Cell Surface Markers CD44 Research Articles

A transcriptomic analysis of dental pulp stem cell senescence in vitro

Background/purposeThe use of human dental pulp stem cells (hDPSCs) as autologous stem cells for tissue repair and regenerative techniques is a significant area of global research. The objective of this study was to investigate the effects of long-term in vitro culture on the multidifferentiation potential of hDPSCs and the potential molecular mechanisms involved.Materials and methodsThe tissue block method was used to extract hDPSCs from orthodontic-minus-extraction patients, which were then expanded and cultured in vitro for 12 generations. Stem cells from passages three, six, nine, and twelve were selected. Flow cytometry was used to detect the expression of stem cell surface markers, and CCK-8 was used to assess cell proliferation. β-Galactosidase staining was employed to detect cellular senescence, Alizarin Red S staining to assess osteogenic potential, and Oil Red O staining to evaluate lipogenic capacity. RNA sequencing (RNA-seq) was conducted to identify differentially expressed genes in DPSCs and investigate their potential mechanisms.ResultsWith increasing passage numbers, pulp stem cells showed an increase in senescence and a decrease in proliferative capacity and osteogenic–lipogenic multidifferentiation potential. The expression of stem cell surface markers CD34 and CD45 was stable, whereas the expression of CD73, CD90, and CD105 decreased with increasing passages. According to the RNA-seq analysis, the differentially expressed genes CFH, WNT16, HSD17B2, IDI1, and COL5A3 may be associated with stem cell senescence.ConclusionIncreased in vitro expansion induced cellular senescence in pulp stem cells, which resulted in a reduction in their proliferative capacity and osteogenic–lipogenic differentiation potential. The differential expression of genes such as CFH, WNT16, HSD17B2, IDI1, and COL5A3 may represent a potential mechanism for the induction of cellular senescence in pulp stem cells.

Role of mitochondria in endogenous renal repair.

Renal tubules have potential to regenerate and repair after mild-to-moderate injury. Proliferation of tubular epithelial cells represents the initial step of this reparative process. Although for many years, it was believed that proliferating cells originated from a pre-existing intra-tubular stem cell population, there is now consensus that surviving tubular epithelial cells acquire progenitor properties to regenerate the damaged kidney. Scattered tubular-like cells (STCs) are dedifferentiated adult renal tubular epithelial cells that arise upon injury and contribute to renal self-healing and recovery by replacing lost neighboring tubular epithelial cells. These cells are characterized by the co-expression of the stem cell surface markers CD133 and CD24, as well as mesenchymal and kidney injury markers. Previous studies have shown that exogenous delivery of STCs ameliorates renal injury and dysfunction in murine models of acute kidney injury, underscoring the regenerative potential of this endogenous repair system. Although STCs contain fewer mitochondria than their surrounding terminally differentiated tubular epithelial cells, these organelles modulate several important cellular functions, and their integrity and function are critical to preserve the reparative capacity of STCs. Recent data suggest that the microenviroment induced by cardiovascular risk factors, such as obesity, hypertension, and renal ischemia may compromise STC mitochondrial integrity and function, limiting the capacity of these cells to repair injured renal tubules. This review summarizes current knowledge of the contribution of STCs to kidney repair and discusses recent insight into the key role of mitochondria in modulating STC function and their vulnerability in the setting of cardiovascular disease.

Decellularized leaf-based biomaterial supports osteogenic differentiation of dental pulp mesenchymal stem cells.

Decellularized tissues are an attractive scaffolds for 3D tissue engineering. Decellularized animal tissues have certain limitations such as the availability of tissue, high costs and ethical concerns related to the use of animal sources. Plant-based tissue decellularized scaffolds could be a better option to overcome the problem. The leaves of different plants offer a unique opportunity for the development of tissue-specific scaffolds, depending on the reticulate or parallel veination. Herein, we decellularized spinach leaves and employed thesefor the propagation and osteogenic differentiation of dental pulp stem cells (DPSCs). DPSCs were characterized by using mesenchymal stem cell surface markers CD90, CD105 and CD73 and CD34, CD45 and HLA-DR using flow cytometry. Spinach leaves were decellularized using ethanol, NaOH and HCL. Cytotoxicity of spinach leaf scaffolds were analysed by MTT assay. Decellularized spinach leaves supported dental pulp stem cell adhesion, proliferation and osteogenic differentiation. Our data demonstrate that the decellularized spinach cellulose scaffolds can stimulate the growth, proliferation and osteogenic differentiation of DPSCs. In this study, we showed the versatile nature of decellularized plant leaves as a biological scaffold and their potential for bone regeneration in vitro.

