Mesenchymal Stem Cell Line Research Articles

The effect and mechanism of microRNA-83a targeting on differentiation of bone marrow mesenchymal stem cells in vitro

Objective To study the anti-proliferation and inducing apoptosis effects of microRNA (miRNA, miR)-83a on bone marrow mesenchymal stem cells. Methods The experiment was divided into 3 groups: group A (PEF miR-83a and PSV2neo co-transfection group), group B (non-transfection group), and group C (PEF and PSV2neo co-transfection group). Group A served as the experimental group, and groups B and C group as the control groups. Transfection according to instructions, effects of application of expression detected miR-83a transcription of bone marrow mesenchymal stem cells lines in vitro antiproliferative effect of and biological function; by detecting the expression level of miR-83a immunohistochemical method and real-time quantitative polymerase chain reaction (Real-time PCR) technology in cells, Real-time PCR was measured after transfection, bone marrow mesenchymal stem cells lines Ras, Raf, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK): mRNA expression changes, and through the methyl thiazol tetrazolium (MTT) method, the scratch test, hematoxylin and eosin (HE) staining, microscope observation and comparison of miR-83a transfection on the migration ability of cell proliferation, apoptosis and morphological changes. To analyse by using SPSS 14.0 statistical software. Results Real-time PCR and immunohistochemistry confirmed in miR-83a transfected cells with stable expression; Assingland staining results: after seventh days culture, the cells of the induction differentiation group were full of brown specific staining granules, and no specific staining granules were found in the undifferentiated differentiation group. Real-time PCR results: The expression of Ras, Raf and Akt mRNA in group A were 0.97±0.27, 0.54±0.18 and 0.99±0.31, in group B were 1.68±0.30, 1.58±0.25 and 2.06±0.42, in group C were 1.68±0.30, 1.58±0.25 and 1.89±0.31. Compared with group B and C, the levels of Ras, Raf and Akt mRNA in group A were significantly lower (t=1.531 and 1.838, P=0.016 and 0.011). MTT assay showed that the apoptotic rates of the cells were (23.49±4.64)%, (45.73±8.72)% and (59.25±13.16)% after 24, 48 and 72 h, respectively. There was significant difference between the two groups (χ2= 5.203 and 2.146, P =0.013 and 0.045). The scratch test showed that miR-83a transfected cell migration ability is lower than the normal cells. The effect of miR-83a on lung cancer cell 24 h, morphological observation of lung cancer cell apoptosis or necrosis. Conclusion Transfection of miR-83a can inhibit proliferation and promote apoptosis effect on pituitary adenoma cells in vitro cells, which is likely to play a role through inhibition of epidermal growth factor receptor (EGFR) and EGFRvⅢ signal transduction pathway in the RAS-RAF-mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K)-Akt of two important signaling pathways activated. Key words: Bone marrow mesenchymal stem cells; MicroRNA-83a; Proliferation; Apoptosis; Biological function

Abstract 792: Development of high-throughput cell-based co-culture angiogenesis assay system using hTERT immortalized cells

