Genes In Mesenchymal Stem Cells Research Articles

The Phosphonate Derivative of C60 Fullerene Induces Differentiation towards the Myogenic Lineage in Human Adipose-Derived Mesenchymal Stem Cells

Inductors of myogenic stem cell differentiation attract attention, as they can be used to treat myodystrophies and post-traumatic injuries. Functionalization of fullerenes makes it possible to obtain water-soluble derivatives with targeted biochemical activity. This study examined the effects of the phosphonate C60 fullerene derivatives on the expression of myogenic transcription factors and myogenic differentiation of human mesenchymal stem cells (MSCs). Uptake of the phosphonate C60 fullerene derivatives in human MSCs, intracellular ROS visualization, superoxide scavenging potential, and the expression of myogenic, adipogenic, and osteogenic differentiation genes were studied. The prolonged MSC incubation (within 7–14 days) with the C60 pentaphoshonate potassium salt promoted their differentiation towards the myogenic lineage. The transcription factors and gene expressions determining myogenic differentiation (MYOD1, MYOG, MYF5, and MRF4) increased, while the expression of osteogenic differentiation factors (BMP2, BMP4, RUNX2, SPP1, and OCN) and adipogenic differentiation factors (CEBPB, LPL, and AP2 (FABP4)) was reduced or did not change. The stimulation of autophagy may be one of the factors contributing to the increased expression of myogenic differentiation genes in MSCs. Autophagy may be caused by intracellular alkalosis and/or short-term intracellular oxidative stress.

CXCL2 Impairs Functions of Bone Marrow Mesenchymal Stem Cells and Can Serve as a Serum Marker in High-Fat Diet-Fed Rats.

In modern society excessive consumption of a high-fat diet (HFD) is a significant risk factor for many diseases such as diabetes, osteoarthritis and certain cancers. Resolving cellular and molecular mechanisms underlying HFD-associated disorders is of great importance to human health. Mesenchymal stem cells (MSCs) are key players in tissue homeostasis and adversely affected by prolonged HFD feeding. Low-grade systemic inflammation induced by HFD is characterized by increased levels of pro-inflammatory cytokines and alters homeostasis in many organs. However, whether, which and how HFD associated inflammatory cytokines impair MSCs remain unclear. Here we demonstrated that HFD induced serum cytokines disturbances, especially a continuous elevation of serum CXCL2 level in rats. Coincidentally, the differentially expressed genes (DEGs) of bone marrow MSCs (BMSCs) which functions were impaired in HFD rats were enriched in cytokine signaling. Further mechanism analysis revealed that CXCL2 treatment in vitro suppresses the adipogenic potential of BMSCs via Rac1 activation, and promoted BMSC migration and senescence by inducing over-production of ELMO1 and reactive oxygen species (ROS) respectively. Moreover, we found that although glycolipid metabolism indicators can be corrected, the CXCL2 elevation and BMSC dysfunctions cannot be fully rescued by diet correction and anti-inflammatory aspirin treatment, indicating the long-lasting deleterious effects of HFD on serum CXCL2 levels and BMSC functions. Altogether, our findings identify CXCL2 as an important regulator in BMSCs functions and may serve as a serum marker to indicate the BMSC dysfunctions induced by HFD. In addition, our findings underscore the intricate link among high-fat intake, chronic inflammation and BMSC dysfunction which may facilitate development of protective strategies for HFD associated diseases.

Identifying a possible new target for diagnosis and treatment of postmenopausal osteoporosis through bioinformatics and clinical sample analysis.