Preliminary study on the mechanism by which exosomes derived from human exfoliated deciduous teeth improve the proliferation and osteogenic inhibitory effect of glucocorticoid-induced BMSCs

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a disease characterized by a collapsed femoral head caused by the overuse of glucocorticoids. Dysfunction of bone marrow mesenchymal stem cells (BMSCs) is an important pathological feature of SONFH. In this study, we investigated whether exosomes from SHEDs (stem cells from human exfoliated deciduous teeth) have a therapeutic effect on glucocorticoid-induced inhibition of proliferation and osteogenesis in BMSCs, and elucidated the underlying mechanisms involved. MethodsPrimary dental pulp cells were isolated and cultured from human deciduous tooth pulp, SHEDs were isolated and purified by the limiting dilution method and exosomes were isolated from the supernatants of SHEDs by ultracentrifugation. The cell surface markers CD31, CD34, CD45, CD73, CD90 and CD105 were detected by flow cytometry. A Cell-Counting-Kit-8 assay was used to detect cell activity. ALP and Alizarin Red staining were used to identify osteogenic differentiation ability, and exosomes were identified using transmission electron microscopy, NanoFCM and Western blotting. PKH67 fluorescence was used to track the uptake of exosomes by BMSCs. Transcriptome analysis combined with quantitative real-time PCR was used to explore the underlying mechanism involved. ResultsExosomes secreted by SHEDs can be endocytosed by BMSCs, and can partially reverse the inhibitory effects of glucocorticoids on the viability and osteogenic differentiation of BMSCs. Transcriptome sequencing analysis revealed that the differentially expressed mRNAs regulated by SHED-derived exosomes were enriched mainly in signaling pathways such as the apoptosis pathway, the PI3K-Akt signaling pathway, the Hippo signaling pathway and the p53 signaling pathway. qPCR showed that SHED-derived exosomes reversed the dexamethasone-induced upregulation of HGF and ITGB8 expression and the inhibition of EFNA1 expression, but further increased the dexamethasone-induced downregulation of IL7 expression. In conclusion, SHED-derived exosomes partially reversed the inhibitory effects of glucocorticoids on BMSC proliferation and osteogenesis by inhibiting the expression of HGF, ITGB8 and IL7, and upregulating the expression of EFNA1.

Human Cord Blood Endothelial Progenitor Cells and Pregnancy Complications (Preeclampsia, Gestational Diabetes Mellitus, and Fetal Growth Restriction).

Hemangioblasts give rise to endothelial progenitor cells (EPCs), which also express the cell surface markers CD133 and c-kit. They may differentiate into the outgrowth endothelial cells (OECs) that control neovascularization in the developing embryo. According to numerous studies, reduced levels of EPCs in circulation have been linked to human cardiovascular disorders. Furthermore, preeclampsia and senescence have been linked to levels of EPCs produced from cord blood. Uncertainties surround how preeclampsia affects the way EPCs function. It is reasonable to speculate that preeclampsia may have an impact on the function of fetal EPCs during the in utero period; however, the present literature suggests that maternal vasculopathies, including preeclampsia, damage fetal circulation. Additionally, the differentiation potential and general activity of EPCs may serve as an indicator of the health of the fetal vascular system as they promote neovascularization and repair during pregnancy. Thus, the purpose of this review is to compare-through the assessment of their quantity, differentiation potency, angiogenic activity, and senescence-the angiogenic function of fetal EPCs obtained from cord blood for normal and pregnancy problems (preeclampsia, gestational diabetes mellitus, and fetal growth restriction). This will shed light on the relationship between the angiogenic function of fetal EPCs and pregnancy complications, which could have an effect on the management of long-term health issues like metabolic and cardiovascular disorders in offspring with abnormal vasculature development.

The biological characteristics of chicken embryo mesenchymal stem cells isolated from chorioallantoic membrane.

Mesenchymal stem cells (MSCs) derived from fetal membranes (FMs) have the potential to exhibit immunosuppression, improve blood flow, and increase capillary density during transplantation. In the field of medicine, opening up new avenues for disease treatment. Chicken embryo chorioallantoic membrane (CAM), as an important component of avian species FM structure, has become a stable tissue engineering material in vivo angiogenesis, drug delivery, and toxicology studies. Although it has been confirmed that chorionic mesenchymal stem cells (Ch-MSCs) can be isolated from the outer chorionic layer of FM, little is known about the biological characteristics of MSCs derived from chorionic mesodermal matrix of chicken embryos. Therefore, we evaluated the characteristics of MSCs isolated from chorionic tissues of chicken embryos, including cell proliferation ability, stem cell surface antigen, genetic stability, and in vitro differentiation potential. Ch-MSCs exhibited a broad spindle shaped appearance and could stably maintain diploid karyotype proliferation to passage 15 in vitro. Spindle cells were positive for multifunctional markers of MSCs (CD29, CD44, CD73, CD90, CD105, CD166, OCT4, and NANOG), while hematopoietic cell surface marker CD34, panleukocyte marker CD45, and epithelial cell marker CK19 were negative. In addition, chicken Ch-MSC was induced to differentiate into four types of mesodermal cells in vitro, including osteoblasts, chondrocytes, adipocytes, and myoblasts. Therefore, the differentiation potential of chicken Ch-MSC in vitro may have great potential in tissue engineering. In conclusion, chicken Ch-MSCs may be an excellent model cell for stem cell regenerative medicine and chorionic tissue engineering.

