Mesenchymal Cell Line Research Articles

The Influence of the Epithelial Sodium Channel in Breast Cancer Cells

Breast cancer is the most common cancer in women worldwide and leads to over half a million deaths annually. Most breast cancer deaths can be attributed to metastasis, in which the cancerous cells spread from the primary site in the breast tissue to other organs throughout the body. Thus, researching how breast cancer cells metastasise is vital. Ion channels, such as the epithelial sodium channel (ENaC) are an emerging area within cancer research due to their possible role in regulating cancer cell functions; such as epithelial- mesenchymal transition (EMT) which involves the cell undergoing phenotype changes before metastasising. Our research aims to investigate the influence of ENaC on breast cancer cell migration, EMT, invasion and metastasis. We hypothesise that high ENaC expression enables the cell to maintain a more epithelial and less migratory phenotype. We have shown mesenchymal cell lines have lower mRNA expression of alpha-ENaC and similarly less alpha-ENaC protein being expressed in mesenchymal cell lines compared to epithelial when examined using western blotting. Using Boyden chamber assays, an established model for cell migration, the influence of overexpressing ENaC subunits on the cells’ ability to migrate was investigated in two mesenchymal breast cancer cell lines, MDAMB231 and BT549. The overexpression of ENaC resulted in a reduction in the number of cells migrating through the Boyden chamber, suggesting a reduced migration ability compared to control cells. These results suggest ENaC has a role in the migration of breast cancer cells and highlights ENaC as a potential target in the future development of breast cancer therapies.

Ginkwanghols A and B, osteogenic coumaric acid-aliphatic alcohol hybrids from the leaves of Ginkgo biloba.

Ginkgo biloba (Ginkgoaceae), commonly known as "ginkgo", is called a living fossil, and it has been cultivated early in human history for various uses in traditional medicine and as a source of food. As part of ongoing research to explore the chemical diversity and biologically active compounds from natural resources, two new coumaric acid-aliphatic alcohol hybrids, ginkwanghols A (1) and B (2) were isolated from the leaves of G. biloba. The coumaric acid-aliphatic alcohol hybrids of natural products have rarely been reported. The structures of the new compounds were determined by extensive NMR spectroscopic analysis, HRESI-MS, and quantum chemical ECD calculations, and by comparing the experimental HRESI-MS/MS spectrum of chemically transformed compound 1a with the predicted HRESI-MS/MS spectra proposed from CFM-ID 3.0, a software tool for MS/MS spectral prediction and MS-based compound identification. Ginkwanghols A (1) and B (2) increased alkaline phosphatase (ALP) production in C3H10T1/2, a mouse mesenchymal stem cell line, in a dose-dependent manner. In addition, ginkwanghols A and B mediated the promotion of osteogenic differentiation as indicated by the induction of the mRNA expression of the osteogenic markers ALP and osteopontin (OPN).

Pay more attention to the application of mesenchymal stem cells in the treatment for immune skin diseases

Mesenchymal stem cells (MSC) represents a heterogeneous group of multipotent non-hematopoietic stem cells with self-renewal and differentiating into mesenchymal cell lineage. Recent studies have demonstrated that MSC has low immunogenicity, which means it does not express major histocompatibility complex (MHC) class Ⅱand other co-stimulating factors. Thus, due to the capability of anti-inflammatory and immunomodulatory effects, MSC therapy provides many possibilities for the treatment for immune skin diseases.

Role of Discoidin Domain Receptor 2 in Craniofacial Bone Regeneration

Bone loss caused by trauma, neoplasia, congenital defects, or periodontal disease is a major cause of disability and human suffering. Skeletal progenitor cell–extracellular matrix interactions are critical for bone regeneration. Discoidin domain receptor 2 (DDR2), an understudied collagen receptor, plays an important role in skeletal development. Ddr2 loss-of-function mutations in humans and mice cause severe craniofacial and skeletal defects, including altered cranial shape, dwarfing, reduced trabecular and cortical bone, alveolar bone/periodontal defects, and altered dentition. However, the role of this collagen receptor in craniofacial regeneration has not been examined. To address this, calvarial subcritical-size defects were generated in wild-type (WT) and Ddr2-deficient mice. The complete bridging seen in WT controls at 4 wk postsurgery was not observed in Ddr2-deficient mice even after 12 wk. Quantitation of defect bone area by micro–computed tomography also revealed a 50% reduction in new bone volume in Ddr2-deficient mice. Ddr2 expression during calvarial bone regeneration was measured using Ddr2-LacZ knock-in mice. Expression was restricted to periosteal surfaces of uninjured calvarial bone and, after injury, was detected in select regions of the defect site by 3 d postsurgery and expanded during the healing process. The impaired bone healing associated with Ddr2 deficiency may be related to reduced osteoprogenitor or osteoblast cell proliferation and differentiation since knockdown/knockout of Ddr2 in a mesenchymal cell line and primary calvarial osteoblast cultures reduced osteoblast differentiation while Ddr2 overexpression was stimulatory. In conclusion, Ddr2 is required for cranial bone regeneration and may be a novel target for therapy.

