbody fusions by measuring metabolic activity, DNA content, glycosaminoglycan (GAG) content, and cellular phenotype using qRT-PCR profiling with a custom array of 42 genes. NP cells were cultured in alginate for 7 days with 4 treatment groups: transforming growth factor beta 3 (TGFβ3) + dexamethasone (Dex), soluble factors released from notochordal cells (NCs) cultured in alginate (NCA), soluble factors released from NCs in their native tissue environment (NCT), and basal media. TGFβ3 + Dex stimulated degenerated human NP cells to proliferate and exhibit an anti-catabolic gene expression profile (with a decrease in ADAMTS5 and MMP1 compared to basal, and an increase in SOX9, decrease in ADAMTS5, MMP1, collagen I and collagen III compared to day 0), while NCA stimulated the greatest GAG per cell. We conclude that degenerated human NP cells exhibit regenerative potential, and that an optimal treatment will likely require treatments, such as TGFβ3 + Dex, which were able to increase cell metabolism and reduce catabolism, as well as treatments with factors found in NC conditioned medium, that were able to produce high amounts of GAG per cell. Additional studies to optimize NC culture conditions are required to determine if NC conditioned medium can be made with the capacity to enhance NP cell proliferation and metabolism.

Atorvastatin Attenuates Sox9 Joint and Cardiac Calcification in ApoE null mice

BackgroundDegenerative joint disease is responsible for high morbidity in patients. The mechanisms of osteoarthritis and cardiac calcification are under investigation. We hypothesize that lipids may play a role via chondrogenesis.MethodsWe tested lipids with and without statins in the ApoE mouse to determine the Sox9 expression in the joints and valves. ApoE−/− mice (n=60). Group I (n=20) normal diet, Group II (n=20) 0.25% chol diet (w/w), and Group III (n=20) 0.25% (w/w) chol diet+atorv for the development of abnormal matrix synthesis in the joints over 24 weeks. The aortic valve (AVA) was examined for Bone formation and was assessed by microCT. Aortic Valve Disease was assessed by echo.ResultsThe chol diet induced bone in the AV and thickened knee joints with an increase in Sox9 and Cyclin as compared to the Control(&gt;3‐fold(p&lt;05). Atorvastatin attenuated all markers. Echo demonstrated a mild increase in the peak jet velocity. Masson Trichrome demonstrated a marked increase in the matrix synthesis.ConclusionWe demonstrated that chondrogenic mediated bone formation in the hypercholesterolemic aortic valves and abnormal matrix synthesis in the knee joints is attenuated with atorvastatin.

Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

The molecular mechanisms controlling differentiation of mesenchymal precursor cells into chondrocytes (chondrogenesis) are not completely understood. We have recently shown that the small GTPase RhoA inhibits this process. Here we demonstrate that a different Rho GTPase family member, Rac1, promotes chondrogenesis. Pharmacological inhibition of Rac1 expression in micromass culture resulted in reduced mRNA levels of the chondrogenic markers collagen II and aggrecan, and decreased accumulation of glycosaminoglycans. Expression of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6 was also reduced upon inhibition of Rac1 signaling. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased mRNA transcripts of Sox9, 5, and 6, collagen II, and aggrecan. Inhibition of Rac1 resulted in a reduction in the number, size, and organization of cellular condensations and decreased expression of N-cadherin. Overexpression of Rac1 resulted in an increase in N-cadherin expression levels. Furthermore, genetic ablation of Rac1 in primary micromass cultures resulted in reduced expression of chondrogenic markers. Additionally, we provide evidence that Cdc42 also promotes chondrogenesis. Overexpression of Cdc42 in ATDC5 cells resulted in increased expression of Sox5, Sox9, and collagen II but not Sox6, aggrecan, or N-cadherin. Therefore, we demonstrate that Rac1 and Cdc42 are positive regulators of chondrogenesis, but act at least in part through different cellular and molecular mechanisms.