Abstract 122: Fusobacterium nucleatum in saliva of oral cancer patients induces tumor stemness defense phenotype in cancer stem cells

Abstract Background: Oral squamous cell carcinoma (OSCC) is a highly aggressive cancer. Cancer Stem Cells (CSCs) having cell surface markers CD44, ALDH, and EpCAM+ have been identified in OSCC. However, the host pathogen interaction between CSCs and oral microbiome has not yet been studied. A number of studies have recently demonstrated the presence of specific oral bacteria populations and their lipopolysaccharides (LPS) in the tumor microenvironment (1). We previously reported pathogenic bacterial internalization in the CSCs (2). Based on the findings, we have developed an in-vitro model to investigate how oral microbiota may integrate into the tumor microenvironment's CSC population and control its activity. Here, we investigated the host/pathogen interaction between CSCs and FN. Methods: Oral bacteria FN was selectively isolated using FEA agar plates from oral saliva of oral cancer patients (n=10) at stage 3 or 4. The isolated bacteria was co cultured with SCC 25 cells at MOI of 1:50. After co culturing the stemness genes such as SOX 2, OCT 4, Nanog,LPS and ABCG2+ cancer stem cell markers were analysed. ABCG2+ CSCs and ABCG2- cells were immunomagnetically sorted and lysed followed by RNA extraction and RT QPCR was performed to observe if FN has selectively internalised in the CSCs. Also ABCG2+ and ABCG 2- cells were used to perform clonogenic assay. Results: Coculture of FN with SCC 25 cell line induced high expression of stemness genes SOX, OCT 4 and Nanog along with CSC markers ABCG2+. Moreover high expression of LPS was observed indicating the presence of FN in the coculture. Importantly live bacteria was found internalized in ABCG2+ cells but not in ABCG2- cells. High clonogenicity was observed in ABCG2+ CSCs compared to ABCG2- cells. Notably, we found that live bacteria and their LPS, mostly Fusobacterium nucleatum isolated (FN) from clinical subjects, were capable of invading CSCs in the in-vitro setting. Post the host-pathogen interaction; it enabled the activation of a niche modulatory tumor stemness defense (TSD) phenotype in the CSCs. These aggressive CSCs with the TSD phenotype have been found to have a critical role in the progression and relapse of oral cancer. Conclusion: Expression of stemness genes infers the niche defence mechanism of the CSCs of the tumour microenvironment due to infection of FN. Importantly presence of live bacteria in ABCG2+ CSCs indicates that FN selectively infects ABCG2+ CSCs but not ABCG2- cells. High clonogenicity of ABCG2+ CSCs confirms the TSD phenotypic attributes of the CSCs.

Astragali Radix-Curcumae Rhizoma inhibits colon cancer progression and enhances 5-FU efficacy by regulating hypoxia-inducible factors and tumor stem cells

The animal and cell models were used in this study to investigate the mechanism of Astragali Radix-Curcumae Rhizoma(HQEZ) in inhibiting colon cancer progression and enhancing the efficacy of 5-fluorouracil(5-FU) by regulating hypoxia-inducible factors and tumor stem cells. The animal model was established by subcutaneous transplantation of colon cancer HCT116 cells in nude mice, and 24 successfully modeled mice were randomized into model, 5-FU, HQEZ, and 5-FU+HQEZ groups. The tumor volume was measured every two days. Western blot was employed to measure the protein levels of epidermal growth factor receptor(EGFR), dihydropyrimidine dehydrogenase(DPYD), and thymidylate synthase(TYMS), the key targets of the hypoxic core region, as well as the hypoxia-inducible factors HIF-1α and HIF-2α and the cancer stem cell surface marker CD133 and SRY-box transcription factor 2(SOX2). The results of animal experiments showed that HQEZ slowed down the tumor growth and significantly increased the tumor inhibition rate of 5-FU. Compared with the model group, HQEZ significantly down-regulated the protein levels of EGFR and DPYD, and 5-FU+HQEZ significantly down-regulated the protein levels of EGFR and TYMS in tumors. Compared with the model group, HQEZ significantly down-regulated the protein levels of HIF-1α, HIF-2α, SOX2, and CD133 in the hypoxic core region. Compared with the 5-FU group, 5-FU+HQEZ lowered the protein levels of HIF-1α, HIF-2α, and SOX2. The cell experiments showed that the protein le-vels of HIF-1α and HIF-2α in HCT116 cells elevated significantly after low oxygen treatment. Compared with 5-FU(1.38 μmol·L~(-1)) alone, HQEZ(40 mg·mL~(-1)) and 5-FU+HQEZ significantly down-regulated the protein levels of HIF-1α, HIF-2α, and TYMS. In conclusion, HQEZ can inhibit the expression of hypoxia-responsive molecules in colon cancer cells and reduce the properties of cancer stem cells, thereby enhancing the therapeutic effect of 5-FU on colon cancer.