Abstract Angiogenesis is a multi-step physiological process which is involved in a large number of normal and disease state processes; In vitro angiogenesis models provide very useful tools to study these processes, one of which is the analysis of tubule formation. Tubules formed in co-culture assays were significantly more heterogeneous and more closely resembled capillaries than Matrigel® tubules. Current co-culture models using primary cells have donor variability, and inconsistent results due to lot to lot variation. In this study, we established an in vitro co-culture model system consisting of an assay ready mixture of an aortic endothelial cell line TeloHAEC-GFP (hTERT immortalized human aortic endothelial cell line) and a hTERT immortalized adipose-derived mesenchymal stem cell line (hTERT-MSCs) in a specially formulated medium containing VEGF supplement (Angio-Ready™ Angiogenesis Assay System). Both cell lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and have been well-characterized showing that the cells retain the most important characteristic of their parental counterparts. The new co-culture system forms functional tubular structures in less than 7 days, and in addition, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by elevated positive αSMA staining (a marker of smooth muscle cells), indicating that the system has physiological relevance. Notably, our results showed the co-culture system has minimal lot-to-lot variation indicated by the treatment of three lots with the anti-cancer drug, Ramucirumab (Cyramza®), which also targets the VEGF pathway. Next, we tested the new system with compounds that impact angiogenesis, results demonstrated that the angiogenesis system responds positively to elevated doses of VEGF and negatively to increasing concentrations of suramin; more importantly, the tubular formation efficiency is reduced or blocked by well-known anti-cancer drugs such as Sunitinib (SUTENT®) and Bevacizumab (Avastin®), both of which target the VEGF pathway. Finally, we used the Angio-Ready™ system validated 4 HIF-1(hypoxia inducible factors-1) inhibitors which have anti-angiogenic properties identified by high-throughput screening methods; data showed the results of the new system match with other screening methods including a system screening time as short as 3 days. Therefore, the co-culture model developed by using hTERT-immortalized cell lines described in this report provide a consistent and robust in vitro system for studying cardiovascular biology, drug screening and tissue engineering. Citation Format: CHAOZHONG ZOU, Chia-Wen Hsu, Menghang Xia, Metewo S Enuameh. Development of high-throughput cell-based co-culture angiogenesis assay system using hTERT immortalized cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 792. doi:10.1158/1538-7445.AM2017-792

RUNX1 Plays an Important Role in Mediating BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells Line C3H10T1/2, Murine Multi-Lineage Cells Lines C2C12 and MEFs.

As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is highly capable of promoting osteogenic differentiation. However, the underlying mechanism involved remains largely unknown. Recent studies have demonstrated that RUNX1 (runt-related transcription factor 1) is essential in osteoblast/chondrocyte maturation. In this study, we investigated the function of RUNX1 in BMP9-induced osteogenic of murine mesenchymal stem cell line (C3H10T1/2) and murine multi-lineage cell lines (C2C12 and MEFs). Our data showed that BMP9 promoted the endogenous expression of RUNX1 in C3H10T1/2, C2C12 and MEFs. Moreover, RUNX1 was probably a direct target of BMP9/Smad signaling. BMP9-induced osteogenic differentiation was enhanced by overexpression of RUNX1, whereas inhibited by knockdown RUNX1 in C3H10T1/2, C2C12 and MEFs. Further mechanism studies demonstrated that RUNX1 might affect BMP9-induced phosphorylation of Smad1/5/8, but not the phosphorylation of p38 and ERK1/2.Our results suggest that RUNX1 may be an essential modulator in BMP9- induced osteogenic differentiation of MSCs (Mesenchymal stem cells).

On-command on/off switching of progenitor cell and cancer cell polarized motility and aligned morphology via a cytocompatible shape memory polymer scaffold.

Invitro biomaterial models have enabled advances in understanding the role of extracellular matrix (ECM) architecture in the control of cell motility and polarity. Most models are, however, static and cannot mimic dynamic aspects of invivo ECM remodeling and function. To address this limitation, we present an electrospun shape memory polymer scaffold that can change fiber alignment on command under cytocompatible conditions. Cellular response was studied using the human fibrosarcoma cell line HT-1080 and the murine mesenchymal stem cell line C3H/10T1/2. The results demonstrate successful on-command on/off switching of cell polarized motility and alignment. Decrease in fiber alignment causes a change from polarized motility along the direction of fiber alignment to non-polarized motility and from aligned to unaligned morphology, while increase in fiber alignment causes a change from non-polarized to polarized motility along the direction of fiber alignment and from unaligned to aligned morphology. In addition, the findings are consistent with the hypothesis that increased fiber alignment causes increased cell velocity, while decreased fiber alignment causes decreased cell velocity. On-command on/off switching of cell polarized motility and alignment is anticipated to enable new study of directed cell motility in tumor metastasis, in cell homing, and in tissue engineering.