BackgroundPostmenopausal osteoporosis, a common yet chronic systemic metabolic disease, has become a major public health problem due to life expectancy increasing around the world. The differentiation of mesenchymal stem cells (MSCs) into osteoblasts, and the differentiation of circulating monocyte cells into osteoclasts, play an important role in the balance of bone metabolism. However, when both undergo pathological changes, it can lead to abnormalities, resulting in osteoporosis. This study aims to explore a new biomarker for postmenopausal osteoporosis, thereby providing a new entry point for bioinformatic research into the clinical diagnosis and treatment of the disease.MethodsUsing the Gene Expression Omnibus (GEO) database, microarray analysis was conducted to identify differentially expressed genes in MSCs and monocytes in both postmenopausal osteoporosis patients and a healthy control group. The Database for Annotation, Visualization and Integrated Discovery (DAVID) database was used to analyze the function and enrichment of the selected genes, and a protein-protein interaction (PPI) network was constructed from the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) website and displayed in Cytoscape. To achieve the final results, module analysis of the PPI network was performed by using Molecular Complex Detection (MCODE).ResultsWe identified 45 high-expression and 26 low-expression genes through the study, all of which underwent pathway enrichment analysis. This enrichment was observed in the cell cycle regulation, osteoclast differentiation, tumor necrosis factor (TNF) signaling pathway, and RNA transport. The top 10 hub genes of the PPI network were SF3B1, SRSF5, FUBP1, SRSF3, TIA1, KHSRP, LUC7L3, PNN, SRC, and ATRX. Comparing the MSCs and monocytes between the postmenopausal osteoporosis patients and the healthy control group, we noted that the expression of the above genes differed greatly.ConclusionsThrough bioinformatic analysis and clinical specimen validation, our study provides a new way for exploring the pathogenesis of postmenopausal osteoporosis. Most importantly, it suggests that the hub genes, SF3B1, SRSF5, FUBP1, KHSRP, and SRC, may become new diagnostic markers and therapeutic targets for diagnosing and treating postmenopausal osteoporosis in the future.

Randomized, Placebo-Controlled Analysis of the Knee Synovial Environment Following Platelet-Rich Plasma Treatment for Knee Osteoarthritis.

Platelet-rich-plasma (PRP) is used to treat knee osteoarthritis; however, mechanistic evidence of PRP effectiveness for pain relief is limited. To assess molecular biomarkers and mesenchymal stem cells (MSCs) in synovial fluid during PRP treatment of the osteoarthritic knee joint. Single blinded, randomized, placebo controlled pilot study. Veterans Affairs Medical Center. Seventeen participants with mild to moderate knee osteoarthritis were randomized in a 2:1 placebo-controlled ratio, receiving PRP or saline (placebo) intra-articular injection into the knee joint. Knee synovial fluid was analyzed before the respective injections and again 10 days following injection. Participants were followed up to 12 months completing visual analog scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaires at intervals over that period. The effects of PRP on synovial protein and MSC gene expression levels were measured by multiplex enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Novel biomarkers including levels of interleukin (IL)-5, IL-6, IL-10, and tumor necrosis factor-α were measured in synovial fluid 10 days after PRP treatment. Altered gene expression profiles in MSCs from patients treated with PRP were observed for matrix metalloproteinases and inflammatory markers (IL-6, IL-8, CCL2, TNF-α). A2M protease was significantly increased following PRP treatment (P = .005). WOMAC scores declined for up to 3 months from baseline levels and remained low at 6 and 12 months in the PRP group. In contrast, WOMAC scores for patients receiving the saline injection were relatively unchanged for up to 12 months. We report significant changes for the biomarker A2M (P = .005) as well as differences in expression of cellular markers and postulate that PRP modulates the local knee synovial environment by altering the inflammatory milieu, matrix degradation, and angiogenic growth factors. The PRP treatment group had less pain and stiffness and improved function scores.

General gene expression patterns and stemness of the gingiva and dental pulp

Background/purposeDue to the unique properties of healing processes and cellular differentiation, the gingiva and dental pulp have attracted attention as a potential source of mesenchymal stem cells (MSCs). The purpose of this study was to obtain molecular-level information on these tissues in terms of their function and differentiation processes and investigate stemness. Materials and methodsHealthy gingival tissues were collected from patients (n = 9; aged 7–12 years) who underwent simple surgical procedures, and normal dental pulp tissues were obtained from patients (n = 25; aged 11–25 years) undergoing tooth extraction for orthodontic reasons. Complementary DNA microarray, qRT-qPCR, and immunohistochemical staining were performed to assess general and MSC gene expression patterns. ResultsIn the gingival tissue, genes related to keratinization, the formation of epithelial cells and ectoderm, and immune and/or inflammatory responses were highly expressed. Meanwhile, in the dental pulp tissue, genes related to ion transport, neuronal development and axon guidance, bone and enamel mineralization, extracellular matrix organization, and angiogenesis were highly expressed. When focusing on the expression of MSC genes, induced pluripotent stem (iPS) cell genes, such as Sox2, c-Myc, and KLF4, were expressed at higher levels in the gingival tissue, whereas dental stem cell genes, such as NT5E and VCAM1, were expressed in dental pulp tissue. ConclusionWe found different general and MSC gene expression patterns between the gingival and dental pulp tissue. These results have implications for future regenerative medicine, considering the application of gingival tissue as a potential source of iPS cells.