Differentiation Potential Analysis of Bone Marrow Mesenchymal Stem Cells from Guizhou Miniature Pigs

In this study, different growth characteristics of hematopoietic stem cells and bone marrow mesenchymal stem cells were used to isolate and culture BMSCs by whole bone marrow adhesion method, and RT-PCR was used to detect cell surface markers CD29, CD31, CD34, CD44, CD71, CD73, CD90. The differentiation of osteocytes into adipocytes and osteoblasts was induced in vitro, and the differentiation ability was observed by staining with oil red O and rubinin. To investigate the isolation and culture methods of BMSCs from Guizhou miniature pigs (Sussa lvanius) in vitro, and to study their biological characteristics and potential for multi-differentiation. The results showed that a small number of adherent cells could be seen in the primary BMSCs cultured in vitro within 48 hours, and the confluent cells reached 90% in 5~6 days. The cell growth state was good, and the shape was spindle and polygonal. RT-PCR was used to detect the positive expression of CD29, CD44, CD71, CD73 and CD90 on the cell surface, and the negative expression of CD31 and CD34. Oil red O staining showed the formation of lipid droplets in the cells, and alizarine red staining showed calcium nodules in the cells, indicating that the cells isolated and cultured in this experiment had the ability of multidirection differentiation. It is consistent with the characteristics of BMSCs. The growth of BMSCs cultured in vitro was stable, and the expanded BMSCs had multidirectional differentiation potential, which was an ideal seed cell for tissue engineering.

Deer antler stem cells immortalization by modulation of hTERT and the small extracellular vesicles characters.

Deer antler stem cells (AnSCs) exhibit properties of both embryonic and mesenchymal stem cells, with superior self-renewal and proliferation, which drive rapid antler growth and regeneration. AnSCs and their derived small extracellular vesicles (sEVs) hold promising potential for applications in regeneration medicine. Due to the restricted proliferative capacity inherent in primary cells, the production capacity of AnSCs and their sEVs are limited. Human telomerase reverse transcriptase (hTERT) is the most important telomerase subunit, hTERT gene insertion has been successfully employed in generating immortalized cell lines. In this study, we successfully established immortalized AnSCs by transducing the hTERT gene using lentivirus. Compared to primary AnSCs, hTERT-AnSCs demonstrated extended passage potential and accelerated proliferation rates while maintaining the mesenchymal stem cell surface markers CD44 and CD90. Additionally, hTERT-AnSCs retained the capacity for osteogenic, adipogenic, and chondrogenic differentiation. sEVs derived from hTERT-AnSCs exhibited a particle size distribution similar to that of AnSCs, both displaying a cup-shaped morphology and expressing CD81, ALIX, and TSG101, while notably lacking GM130 expression. We successfully isolated primary stem cells from deer antler and established the immortalized hTERT-AnSCs. Remarkably, this cell line maintains its stem cell characteristics even after 40 passages. The sEVs derived from these cells exhibit identical morphological and structural features to those of primary AnSCs. This research provides essential technical support for the application of AnSCs and their sEVs in regenerative medicine.

A novel approach for transforming breast cancer stem cells into endothelial cells.