The intra-articular injection of RANKL-binding peptides inhibits cartilage degeneration in a murine model of osteoarthritis

We recently found that the receptor activator of NF-κB ligand (RANKL)-binding peptide, OP3-4 stimulated the differentiation of both chondrocytes and osteoblasts. OP3-4 is also shown to inhibit cartilage degeneration. To clarify whether the peptide can inhibit cartilage degeneration without stimulating bone formation, we first performed a proliferation assay using C3H10T1/2 (the murine mesenchymal stem cell line), which is the common origin of both chondrocytes and osteoblasts. The RANKL-binding peptides, OP3-4 and W9, promoted cellular proliferation at 24 and 48 h, respectively. Next, we injected both peptides into the intra-articular space of the knee joints of mice with monosodium-iodoacetate (MIA)-induced osteoarthritis to clarify the effects of the peptides on cartilage tissue. Twenty-five nine-week-old male C57BL/6J mice received injections of vehicle, or the same molar amount of W9, OP3-4, or a control peptide (which could not stimulate osteoblast differentiation) on days 7, 14, and 21 after the injection of MIA. The mice were sacrificed on day 28. The histomorphometric analyses revealed that both peptides inhibited the degeneration of cartilage without enhancing bone formation activity. Our data suggest that the stimulation of mesenchymal cell proliferation by the RANKL-binding peptides might lead to the inhibition of cartilage degeneration.

Antitumor Activity of a Mesenchymal Stem Cell Line Stably Secreting a Tumor-Targeted TNF-Related Apoptosis-Inducing Ligand Fusion Protein.

Mesenchymal stem cells (MSCs) are currently exploited as gene delivery systems for transient in situ expression of cancer therapeutics. As an alternative to the prevailing viral expression, we here describe a murine MSC line stably expressing a therapeutic protein for up to 42 passages, yet fully maintaining MSC features. Because of superior antitumoral activity of hexavalent TNF-related apoptosis-inducing ligand (TRAIL) formats and the advantage of a tumor-targeted action, we choose expression of a dimeric EGFR-specific diabody single-chain TRAIL (Db-scTRAIL) as a model. The bioactivity of Db-scTRAIL produced from an isolated clone (MSC.TRAIL) was revealed from cell death induction in Colo205 cells treated with either culture supernatants from or cocultured with MSC.TRAIL. In vivo, therapeutic activity of MSC.TRAIL was shown upon peritumoral injection in a Colo205 xenograft tumor model. Best antitumor activity in vitro and in vivo was observed upon combined treatment of MSC.TRAIL with bortezomib. Importantly, in vivo combination treatment did not cause apparent hepatotoxicity, weight loss, or behavioral changes. The development of well characterized stocks of stable drug-producing human MSC lines has the potential to establish standardized protocols of cell-based therapy broadly applicable in cancer treatment.

Gelatin-Derived Graphene-Silicate Hybrid Materials Are Biocompatible and Synergistically Promote BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells.

Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased the ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPARγ was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms.

TRIM33 is essential for osteoblast proliferation and differentiation via BMP pathway.

Tripartite motif containing 33 (TRIM33) functions both as a positive and negative regulator of the TGF-β/BMP pathway in tumors; however, its effect and mechanism during osteoblast proliferation and differentiation, which involves the TGF-β/BMP pathway is not defined. In this study, we used mouse C3H10T1/2 mesenchymal stem cell line and MC3T3-E1 preosteoblasts to investigate the role of TRIM33 during this process. The results demonstrated that the expression of TRIM33 increased during the differentiation. Moreover, the overexpression or knockdown of TRIM33 resulted in both an augmentation or decrease in osteoblast differentiation, which were measured by the expression of alkaline phosphatase (ALP) at the mRNA level, both Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) at the protein level, and the formation of mineral modules. To further demonstrate the mechanism of TRIM33 in this process, we found that TRIM33 could positively mediate the BMP pathway by forming TRIM33-Smad1/5 complex. This interaction between TRIM33 and Smad1/5 triggered the phosphorylation of Smad1/5. In addition, the essential role of TRIM33 in osteoblast proliferation was determined in this study by CellCounting Kit (CCK) -8 and cell cycle assays. In summary, we establish the function of TRIM33 as a positive regulator of osteoblast differentiation in BMP pathway, which mediates its effect through its interaction with and activation of Smad1/5. In addition, the results clearly demonstrate that TRIM33 is necessary for osteoblast proliferation by regulating cell cycle. These results suggest that TRIM33 can be a positive target of osteoblast proliferation and differentiation through BMP pathway.