Expression of SOX9 and SOX10 in Central Neuroepithelial Tumor

The SOX group E transcription factors play an integral role in the specification and differentiation of astrocytes and oligodendrocytes. We have examined the pattern of expression for SOX9 and SOX10 in primary brain tumors by immunohistochemistry. Pediatric and adult high grade tumors display strong nuclear staining for both SOX9 and SOX10 (astrocytic, oligodendroglial and primitive neuroectodermal tumors). In comparison pediatric pilocytic astrocytoma express much less SOX9 and SOX10. Reactive astrogliosis is characterized by an increase of SOX9 only.

The effect of bone morphogenetic protein-2 on rat intervertebral disc cells in vitro.

An in vitro experiment to determine the molecular and cellular effect of recombinant human bone morphogenetic protein-2 on cultured rat intervertebral disc cells was performed. To determine the effect of recombinant human bone morphogenetic protein-2 on cell proliferation, production of sulfated-glycosaminoglycan, and the expression of genes specific for chondrocytes (Type II collagen, aggrecan, and Sox9) in cultured rat intervertebral disc cells. Intervertebral disc degeneration is associated with cellular and biochemical changes, which include decreased synthesis of cartilage specific gene products such as Type II collagen and aggrecan. Although bone morphogenetic protein-2 is known to induce chondrogenesis during new bone formation, the effects on intervertebral disc cells have not been characterized. Cells were isolated from the anulus fibrosus and transition zones of lumbar discs from Sprague-Dawley rats. The cells were grown in monolayer and treated with recombinant human bone morphogenetic protein-2 (0, 10, 100, 1000 ng/mL) in Dulbecco's Modified Eagle Medium/F-12 with 1% fetal bovine serum (day 0). On days 2, 4, and 7 after recombinant human bone morphogenetic protein-2 treatment, sulfated-glycosaminoglycan content in the media was quantified using 1,9-dimethylmethylene blue staining. The results were normalized according to culture duration and cell number. On day 7, mRNA was extracted for reverse transcriptase-polymerase chain reaction and real-time polymerase chain reaction to quantitate mRNAs of Type I collagen, Type II collagen, aggrecan, Sox9, osteocalcin, and glyceraldehyde phosphate dehydrogenase. Cell number was determined with a hemocytometer. Recombinant human bone morphogenetic protein-2 at 100 and 1000 ng/mL yielded a 17% and 42% increase in cell number on day 4, and a 59% and 79% on day 7, respectively. Recombinant human bone morphogenetic protein-2 at 10 ng/mL had no effect on cell number. Sulfated-glycosaminoglycan increase was greatest at day 7, increasing by 1.3-, 2.1-, and 3.6-fold with recombinant human bone morphogenetic protein-2 treatments of 10, 100, and 1000 ng/mL, respectively. Increases in mRNA levels of Type II collagen, aggrecan, Sox9, and osteocalcin were observed with recombinant human bone morphogenetic protein-2 concentrations of 100 and 1000 ng/mL on day 7 as determined by reverse transcriptase-polymerase chain reaction. No detectable increase in mRNA level of Type I collagen was observed with any levels of recombinant human bone morphogenetic protein-2. Real-time polymerase chain reaction showed the greatest effect at 1000 ng/mL recombinant human bone morphogenetic protein-2, leading to an 11.5-fold increase in aggrecan, a 4.6-fold increase in Type II collagen, a 5.3-fold increase in Sox9, and a 1.9-fold increase in osteocalcin mRNA above untreated controls at day 7. The results of this study show that recombinant human bone morphogenetic protein-2 enhances disc matrix production and chondrocytic phenotype of intervertebral disc cells. Recombinant human bone morphogenetic protein-2 increases cell proliferation and sulfated-glycosaminoglycan (proteoglycan) synthesis. It increases mRNA of Type II collagen, aggrecan, and Sox9 genes (chondrocyte specific genes), and osteocalcin, but not Type I collagen or glyceraldehyde phosphate dehydrogenase.