3D cell sheet structure augments mesenchymal stem cell cytokine production

Mesenchymal stem cells (MSCs) secrete paracrine factors that play crucial roles during tissue regeneration. An increasing body of evidence suggests that this paracrine function is enhanced by MSC cultivation in three-dimensional (3D) tissue-like microenvironments. Toward this end, this study explored scaffold-free cell sheet technology as a new 3D platform. MSCs cultivated on temperature-responsive culture dishes to a confluent 2D monolayer were harvested by temperature reduction from 37 to 20 °C that induces a surface wettability transition from hydrophobic to hydrophilic. Release of culture-adherent tension induced spontaneous cell sheet contraction, reducing the diameter 2.4-fold, and increasing the thickness 8.0-fold to render a 3D tissue-like construct with a 36% increase in tissue volume. This 2D-to-3D transition reorganized MSC actin cytoskeleton from aligned to multidirectional, corresponding to a cell morphological change from elongated in 2D monolayers to rounded in 3D cell sheets. 3D culture increased MSC gene expression of cell interaction proteins, β-catenin, integrin β1, and connexin 43, and of pro-tissue regenerative cytokines, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10), and increased VEGF secretion per MSC 2.1-fold relative to 2D cultures. Together, these findings demonstrate that MSC therapeutic potency can be enhanced by 3D cell sheet tissue structure.

Natural cellulose-based scaffold for improvement of stem cell osteogenic differentiation

The challenge of tissue engineering remains for introducing the best substrate to support stem cell growth and proliferation, and also repair of damaged tissues. Scaffolds made of plant tissues can be considered as suitable substitutes for extracellular matrix. In this study, the outer skin of the onion was decellularized and after characterization, its supportive role on osteogenic differentiation was investigated by the culture of human mesenchymal stem cells (MSCs). Structural characterizations by Atomic Force and Scanning electron microscopes, Infrared spectroscopy and Brunauer-Emmett-Teller, Tensile, and Contact angle tests demonstrated that the decellularized scaffold has a proper 3D structure with interconnected pores, and moderate surface roughness. The Osteogenic differentiation evaluation revealed that alkaline phosphatase activity and calcium deposition in MSCs differentiated on the decellularized onion scaffold was significantly higher than those cells differentiated on tissue culture polystyrene as a 2D control. In addition, the expression level of common bone-related genes in MSCs differentiated on the decellularized onion scaffold was also increased significantly higher than those cells cultured on the 2D control group. These data demonstrated that 3D structure of the decellularized onion scaffold with supporting stem cell proliferation and differentiation has promising potential for use in tissue engineering as a new cost-effective and green scaffold technology.

Nucleus Pulposus Cell Conditioned Medium Promotes Mesenchymal Stem Cell Differentiation into Nucleus Pulposus-Like Cells under Hypoxic Conditions.

Low back pain (LBP) is a major physical and socioeconomic challenge worldwide. Nucleus pulposus (NP) is directly associated with LBP due to intervertebral disc (IVD) degeneration. IVD degeneration is mainly caused by structural and matrix-related changes within the IVD occurring during aging and degeneration. Mesenchymal stem cells (MSCs) can differentiate into multiple mesenchymal lineages under specific stimulatory conditions. This study is aimed at evaluating the effectiveness of the nucleus pulposus cell (NPC) conditioned medium for promoting the expression of MSCs and at confirming the expression of healthy NP phenotypic markers recently recommended by the Spine Research Interest Group. Expression was investigated using quantitative polymerase chain reaction (qPCR) and western blotting under normoxic and hypoxic conditions. qPCR and western blotting demonstrated significant upregulation of NP marker expression in MSCs cultured under hypoxic conditions and treated with the 50% or 100% NPC conditioned medium, compared with those cultured under normoxic conditions. Upregulation was highest in the presence of the 100% NPC conditioned medium compared with the control group (aggrecan, p < 0.01; brachyury, p < 0.05; collagen II, p < 0.001; KRT8, p < 0.01; KRT19, p < 0.001; and Shh, p < 0.01). The expression levels of genes in MSCs treated with the 50% NPC conditioned medium also showed upregulation compared with the control group (collagen II, p < 0.05; KRT8, p < 0.05; and KRT19, p < 0.01). These findings suggested that the NPC conditioned medium stimulated MSC differentiation into an NP-like phenotype with distinct characteristics. The results could inform strategies for IVD regeneration.