Establishment of mouse stably knockout of MYSM1 in MSC cell line C3H10T1/2 and its effect on the immune regulation in vitro

To aimed at the establishment of mouse stably knockout of MYSM1 mesenchymal stem cell(MSC) line C3H10T1/2, and to investigate its immunological capacity of MSC in vitro. To establish the stably transfected MSC cell line by using CRISPR-Cas9 technology. Then the Flow cytometry, quantitative PCR and Western blot were employed to detect whether the MYSM1 have been knockout yet. Furthermore, the immune modulatory effect of MYSM1-/- MSC was tested by addition of MYSM1-/- MSC supernatant into spleen lymphocyte and Foxp3 culture. The mRNA expression of inflammatory cytokines such as interleukin-4, interferon-γ and interleukin-17 were detected by quatitatine PCR. The expression of MYSM1 was steadily knock out in MSC. In addition, MYSM1-/- MSC showed a stronger inhibitory effect on the expression of inflammatory cytokines. Therefore, the MYSM1 has been stably knocked out in C3H10T1/2. The mouse stably knockout of MYSM1 mesenchymal stem cells has been successfully established, the knock-out of MYSM1 in MSC can induce more potent immunosuppressive effects on cellular immune reaction in vitro. Our data laid a foundation for the further MSC-based applications in immune related diseases.

"Neuronal-Like Differentiation of Murine Mesenchymal Stem Cell Line: Stimulation by Juglans regia L. Oil".

Mesenchymal stem cells have been extensively used for cell-based therapies especially in neuronal diseases. Studies still continue to delineate mechanisms involved in differentiating mesenchymal stem cells into neuronal cells under experimental conditions as they have low mortality rate and hence, the number of cells available for experiments is much more limited. Culturing and differentiating of neuronal cell is more challenging as they do not undergo cell division thus, bringing them to differentiate proves to be a difficult task. Here, the aim of this study is to investigate whether Juglans regia L. (walnut oil) differentiates multipotent, C3H10T1/2 cells, a murine mesenchymal stem cell line, into neuronal cells. A simple treatment protocol induced C3H10T1/2 cells to exhibit a neuronal phenotype. With this optimal differentiation protocol, almost all cells exhibited neuronal morphology. The cell bodies extended long processes. C3H10T1/2 cells were plated and treated with walnut oil post 24h of plating. The treatment was given (with walnut oil treated cultures with or without control cultures) at different concentrations. The cultured cells were then stained with cresyl violet acetate solution which was used to stain the Nissl substance in the cytoplasm of the induced neuronal culture. The results indicated that the C3H10T1/2 cells differentiated into neuronal-like cells with long outgrowths of axon-like structures able to take up the cresyl violet acetate stain indicating their preliminary differentiation into neuronal-like morphology with walnut oil treatment. Treating the mesenchymal stem cells can in future establish a cultured mesenchymal stem cell line as neuronal differentiating cell line model.

Long Term Exposure to Myrtucommulone-A Changes CD105 Expression and Differentiation Potential of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) represent a heterogeneous group of multipotent stem cells that could be found in various somatic tissues. MSCs are defined by molecular and functional features including spindle-shape morphology, adherence to plastic surfaces, expression of specific surface markers and differentiation potential to chondrocytes, adipocytes and osteocytes. The surface markers were proposed to affect the differentiation potential of MSCs by a limited number of studies. Endoglin (CD105) is defined to be a significant marker for osteogenic and chondrogenic differentiation ability of MSCs. Low CD105 expression is associated with increased osteogenic potential while high CD105 expression is correlated with strong chondrogenic potential. Myrtucommulone-A (MC-A) is an active compound with various biological effects on different cell types but its effect on MSC differentiation has not been described yet. In the present study we aimed at investigating the long-term effects of MC-A on hMSCs. MC-A-treatment reduced CD105 expression in distinct human mesenchymal stem cell (hMSC) lines and gave rise to CD105low population but did not change CD44, CD90 or CD73 expression. The decrease in CD105 expression reduced the chondrogenic potential of hMSCs subsequently while adipogenic or osteogenic differentiation was not affected dramatically. MC-A-treatment also suppressed the NF-κB p65 activation which might be responsible for the reduced chondrogenic potential. Our findings suggest that MC-A could be used to enrich CD105low hMSCs without the need for cell sorting or changing culture conditions which could be utilised in targeted differentiation studies.