Tumor vascular endothelial cells play a pivotal in the tumor microenvironment, influencing the proliferation, invasion, and metastasis of tumor progression. The present study investigated a novel method for inducing the transformation of breast cancer stem cells into endothelial cells, providing a cellular model investigating anti-angiogenic mechanisms in vitro. The breast cancer cell line MCF-7 was used, and the expression of CD133 was initially detected using flow cytometry. CD133+ breast cancer cells were purified using immunomagnetic bead sorting technology, yielding an MCF-7CD133+ subpopulation. The proliferation ability of these cells was assessed using an MTT assay, while their microsphere formation ability was evaluated using a microsphere formation assay. Post-transformation in an optimized endothelial cell culture medium, expression of endothelial cell markers CD31 and CD105 were detected using flow cytometry. Endothelial cell tube formation assays and DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) assays were employed to analyze the endothelial cell function of the MCF-7CD133+ cells. MDM2/CEN12 gene amplification was detected through fluorescence in situ hybridization (FISH). The MCF-7 breast cancer cell line exhibited 1.7±0.3% trace cells expressing the stem cell surface marker CD133. After anti-CD133 immunomagnetic bead sorting, MCF-7CD133+ and MCF-7CD133- subpopulation cells were obtained, with CD133 expression rates of 85.6±2.8 and 0.18±0.08%, respectively. MTT assay results demonstrated that, after 7 days, the proliferation rate of MCF-7CD133+ cells was significantly higher compared with MCF-7CD133- cells. MCF-7CD133+ subpopulation cells displayed strong stem cell characteristics, growing in suspension in serum-free media and forming tumor cell spheres. In contrast, MCF-7CD133- cells failed to form microspheres. After culturing cells in endothelial cell differentiation and maintenance media, the percentage of MCF-7CD133+ cells before and after endothelial cell culture was 0.3±0.16 and 81.4±8.37% for CD31+ cells and 0.2±0.08 and 83.8±7.24% for CD105+ cells, respectively. Vascular-like structure formation and Ac-LDL phagocytosis with red fluorescence in the tube formation assays confirmed endothelial cell function in the MCF-7CD133+ cells. FISH was used to verify MDM2/CEN12 gene amplification in the induced MCF-7CD133+ cells, indicating tumor cell characteristics. The modified endothelial cell transformation medium effectively induced differentiated tumor stem cells to express vascular endothelial cell markers and exhibit endothelial functions, ideal for in vitro anti-angiogenesis research.

ANGI-01. INDUCIBLE CAS9 IN PATIENT-DERIVED CULTURES OF IDH WILDTYPE GLIOBLASTOMA WITH DISTINCT GROWTH CHARACTERISTICS

Abstract To identify new treatment opportunities for IDH wildtype glioblastoma, it is essential to establish robust methods to evaluate candidate genes in patient-derived cells. Here, we report initiative to establish large-scale candidate gene evaluation by CRISPR-Cas9 using well characterized cells from the Uppsala Human Glioma Cell Culture biobank. Previously, we have characterized fully the HGCC cultures (n = 100) in terms of multi-omics, and by systematic injection as patient-derived orthotopic xenografts. Here, we report a procedure to introduce doxycycline-inducible Cas9 into cultures from our biobank. This allows for temporally controlled target gene ablation in patient-derived cells with known growth characteristics. As proof-of-concept, we describe the derivation of clonal Cas9-inducible cells in the cell lines U3013MG (proneural, bulk forming) and U3180MG (classical, diffuse-growing). The Cas9 induction and editing efficiency was evaluated by ablation of the gene encoding the cell surface marker CD44, for which we transduced the Cas9-inducible clones with CD44 dual sgRNA vector and treated then with doxycycline over time, evaluating CD44 knockout by longitudinal flow cytometry. From a library of 106 clones, we identified the best performing clones, as evaluated by CD44 loss at day 21. Ten out of 64 clones assessed, showed potent Cas9 activity, estimating a drop in CD44 levels by 40-60% after 21 days of doxycycline treatment. The amount of editing by inducible Cas9 was higher with CD44 sgRNA administered by lentivirus than by electroporation. To ensure that the Cas9-inducible patient-derived cell lines have reproducible tumor phenotypes and did not divert from their parental growth pattern, they were transplanted orthotopically into athymic nude mice. We also validated the activity of Cas9ind in vivo by checking the editing of CD44 expression in one of our clones. The established procedure provides a basis for large-scale single-gene and pooled in vivo CRISPR studies of IDH wildtype glioblastomas with distinct invasive characteristics.

The Effect of Probiotics in a Milk Replacer on Leukocyte Differential Counts, Phenotype, and Function in Neonatal Dairy Calves.