Mesenchymal Stem Cells as a Bio Organ for Treatment of Female Infertility.

Female infertility is a global medical condition that can be caused by various disorders of the reproductive system, including premature ovarian failure (POF), polycystic ovary syndrome (PCOS), endometriosis, Asherman syndrome, and preeclampsia. It affects the quality of life of both patients and couples. Mesenchymal stem cells (MSCs) have received increasing attention as a potential cell-based therapy, with several advantages over other cell sources, including greater abundance, fewer ethical considerations, and high capacity for self-renewal and differentiation. Clinical researchers have examined the therapeutic use of MSCs in female infertility. In this review, we discuss recent studies on the use of MSCs in various reproductive disorders that lead to infertility. We also describe the role of microRNAs (miRNAs) and exosomal miRNAs in controlling MSC gene expression and driving MSC therapeutic outcomes. The clinical application of MSCs holds great promise for the treatment of infertility or ovarian insufficiency, and to improve reproductive health for a significant number of women worldwide.

The effect of microglial ablation and mesenchymal stem cell transplantation on a cuprizone-induced demyelination model.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system with symptoms such as neuroinflammation, astrocytosis, microgliosis, and axonal degeneration. Mesenchymal stem cells (MSCs) with their immunomodulation, differentiation, and neuroprotection abilities can influence the remyelination process. The goal of this study is to investigate the impact of microglial ablation and MSCs transplantation on remyelination processes in the corpus callosum (CC) of the cuprizone demyelination model. For the induction of a chronic demyelination model, C57BL6 mice were fed with chow containing 0.2% cuprizone (wt/wt) for 12 weeks. For the depletion of microglia, PLX3397 was used as a colony-stimulating factor 1 receptor inhibitor for 21 days. MSCs were injected to the right lateral ventricle and after 2 weeks, the mice were killed. We assessed glial cells using specific markers such as APC, Iba-1, and GFAP using the immunohistochemistry method. Remyelination was evaluated by Luxol fast blue (LFB) staining and transmission electron microscope (TEM). The specific genes of microglia and MSCs were evaluated by a quantitative real-time polymerase chain reaction. According to the results of the study, 21 days of PLX3397 treatment significantly reduced microglial cells, and MSCs transplantation decreased the number of astrocytes, whereas the oligodendrocytes population increased significantly in PLX + MSC group in comparison with the cuprizone mice. Furthermore, PLX and MSC treatment elevated levels of remyelination compared with the cuprizone group, as confirmed by LFB staining and TEM analysis. The molecular results showed that MSC transplantation significantly decreased the number of microglia through the CX3CL1/CX3CR1 axis. These results revealed that PLX3397 treatment and MSCs injection reduced microgliosis and astrocytosis. It also increased the oligodendrocytes population by enhancing remyelination in the CC of the cuprizone model of MS.

HLA-G Expression in Human Mesenchymal Stem Cells (MSCs) Is Related to Unique Methylation Pattern in the Proximal Promoter as well as Gene Body DNA.

Multipotent human mesenchymal stem cells (MSCs) harbor clinically relevant immunomodulation, and HLA-G, a non-classical MHC class I molecule with highly restricted tissue expression, is one important molecule involved in these processes. Understanding of the natural regulatory mechanisms involved in expression of this elusive molecule has been difficult, with near exclusive reliance on cancer cell lines. We therefore studied the transcriptional control of HLA-G in primary isolated human bone marrow- (BM), human embryonic stem cell-derived (hE-), as well as placenta-derived MSCs (P-MSCs), and found that all 3 types of MSCs express 3 of the 7 HLA-G isoforms at the gene level; however, fibroblasts did not express HLA-G. Protein validation using BM- and P-MSCs demonstrated expression of 2 isoforms including a larger HLA-G-like protein. Interferon-γ (IFN-γ) stimulation upregulated both gene and protein expression in MSCs but not the constitutively expressing JEG-3 cell line. Most interestingly in human MSCs and placental tissue, hypomethylation of CpG islands not only occurs on the HLA-G proximal promoter but also on the gene body as well, a pattern not seen in either of the 2 commonly used choriocarcinoma cell lines which may contribute to the unique HLA-G expression patterns and IFN-γ-responsiveness in MSCs. Our study implicates the importance of using normal cells and tissues for physiologic understanding of tissue-specific transcriptional regulation, and highlight the utility of human MSCs in unraveling the transcriptional regulation of HLA-G for better therapeutic application.