Modeling in vitro osteoarthritis phenotypes in a human vascularized bone construct based on a bone-marrow derived mesenchymal cell line and endothelial cells

Purpose: The main clinical indicators of osteoarthritis (OA) include loss of articular cartilage, altered subchondral bone remodeling, increased vascularization, and inflammation. Although cartilage degeneration has been always considered as the hallmark of OA, there is growing evidence that the subchondral bone and its associated vasculature could play an important role in OA onset. Reliable multi-cellular and complex human in vitro models are needed to resemble diverse tissues and to tackle different aspects of the pathology.

The AXL-PYK2-PKCα axis as a nexus of stemness circuits in TNBC.

Cancer stem cells (CSCs) are implicated in tumor initiation, metastasis and drug resistance, and considered as attractive targets for cancer therapy. Here we identified a clinically relevant signaling nexus mediated by AXL receptor, PYK2 and PKCα and show its impact on stemness in TNBC. AXL, PYK2, and PKCα expression correlates with stemness signature in basal-like breast cancer patients, and their depletion in multiple mesenchymal TNBC cell lines markedly reduced the number of mammosphere-forming cells and cells harboring CSCs characteristic markers. Knockdown of PYK2 reduced the levels of AXL, PKCα, FRA1, and PYK2 proteins, and similar trend was obtained upon PKCα depletion. PYK2 depletion decreased AXL transcription through feedback loops mediated by FRA1 and TAZ, whereas PKCα inhibition induced redistribution of AXL to endosomal/lysosomal compartment and enhanced its degradation. PYK2 and PKCα cooperate at a convergence point of multiple stemness-inducing pathways to regulate AXL levels and concomitantly the levels/activation of STAT3, TAZ, FRA1, and SMAD3 as well as the pluripotent transcription factors Nanog and Oct4. Induction of stemness in TNBC sensitized cells to PYK2 and PKCα inhibition suggesting that targeting the AXL-PYK2-PKCα circuit could be an efficient strategy to eliminate CSCs in TNBC.

Progressive and Coordinated Mobilization of the Skeletal Muscle Niche throughout Tissue Repair Revealed by Single-Cell Proteomic Analysis.

Background: Skeletal muscle is one of the only mammalian tissues capable of rapid and efficient regeneration after trauma or in pathological conditions. Skeletal muscle regeneration is driven by the muscle satellite cells, the stem cell population in interaction with their niche. Upon injury, muscle fibers undergo necrosis and muscle stem cells activate, proliferate and fuse to form new myofibers. In addition to myogenic cell populations, interaction with other cell types such as inflammatory cells, mesenchymal (fibroadipogenic progenitors—FAPs, pericytes) and vascular (endothelial) lineages are important for efficient muscle repair. While the role of the distinct populations involved in skeletal muscle regeneration is well characterized, the quantitative changes in the muscle stem cell and niche during the regeneration process remain poorly characterized. Methods: We have used mass cytometry to follow the main muscle cell types (muscle stem cells, vascular, mesenchymal and immune cell lineages) during early activation and over the course of muscle regeneration at D0, D2, D5 and D7 compared with uninjured muscles. Results: Early activation induces a number of rapid changes in the proteome of multiple cell types. Following the induction of damage, we observe a drastic loss of myogenic, vascular and mesenchymal cell lineages while immune cells invade the damaged tissue to clear debris and promote muscle repair. Immune cells constitute up to 80% of the mononuclear cells 5 days post-injury. We show that muscle stem cells are quickly activated in order to form new myofibers and reconstitute the quiescent muscle stem cell pool. In addition, our study provides a quantitative analysis of the various myogenic populations during muscle repair. Conclusions: We have developed a mass cytometry panel to investigate the dynamic nature of muscle regeneration at a single-cell level. Using our panel, we have identified early changes in the proteome of stressed satellite and niche cells. We have also quantified changes in the major cell types of skeletal muscle during regeneration and analyzed myogenic transcription factor expression in satellite cells throughout this process. Our results highlight the progressive dynamic shifts in cell populations and the distinct states of muscle stem cells adopted during skeletal muscle regeneration. Our findings give a deeper understanding of the cellular and molecular aspects of muscle regeneration.