Probiotics have been investigated for many health benefits; however, few studies have been performed to determine the effects of oral probiotics on peripheral blood and respiratory immune cells in cattle. Our objectives were to determine changes in health and growth status, differential blood cell counts and function, and blood and lung cell function using flow cytometry and PCR in dairy calves fed a milk replacer with (PRO, N = 10) or without (CON, N = 10) the addition of probiotics to the milk replacer and dry rations from birth to weaning. Performance and clinical scores were not different between the treatment groups. Treatment-by-day interactions for peripheral blood leukocyte populations differed in cell number and percentages. A greater percentage of leukocytes expressed the cell surface markers CD3, CD4, CD8, CD11b, and CD205 on d 21 in CON animals. Lung lavages were performed on five animals from each treatment group on d 52. There were no differences between treatment groups for the expression of cytokines and Toll-Like Receptors as measured using Polymerase Chain Reaction, possibly due to the small sample size. Oral probiotics appear to affect peripheral blood immune cells and function. Their effect on overall calf health remains to be determined.

Biological evaluation of [4-(4-aminophenyl)-1-(4-fluorophenyl)-1H-pyrrol-3-yl](3,4,5-trimethoxyphenyl)methanone as potential antineoplastic agent in 2D and 3D breast cancer models.

Targeting tubulin polymerization and depolymerization represents a promising approach to treat solid tumors. In this study, we investigated the molecular mechanisms underlying the anticancer effects of a structurally novel tubulin inhibitor, [4-(4-aminophenyl)-1-(4-fluorophenyl)-1H-pyrrol-3-yl](3,4,5-trimethoxyphenyl)methanone (ARDAP), in two- and three-dimensional MCF-7 breast cancer models. At sub-cytotoxic concentrations, ARDAP showed a marked decrease in cell proliferation, colony formation, and ATP intracellular content in MCF-7 cells, by acting through a cytostatic mechanism. Additionally, drug exposure caused blockage of the epithelial-to-mesenchymal transition (EMT). In 3D cell culture, ARDAP negatively affected tumor spheroid growth, with inhibition of spheroid formation and reduction of ATP concentration levels. Notably, ARDAP exposure promoted the differentiation of MCF-7 cells by inducing: (i) expression decrease of Oct4 and Sox2 stemness markers, both in 2D and 3D models, and (ii) downregulation of the stem cell surface marker CD133 in 2D cell cultures. Interestingly, treated MCF7 cells displayed a major sensitivity to cytotoxic effects of the conventional chemotherapeutic drug doxorubicin. In addition, although exhibiting growth inhibitory effects against breast cancer cells, ARDAP showed insignificant harm to MCF10A healthy cells. Collectively, our results highlight the potential of ARDAP to emerge as a new chemotherapeutic agent or adjuvant compound in chemotherapeutic treatments.

P071 Exercise as an anti-inflammatory treatment in axial spondyloarthritis (axSpA): a proof-of-concept study

Abstract Background/Aims Axial spondyloarthritis (axSpA) is an inflammatory arthritis affecting 0.5-1.0% of people in the UK. The inflammatory profile with axSpA causes pain and loss of mobility but can also contribute to other manifestations such as cardiovascular disease (CVD). Current management of axSpA relies on biologic medication; however, they are expensive, and effectiveness is influenced by disease duration, baseline inflammation and age. Monocytes, a type of white blood cell, express the cell surface markers CD14 ++CD16- (classical), CD14 ++CD16 + (intermediate), and CD14+CD16 ++ (non-classical). CD16+ monocytes are pro-inflammatory and are elevated in populations with CVD. Importantly, regular exercise reduces the relative proportion of CD16+ cells in other populations, but this is unknown in axSpA. Therefore, the present randomised control trial investigated whether exercise reduced the relative percentage of CD16+ monocytes in response to 12-weeks of home-based walking. Subjective measures of disease activity, physical function, spinal pain, and work productivity are included to supplement the findings. Methods 20 participants (10 control, 10 exercise; 8 vs 7 presented here due to ongoing data collection) were recruited, provided written informed consent, and completed baseline assessments before being assigned into a control or exercise group. The control group carried on as normal. The exercise group completed 5 x 30-minute bouts of ‘somewhat hard’ walking per week which was confirmed via heart rate monitoring and a rating of perceived exertion scale. At baseline (week 0) and follow-up (week 12), participants completed questionnaires to assess BASDAI, BASFI, ASAS-HI, WPAI, and spinal pain. Blood samples were also collected for the analysis of monocyte subsets. Data were analysed using a two-way mixed analysis of variance (ANOVA) with time as the within factor. Statistical significance was accepted as P < 0.05. However, description of trends is used throughout due to this being preliminary data with a small sample size. Results Group-by-time interactions revealed a significant reduction in the proportion of non-classical monocytes in the exercise group and an increase in the control group (P < 0.001). This coincided with a significant interaction for the proportion of less inflammatory classical monocytes, with an increase in the exercise group and decrease in the control group (P < 0.001). Overall, the exercise group also responded more favourably for BASDAI (P = 0.026) and spinal pain (P = 0.088) than the control group. Conclusion Preliminary findings suggest regular walking reduces pro-inflammatory immune cell populations in axSpA which coincides with favourable BASDAI and spinal pain responses compared to a control group. This highlights the importance of regular exercise to improve underlying inflammatory profiles in patients with axSpA. Disclosure M. Roberts: None. M. Hamrouni: None. V. Linsley: None. A. Moorthy: Consultancies; A.M has received speaker fees from Lilly, Novartis, Galapagos and UCB. Grants/research support; A.M has received funding from the National Axial Spondyloarthritis Society for this work. N. Bishop: Grants/research support; N.B has received funding from the National Axial Spondyloarthritis Society for this work.