Nanovibrational Stimulation of Mesenchymal Stem Cells Induces Therapeutic Reactive Oxygen Species and Inflammation for Three-Dimensional Bone Tissue Engineering.

There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts, combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilize these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in two- and three-dimensional, but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz and assess MSC metabolite, gene, and protein changes. These approaches reveal that dose-dependent changes occur in MSCs’ responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level inflammation as well as to ROS- and inflammation-balancing pathways. These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells’ mechanical memory, driven by the stronger N90 stimulation. These mechanistic insights and cell-scaffold design are underpinned by a process that is free of inductive chemicals.

Aging-Related Reduced Expression of CXCR4 on Bone Marrow Mesenchymal Stromal Cells Contributes to Hematopoietic Stem and Progenitor Cell Defects.

Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging, however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS), DNA damage, senescence, and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC, as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically, CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress, as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC), improved their niche supporting activity, and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production, driving an HSC aging phenotype. Thus, modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function.

Poly-phosphate increases SMC differentiation of mesenchymal stem cells on PLGA-polyurethane nanofibrous scaffold.

The use of bioactive scaffolds in tissue engineering has a significant effect on the damaged tissue healing by an increase in speed and quality of the process. Herein, electrospinning was applied to fabricate composite nanofibrous scaffolds by Poly lactic-co-glycolic acid (PLGA) and Polyurethane (PU) with and without poly-phosphate (poly-P). Scaffolds were characterized morphologically by scanning electron microscope (SEM), and their biocompatibility was also investigated by SEM, protein adsorption, cell attachment and survival assays. The applicability of the scaffolds for bladder tissue engineering was also evaluated by culturing mesenchymal stem cells (MSCs) on the scaffolds and their differentiation into smooth muscle cell (SMC) was studied at the gene and protein levels. The results demonstrated that scaffold biocompatibility was increased significantly by loading poly-P. SMC related gene and protein expression level in MSCs cultured on poly-P-loaded scaffold was also increased significantly compared to those cells cultured on empty scaffold. It can be concluded that poly-P hasn’t also increased scaffold biocompatibility, but also SMC differentiation potential of MSCs was also increased while cultured on the poly-P containing scaffold compared to the empty scaffold. Taken together, our study showed that PLGA–PU–poly-P alone and in combination with MSCs has a promising potential for support urinary bladder smooth muscle tissue engineering.

Gene expression profiling of progenitor cells isolated from rat rotator cuff musculotendinous junction

BackgroundRotator cuff tendon tears are typically degenerative and usually affect the region of tendon insertion on bone. The remnant torn tendon is degenerative and may not be an ideal source for progenitor cells for cell-based therapies. Therefore, the aim of this study was to determine if musculotendinous junction (MTJ), which is adjacent to tendon would be a viable alternate source of progenitor stem cells. We also sought to study the gene expression profile MTJ progenitors and compare it with progenitors isolated from RC tendon, RC muscle and other existing tissue sources (bone marrow, adipose tissue, and Achilles tendon).MethodsRotator cuff tendon (RCT), muscle (RCM), and RCMTJ as well as Achilles tendon (AT) tissues were harvested from healthy male Lewis rats and progenitor cultures were established from these tissues and also from bone marrow and adipose tissue. Quantitative RT-PCR was performed on RNA extracts from intact tissues and progenitor cells using a custom array for the mesenchymal stem cell (MSC) differentiation marker genes. The gene expression profile of MSC differentiation markers within four tissues types, six progenitor cells, and between tissue and their corresponding progenitors were compared.ResultsProgenitors cells can be isolated from rat rotator cuff musculotendinous tissue and their pattern of MSC gene expression was similar to the rotator cuff tendon progenitors for majority of the genes tested. However, there were significant differences between the MSC gene expression patterns of RCMTJ and RCM progenitors. Furthermore, there were differences in gene expression between the RCMTJ tissue and its progenitor cells with respect to MSC differentiation markers. The gene expression pattern of RCMTJ tissue was similar to RCM tissue with respect to markers of chondrogenesis, myogenesis, tenogenesis, and MSC specific markers.ConclusionWe demonstrate that the musculotendinous junction contains distinct set of progenitor cells and their MSC gene expression pattern is similar to rotator cuff tendon progenitors. RCMTJ progenitors will be an attractive option for cell-based regenerative treatment of chronic rotator cuff tears.