Neprilysin facilitates adipogenesis through potentiation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway.

Neprilysin (NEP) is a zinc metallopeptidase that cleaves a number of small peptides into inactive forms. Despite the recent evidence of a significant correlation between the levels of NEP in plasma and the severity of obesity in humans, a cause-and-effect relationship or a functional role of NEP in obesity has remained uncertain. In this study, we show that NEP has a positive regulatory effect on fat cell formation from precursor cells. NEP increases the accumulation of cytoplasmic triglycerides in 3T3-L1 preadipocytes or the C3H10T1/2 mesenchymal stem cell line in differentiation conditions. Consistently, cells expressing NEP showed an increase in mRNA expression of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), and the adipocyte markers aP2 and adipsin. Furthermore, this NEP-enhanced induction of adipogenesis was found to require the enzymatic activity of NEP, leading to augmentation of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway. In summary, our results indicate that NEP accelerates adipogenesis through enhancement of insulin-mediated PI3K-Akt activation and imply a high therapeutic value of NEP in treating obesity and obesity-related disorders.

A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties

Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo.

Relationships between the expression of hepatocyte nuclear factors and factors essential for lipoprotein production in a human mesenchymal stem cell line, UE7T-13.

To clarify the mechanisms regulating lipoprotein production by hepatocyte nuclear factors (HNFs), we generated four kinds of transfectants in human bone marrow mesenchymal stem cells: UE7T-13, stably expressing FOXA2 (also known as HNF3β), HNF4α, HNF1α or co-expressing HNF4α, and HNF1α (HNF4α/HNF1α). In HNF4α/HNF1α transfectants, cellular contents of triglycerides (TG) and cholesterol were markedly higher than in UE7T-13 cells and comparable to those in human hepatoma HepG2 cells. However, TG and cholesterol, which are secreted from cells as components of lipoproteins, were hardly detected in the medium for any of the transfectants. ApoB100 and MTP, which are essential for the formation and secretion of lipoproteins, were undetectable and detected at low levels, respectively, in HNF4α/HNF1α transfectants. We suggest that enforced co-expression of HNF4α and HNF1α is effective for cellular lipid accumulation, while additional factors are probably required for lipoprotein formation and secretion.

Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes.

BackgroundWhile screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied.Methodology/Principal FindingsOral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT) and Pin1-/- (Pin1-KO) adipose-derived mesenchymal stem cell (ASC) lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not.Conclusion and SignificanceOral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity.

A Cost-Effective Method to Assemble Biomimetic 3D Cell Culture Platforms.

Developing effective stem cell based therapies requires the design of complex in vitro culture systems for more accurate representation of the stem cell niche. Attempts to improve conventional cell culture platforms include the use of biomaterial coated culture plates, sphere culture, microfluidic systems and bioreactors. Most of these platforms are not cost-effective, require industrial technical expertise to fabricate, and remain too simplistic compared to the physiological cell niche. The human amniotic membrane (hAM) has been used successfully in clinical grafting applications due to its unique biological composition and regenerative properties. In this study, we present a combinatorial platform that integrates the hAM with biomolecular, topographic and mechanical cues in one versatile model.MethodsWe utilized the hAM to provide the biological and the three dimensional (3D) topographic components of the prototype. The 3D nano-roughness of the hAM was characterized using surface electron microscopy and surface image analysis (ImageJ and SurfaceJ). We developed additional macro-scale and micro-scale versions of the platform which provided additional shear stress factors to simulate the fluid dynamics of the in vivo extracellular fluids.ResultsThree models of varying complexities of the prototype were assembled. A well-defined 3D surface modulation of the hAM in comparable to commercial 3D biomaterial culture substrates was achieved without complex fabrication and with significantly lower cost. Performance of the prototype was demonstrated through culture of primary human umbilical cord mononuclear blood cells (MNCs), human bone marrow mesenchymal stem cell line (hBMSC), and human breast cancer tissue.ConclusionThis study presents methods of assembling an integrated, flexible and low cost biomimetic cell culture platform for diverse cell culture applications.