Effect of ZEB1 Associated with microRNAs on Tumor Stem Cells in Head and Neck Cancer

Cancer biologists have focused on studying cancer stem cells (CSCs) because of their ability to self-renew and recapitulate tumor heterogeneity, which increases their resistance to chemotherapy and is associated with cancer relapse. Here, we used two approaches to isolate CSCs: the first involved the metabolic enzyme aldehyde dehydrogenase ALDH, and the second involved the three cell surface markers CD44, CD117, and CD133. ALDH cells showed a higher zinc finger E-box binding homeobox 1 (ZEB1) microRNA (miRNA) expression than CD44/CD117/133 triple-positive cells, which overexpressed miRNA 200c-3p: a well-known microRNA ZEB1 inhibitor. We found that ZEB1 inhibition was driven by miR-101-3p, miR-139-5p, miR-144-3p, miR-199b-5p, and miR-200c-3p and that the FaDu Cell Line inhibition occurred at the mRNA level, whereas HN13 did not affect mRNA expression but decreased protein levels. Furthermore, we demonstrated the ability of the ZEB1 inhibitor miRNAs to modulate CSC-related genes, such as TrkB, ALDH, NANOG, and HIF1A, using transfection technology. We showed that ALDH was upregulated upon ZEB1-suppressed miRNA transfection (Mann-Whitney ** p101 = 0.009, t-test ** p139 = 0.009, t-test ** p144 = 0.002, and t-test *** p199 = 0.0006). Overall, our study enabled an improved understanding of the role of ZEB1-suppressed miRNAs in CSC biology.

SPRED2: A Novel Regulator of Epithelial-Mesenchymal Transition and Stemness in Hepatocellular Carcinoma Cells.

The downregulation of SPRED2, a negative regulator of the ERK1/2 pathway, was previously detected in human cancers; however, the biological consequence remains unknown. Here, we investigated the effects of SPRED2 loss on hepatocellular carcinoma (HCC) cell function. Human HCC cell lines, expressing various levels of SPRED2 and SPRED2 knockdown, increased ERK1/2 activation. SPRED2-knockout (KO)-HepG2 cells displayed an elongated spindle shape with increased cell migration/invasion and cadherin switching, with features of epithelial-mesenchymal transition (EMT). SPRED2-KO cells demonstrated a higher ability to form spheres and colonies, expressed higher levels of stemness markers and were more resistant to cisplatin. Interestingly, SPRED2-KO cells also expressed higher levels of the stem cell surface markers CD44 and CD90. When CD44+CD90+ and CD44-CD90- populations from WT cells were analyzed, a lower level of SPRED2 and higher levels of stem cell markers were detected in CD44+CD90+ cells. Further, endogenous SPRED2 expression decreased when WT cells were cultured in 3D, but was restored in 2D culture. Finally, the levels of SPRED2 in clinical HCC tissues were significantly lower than those in adjacent non-HCC tissues and were negatively associated with progression-free survival. Thus, the downregulation of SPRED2 in HCC promotes EMT and stemness through the activation of the ERK1/2 pathway, and leads to more malignant phenotypes.

Hydrogel-based miR-192 delivery inhibits the development of hepatocellular carcinoma by suppressing the GSK3β/Wnt/β-catenin pathway.