Combining Innovative Bioink and Low Cell Density for the Production of 3D-Bioprinted Cartilage Substitutes: A Pilot Study.

3D bioprinting offers interesting opportunities for 3D tissue printing by providing living cells with appropriate scaffolds with a dedicated structure. Biological advances in bioinks are currently promising for cell encapsulation, particularly that of mesenchymal stem cells (MSCs). We present herein the development of cartilage implants by 3D bioprinting that deliver MSCs encapsulated in an original bioink at low concentration. 3D-bioprinted constructs (10 × 10 × 4 mm) were printed using alginate/gelatin/fibrinogen bioink mixed with human bone marrow MSCs. The influence of the bioprinting process and chondrogenic differentiation on MSC metabolism, gene profiles, and extracellular matrix (ECM) production at two different MSC concentrations (1 million or 2 million cells/mL) was assessed on day 28 (D28) by using MTT tests, real-time RT-PCR, and histology and immunohistochemistry, respectively. Then, the effect of the environment (growth factors such as TGF-β1/3 and/or BMP2 and oxygen tension) on chondrogenicity was evaluated at a 1 M cell/mL concentration on D28 and D56 by measuring mitochondrial activity, chondrogenic gene expression, and the quality of cartilaginous matrix synthesis. We confirmed the safety of bioextrusion and gelation at concentrations of 1 million and 2 million MSC/mL in terms of cellular metabolism. The chondrogenic effect of TGF-β1 was verified within the substitute on D28 by measuring chondrogenic gene expression and ECM synthesis (glycosaminoglycans and type II collagen) on D28. The 1 M concentration represented the best compromise. We then evaluated the influence of various environmental factors on the substitutes on D28 (differentiation) and D56 (synthesis). Chondrogenic gene expression was maximal on D28 under the influence of TGF-β1 or TGF-β3 either alone or in combination with BMP-2. Hypoxia suppressed the expression of hypertrophic and osteogenic genes. ECM synthesis was maximal on D56 for both glycosaminoglycans and type II collagen, particularly in the presence of a combination of TGF-β1 and BMP-2. Continuous hypoxia did not influence matrix synthesis but significantly reduced the appearance of microcalcifications within the extracellular matrix. The described strategy is very promising for 3D bioprinting by the bioextrusion of an original bioink containing a low concentration of MSCs followed by the culture of the substitutes in hypoxic conditions under the combined influence of TGF-β1 and BMP-2.

Integrin αvβ3-Mediated Effects of Thyroid Hormones on Mesenchymal Stem Cells in Tumor Angiogenesis.

Background: Several clinical and experimental studies have implicated thyroid hormones in cancer progression. Cancer-relevant effects, including stimulation of tumor growth and new blood vessel formation by angiogenesis, are thought to be mediated by a nonclassical signaling pathway initiated through integrin αvβ3 expressed on cancer cells and proliferating endothelium. In an earlier study, we established mesenchymal stem cells (MSCs), important contributors to the fibrovascular network of tumors, as new thyroid hormone-dependent targets. Here, we evaluated the effects of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) versus Tetrac, an integrin-specific inhibitor of thyroid hormone action, on MSCs in tumor angiogenesis. Methods: Modulation of the expression and secretion of angiogenesis-relevant factors by thyroid hormones in primary human MSCs and their effect on endothelial cell tube formation were tested in vitro. We further engineered MSCs to express the sodium iodide symporter (NIS) reporter gene under control of a hypoxia-responsive promoter and the vascular endothelial growth factor (VEGF) promoter to test effects on these pathways in vitro and, for VEGF, in vivo in an orthotopic hepatocellular carcinoma (HCC) xenograft mouse model by positron emission tomography imaging. Results: T3 and T4 increased the expression of pro-angiogenic genes in MSCs and NIS-mediated radioiodide uptake in both NIS reporter MSC lines in the presence of HCC cell-conditioned medium. Supernatant from thyroid hormone-treated MSCs significantly enhanced endothelial cell tube formation. Tetrac and/or inhibitors of signaling pathways downstream of the integrin reversed all these effects. Tumoral radioiodide uptake in vivo demonstrated successful recruitment of MSCs to tumors and VEGF promoter-driven NIS expression. Hyperthyroid mice showed an increased radioiodide uptake compared with euthyroid mice, while tracer uptake was markedly reduced in hypothyroid and Tetrac-treated mice. Conclusions: Our data suggest that thyroid hormones influence angiogenic signaling in MSCs via integrin αvβ3 and further substantiate the anti-angiogenic activity of Tetrac in the tumor microenvironment.