Comparative Transcriptomic and Epigenomic Analyses Reveal New Regulators of Murine Brown Adipogenesis

Increasing energy expenditure through brown adipocyte recruitment is a promising approach to combat obesity. We report here the comprehensive profiling of the epigenome and transcriptome throughout the lineage commitment and differentiation of C3H10T1/2 mesenchymal stem cell line into brown adipocytes. Through direct comparison to datasets from differentiating white adipocytes, we systematically identify stage- and lineage-specific coding genes, lncRNAs and microRNAs. Utilizing chromatin state maps, we also define stage- and lineage-specific enhancers, including super-enhancers, and their associated transcription factor binding motifs and genes. Through these analyses, we found that in brown adipocytes, brown lineage-specific genes are pre-marked by both H3K4me1 and H3K27me3, and the removal of H3K27me3 at the late stage is necessary but not sufficient to promote brown gene expression, while the pre-deposition of H3K4me1 plays an essential role in poising the brown genes for expression in mature brown cells. Moreover, we identify SOX13 as part of a p38 MAPK dependent transcriptional response mediating early brown cell lineage commitment. We also identify and subsequently validate PIM1, SIX1 and RREB1 as novel regulators promoting brown adipogenesis. Finally, we show that SIX1 binds to adipogenic and brown marker genes and interacts with C/EBPα, C/EBPβ and EBF2, suggesting their functional cooperation during adipogenesis.

The Role of MSCs in the Development of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a malignant disease of the bone marrow. The prognosis of AML is still poor with only 25% of patients living longer than 5 years. Thus, new therapeutic approaches are warranted to improve patients' survival. The growth of AML cells not only depends on cell intrinsic factors, but is also supported by stroma cells, among them mesenchymal stem cells (MSCs). We reported here that MSCs derived from human AML patients or murine models of human AML better supported the in vitro growth of leukemic cells than MSCs from non-leukemic patients or mice and this is dependent on cell-to-cell contact. Similarly, immortalized MSCs lines derived from AML patients supported the growth of human AML cell lines better than a MSC line originating from a healthy donor. In addition, the frequency of MSCsin the BM of leukemic mice was increased. On a molecular level, the polarization of MSCs towards an AML-supporting state depended on the expression of the transcription factor Growth factor independence 1 (Gfi1). Loss of Gfi1 abrogated the tumor-supporting state of AML-associated MSCs in vitro and in vivo. In summary, MSCs from AML patients support the growth of AML cells in vitro in a Gfi1-dependent manner which could open the path to new therapeutic approaches. DisclosuresDührsen:Amgen: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Khandanpour:Hospital of Essen university: Research Funding; Max-Eder: Research Funding.

Guar gum oleate-graft-poly(methacrylic acid) hydrogel as a colon-specific controlled drug delivery carrier

A novel type of ethylene glycol dimethacrylate (EGDMA) cross-linked guar gum oleate-graft-poly(methacrylic acid) (GGO-g-PMAc) hydrogel was prepared as a pH-responsive controlled release carrier for colon-specific drug delivery. The structure of GGO-g-PMAc hydrogel was characterized by FT-IR, 1H NMR and X-ray diffraction (XRD) analysis. The swelling degree of the GGO-g-PMAc hydrogel at pH 7.4 was found to be higher than that at pH 1.2. The drug release studies performed in pH 7.4 and 1.2 buffer solutions at 37°C revealed that the rate and amount of drug released from the GGO-g-PMAc hydrogel at pH 7.4 were higher than that at pH 1.2. The MTT assay revealed that there is no noticeable cytotoxicity of GGO-g-PMAc hydrogel at the concentration range of 0–100μg/ml against the mouse mesenchymal stem cell line (C3H10T1/2). These results suggested that GGO-g-PMAc hydrogel can be a prospective pH-sensitive carrier for colon-targeted drug delivery.

Abstracts from the In Vitro Toxicology Society Annual Meeting Radisson Blu Hotel Glasgow, United Kingdom November 14–15, 2016

Abstracts from the In Vitro Toxicology Society Annual Meeting Radisson Blu Hotel Glasgow, United Kingdom November 14–15, 2016