Hepatocellular carcinoma (HCC) is a primary liver cancer characterized by high invasiveness, metastasis, and poor prognosis, which lacks effective treatments. Although the role of miR-192 in HCC development has been recognized, the underlying molecular mechanism is still poorly understood. This study aimed to explore the impact of mir-192 on HCC and its potential as a therapeutic strategy. Wound healing assay, Transwell assay, CCK-8 assay, and flow cytometry were performed to detect the impact of miR-192 on HCC cell metastasis, invasion, proliferation, and apoptosis, respectively. q-PCR and western blot were applied to measure the relative mRNA and protein expression of the GSK3β/Wnt/β-catenin pathway in miR-192-overexpressing cell lines. Immunofluorescence was carried out to detect the nuclear translocation of β-catenin. starBase website and dual luciferase reporter assay were used to verify the interaction between miR-192 and the target gene WNT10B 3'-untranslated region (3'-UTR) of the Wnt pathway. In addition, we developed algin/polyethyleneimine@miR-192 (AG/PEI@miR-192) nanohydrogel for in vivo delivery of miR-192-agomir. The results revealed that overexpressed miR-192 reduced the expression of HCC cell surface markers CD90, EpCAM, and CD133. Moreover, miR-192 overexpression inhibited HCC cell metastasis, invasion, and proliferation, promoted cell apoptosis, and reduced GSK3β/Wnt/β-catenin pathway expression. Additionally, AG/PEI@miR-192 exhibited good drug release and tumor inhibition. In conclusion, our study suggested that miR-192 inhibits HCC development by suppressing the GSK3β/Wnt/β-catenin pathway and proposed a promising hydrogel-based miR-192 delivery approach to hinder tumor growth.

A comparison study of dental pulp stem cells derived from healthy and orthodontically intruded human permanent teeth for mesenchymal stem cell characterisation.

The objective of this study was to compare the characteristics of Dental Pulp Stem Cells (DPSCs) derived from healthy human permanent teeth with those that were orthodontically-intruded to serve as potential Mesenchymal Stem Cells (MSC). Recruited subjects were treated with orthodontic intrusion on one side of the maxillary first premolar while the opposite side served as the control for a period of six weeks before the dental pulp was extracted. Isolated DPSCs from both the control and intruded samples were analyzed, looking at the morphology, growth kinetics, cell surface marker profile, and multilineage differentiation for MSC characterisation. Our study showed that cells isolated from both groups were able to attach to the cell culture flask, exhibited fibroblast-like morphology under light microscopy, able to differentiate into osteogenic, adipogenic and chondrogenic lineages as well as tested positive for MSCs cell surface markers CD90 and CD105 but negative for haematopoietic cell surface markers CD34 and HLA-DR. Both groups displayed a trend of gradually increasing population doubling time from passage 1 to passage 5. Viable DPSCs from both groups were successfully recovered from their cryopreserved state. In conclusion, DPSCs in the dental pulp of upper premolar not only remained viable after 6 weeks of orthodontic intrusion using fixed appliances but also able to develop into MSCs.

Alzheimer’s Disease risk gene SORL1 affects synaptic function in hiPSC‐derived cortical neurons

AbstractBackgroundThe cellular endo‐lysosomal network (ELN) is implicated in Alzheimer’s Disease pathology and Sortilin‐related receptor 1 (SORL1) is important for normal function of the neuronal ELN. Loss of SORL1 in human induced pluripotent stem cell (hiPSC) derived neurons replicates early AD cytopathology, such as enlarged early endosomes. Work in our laboratory has shown that SORL1 deficiency results in decreased cell‐surface expression of glutamate receptor subunit GLUR1 and neurotrophic receptor TRKB. RNAseq of SORL1 deficient neurons revealed alterations in synaptic‐associated gene ontology transcripts. Together, these data implicate a potential functional consequence for SORL1 deletion. Here we investigate the impact of SORL1 on hiPSC‐derived neuronal synaptic function.MethodNeuronal cultures were differentiated using dual SMAD inhibition of previously generated wild‐type and SORL1 deficient hiPSCs. Following differentiation, neuronal cultures were either purified using cell surface markers CD44, CD184, and CD271 or passaged as mixed cultures for immunocytochemical (ICC) or electrophysiological experiments, respectively. For ICC, neurons were then fixed with paraformaldehyde and stained with experiment‐appropriate antibodies before imaging on a confocal scanning microscope. Mixed cultures were plated onto Axion Biosystems multiple electrode array (MEA) culture plates for electrophysiological analysis. MEA cultures were recorded twice a week for up to ten weeks post‐plating. Recordings and analysis were performed using Axion Biosystems Maestro Pro system.ResultUsing ICC, we find alterations in alterations in synaptic protein and glutamate receptor co‐localization in SORL1 deficient neuronal cultures. When cultured on MEA plates, wild‐type and SORL1 deficient neuronal cultures are active and robust. Neuronal cultures develop glutamate signaling dependent patterns of activity, or network bursting. Neural firing rates are increased in SORL1 deficient neurons. Network bursting properties in SORL1 deficient are altered in comparison to wild‐type cultures, consistent with increased neural excitability.ConclusionLoss of SORL1 alters hiPSC‐derived neuronal activity and synapse‐dependent activity. Further investigation is warranted into the alterations in neuronal physiology and will help us understand the role AD‐related ELN disruption plays in synaptic pathologies.