268O - Genetic variations in hematopoietic and mesenchymal stem cells in de novo myelodysplastic syndromes

BackgroundMyelodysplastic syndromes (MDS) are a bone marrow (BM) stem cell disorders. Studying both hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) in MDS marrow is equally important for the proper understanding of the disease pathogenesis. Comparative genetic variation (GV) profiling of HSCs and MSCs in MDS were conducted in the same patient group and between patients of de novo MDS patients of a South Asian cohort. MethodsDNA was isolated from BM derived HSCs and MSCs from 20 newly diagnosed untreated MDS patients. Next Generation Sequencing was performed using a panel of 54 genes that are commonly mutated in myeloid malignancies. The structural and functional effects of GVs were determined by bioinformatics analysis. A phenotype-genotype correlation was made based on the detected GVs. ResultsThe number of mutated genes in HSCs and MSCs were 42 and 38 respectively. The genetic variants identified in HSCs were distinct from that of MSCs. The most frequently mutated genes in both cell types were TET2, KDM6A, BCOR, EZH2 and ASXL. DNA methylation and chromatin remodeling were affected mostly in both cell types. The genetic variations in genes in the cohesion complex and epigenetic mechanisms were shown to co-exist in both cell types. Our cohort carried previously reported variations in myeloid malignancies in HSCs and MSCs. Novel recurrent variants were: KDM6A(c.619 + 6T>A), KMT2A(D2488V), ASXL1(C1182W & V1539G), STAG2(T1124P), BCORL1(V1268G) and IDH1(E47G) in HSCs and DNMT3A(H588P), KIT(H485P), KDM6A(T181P & T833P), ASXL1 (S767P), ETV6 (E327G), BCOR(H1367P) and CBL(E448V) in MSCs. The insertion (E598DYVDFREYE) of the FLT3 gene and the insertion (L287LCX) in the NPM1 gene were co-occurred in 3 patients. Genetic variations in BCORL1 were significantly associated with refractory anaemia. ConclusionsAltered genome of MSCs supports the hypothesis of a possible role of MSCs in MDS pathogenesis. Detection of distinct variants in HSC and MSC compartments would indicate the genetic variations occur following differentiation into the two cell lineages. The novel recurrent variants identified in the study stand as potential biological markers in diagnosis, prognosis and therapeutic intervention of MDS and needs further study. Legal entity responsible for the studyNA. FundingUniversity Grants Commission of Sri Lanka and National Science Foundation of Sri Lanka. DisclosureAll authors have declared no conflicts of interest.

Abstract 1161: Utility of evidence based mesenchymal gene signatures gene expression panel in predicting outcome in stage matched colorectal cancer patients

Abstract Colorectal cancer (CRC) is third deadliest cancer in United States, only behind lung and prostate cancer. Although several therapies are available to treat colorectal cancer but there is need for new prognostic biomarkers to tailor appropriate personalized therapy. Mesenchymal Stem cells (MSCs) are multipotent stem cells which are attracted toward inflammatory sites. In colon cancer, they have been shown to assist in tumor progression. To assess the MSC gene signatures in colon cancer patients, we picked 5 genes from nCounter plexset Mesenchymal Stem cell panel. These genes named: SMAD4, PTK2, GTF3A, CASP3 and HDAC1 were quantified on Nanostring platform. The aim of the study is to quantify these Mesenchymal gene signatures in colorectal cancer patients. Under the IRB approved protocol, a total of 750 colon cancer patients at Medical College of Georgia with 5 years follow-up were initially selected. The patients were stratified on the basis of overall survival in two groups, with higher (&amp;gt; 5 years) and lower survival (&amp;lt;1 year) along with AJCC staging (I to IV), grade, gender and age. A total of 88 patients fitted in our inclusion criteria on the basis of survival duration after diagnosis. The FFPE blocks were acquired from Medical College of Georgia, Augusta. Total RNA was isolated and quantified through Nanodrop method. The statistical analysis of data was performed using student t-test and Pearson correlation. We found PTK2 levels to be high in stage I patients (p = 0.02*). Also, patients with higher survival in stage I had high expression of PTK2 (p=0.04*). Additionally, PTK2 was expressed in higher amounts in lower surviving patient group (0.00*). GTF3 levels were higher in (p = 0.01) stage I. CASP3 showed higher expression in stage III and IV combined. HDAC1 expression was higher in Grade II patients compared to Grade I. HDAC1 expression was also higher in African American population. Citation Format: Benjamin Johnson, Pankaj Ahluwalia, Chetan Pundkar, Saleh Heneidi, Kimya Jones, Ashis Mondal, Ravindra Kolhe. Utility of evidence based mesenchymal gene signatures gene expression panel in predicting outcome in stage matched colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1161.

PF536 THE EFFECT OF 5‐AZACITIDINE TREATMENT ON THE OF BONE MARROW MESENCHYMAL STEM TRANSCRIPTOME IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES

Background:Myelodysplastic syndromes (MDS) are clonal disorders of the hematopoietic stem cells (HSC), characterized by inefficient bone marrow (BM) haemopoiesis and increased risk for leukaemic evolution. However, MDS pathogenesis is not restricted to the HSC compartment. Emerging evidence suggests a disease permitting or even a disease promoting role for the BM microenviroment (Cogle et al,2015).Mesenchymal stem cells (MSCs) are a key component of the BM niche. MDS derived MSCs (MDS‐MSCs) have been shown to hold intrinsic functional defects and an in ability to sustain normal haemopoesis (Bulycheva et al, 2015). Moreover, recent data suggest that MDS‐MSCs harbor genetic abnormalities (Blau et al,2011) and also an altered DNA methylation status (Poloni et al,2014).5‐Azacitidine (AZA), a hypomethylating agent, is widely used for MDS treatment. Our (Liapis et al,ASH 2017) and other groups have previously shown that AZA is able to partially reverse the proliferative and clonogenic deficiencies of MDS‐MSCs, whereas it is unable to affect their intrinsic genomic instability. However, knowledge of its effect on the MDS‐MSC transcriptome is still lacking.Aims:We aim to provide the first comparative analysis of the MDS‐MSC transcriptome before and after long term AZA treatment.Methods:BM samples were collected from 12 patients with high risk MDS after informed consent. Repeat harvest, after 6 cycles of AZA therapy was possible in 7 of these patients. BM samples were also collected from 7 healthy subjects undergoing orthopedic surgery. MSCs were then isolated, ex vivo expanded and re‐seeded for a total of 5 passages (P). At P2, expanded MSCs, fulfilled the minimal criteria for MSC definition (Dominici et al, 2006) and consisted of a homogenous population. Total RNA was harvested from P2 MSCs and subjected to RNA sequencing assays. We used the QuantSeq 3’ mRNA‐Seq Library Prep Kit FWD for Illumina. It utilizes the 3′prime method of sequencing only the 3′region of the RNAs and also provides quantative measurements.Results:Thus far, data analysis has been completed in 5 paired MDS‐MSC samples, before and after AZA therapy. Proliferation and clonogenicity parameters showed a marked improvement upon AZA treatment.410 genes were shown to have an over 2∗log2 change in their expression. Of these genes, 66 were found to be differently regulated with statistical significance (p &lt; 0.05), in spite of the low sample count.Surprisingly, despite AZA's function as a transcriptional promoter, long termtreatment conferred MSC gene downregulation rather than upregulation: 228 genes exhibited transcriptional suppression. Significantly downregulated genes included members of proliferative signaling cascades, among which,effectors of PI3k‐Akt and Wnt signaling. Other affected cellular processes were those of apoptosis, extracellular matrix synthesis and stromal adherence. Notable mentions of downregulated genes are those of CCND2, encoding for cyclin D2 (‐3.317∗log2, q = 0.013), the pro‐apoptotic protein BAX (‐3.05∗log2, q = 0.013) and FRZ2 (‐2.27∗log2, q = 0.013) a mediator for Wnt signaling.182 genes were shown to be over 2∗log2 fold upregulated. Statistically significant difference was found in just 3 of them and thus, no safe conclusion can be drawn. However, analysis of the remaining MDS‐MSC and normal MSC RNA samples is still in process.Summary/Conclusion:Transcriptome analysis reveals that long term AZA treatment is able to restore MSC stemness. The inhibition of both the mitotic and apoptotic processes contributes to stem cell quiescence and longevity.