Effect of TIMP2/TIMP3 genes on the risk of osteosarcoma in Zhejiang population.

Osteosarcoma is a malignant tumor that develops from a mesenchymal cell line and is caused by gene–environment interactions. This study aimed to explore whether TIMP2/TIMP3 polymorphisms influenced the osteosarcoma risk.The expression of the TIMP2 and TIMP3 genes in osteosarcoma histiocytes was analyzed by immunohistochemistry. In this case-control study, which includes samples from 499 patients and 500 healthy controls, 10 single-nucleotide polymorphisms (SNPs) in TIMP2 and TIMP3 were selected. Furthermore, we used the Agena MassARRAY platform for genotyping. The statistical analysis was performed using χ2 test/Fisher exact test, and logistic regression analysis.The immunohistochemistry results showed that the expression of TIMP2 is obvious higher in osteosarcoma histiocytes than in the normal histiocytes. The association study indicated that the allele of rs2277698 and rs4789936 were protective SNPs reducing the risk of osteosarcoma (odds ratios > 1, P < .05) by the χ2 test. In the genetic model, logistic regression analyses revealed that the rs2277698 and rs4789936 were associated with decreasing the risk of osteosarcoma under the codominant model, dominant model, and log-additive model. Stratification analysis revealed that 2 SNPs (rs2277698 and rs4789936) were significantly associated with a reduced risk of osteosarcoma in allele and genetic model after stratification by gender or age (P < .05). In addition, the haplotype “Trs2277698Crs2009169Crs7342880” of TIMP2 was associated with decreasing the osteosarcoma risk. The “Ars9609634Trs11547635” of TIMP3 was associated with reducing the osteosarcoma risk.This finding shed new light on the high expression of TIMP2 polymorphisms may contribute to decreasing the osteosarcoma risk in Zhejiang populations.

Factor XIII-A: An Indispensable "Factor" in Haemostasis and Wound Healing.

Factor XIII (FXIII) is a transglutaminase enzyme that catalyses the formation of ε-(γ-glutamyl)lysyl isopeptide bonds into protein substrates. The plasma form, FXIIIA2B2, has an established function in haemostasis, with fibrin being its principal substrate. A deficiency in FXIII manifests as a severe bleeding diathesis emphasising its crucial role in this pathway. The FXIII-A gene (F13A1) is expressed in cells of bone marrow and mesenchymal lineage. The cellular form, a homodimer of the A subunits denoted FXIII-A, was perceived to remain intracellular, due to the lack of a classical signal peptide for its release. It is now apparent that FXIII-A can be externalised from cells, by an as yet unknown mechanism. Thus, three pools of FXIII-A exist within the circulation: plasma where it circulates in complex with the inhibitory FXIII-B subunits, and the cellular form encased within platelets and monocytes/macrophages. The abundance of this transglutaminase in different forms and locations in the vasculature reflect the complex and crucial roles of this enzyme in physiological processes. Herein, we examine the significance of these pools of FXIII-A in different settings and the evidence to date to support their function in haemostasis and wound healing.

Green Synthesis of Silver Nanoparticles Using the Lotus lalambensis Aqueous Leaf Extract and Their Anti-Candidal Activity against Oral Candidiasis.

Oral candidiasis is widely spread in both humans and animals, which is caused mainly by Candida albicans. In this study, we aimed to biosynthesize silver nanoparticles (AgNPs) for the first time using the Lotus lalambensis Schweinf leaf extract (L-AgNPs) and investigated their anti-candidal potency alone or in combination with the leaf extract of L. lalambensis (L-AgNPs/LL) against C. albicans. The biosynthesized L-AgNPs were characterized by imaging (transmission electron microscopy, TEM), UV–vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The results of the disk diffusion method showed the potent synergistic anti-candidal activity of L-AgNPs/LL (24 mm inhibition zone). L-AgNPs/LL completely inhibited the morphogenesis of C. albicans and suppressed the adhesion and the formation of the biofilm of C. albicans by 82.5 and 78.7%, respectively. Further, L-AgNPs/LL inhibited the production of antioxidant enzymes of C. albicans by 80%. SEM and TEM revealed deteriorations in the cell wall ultrastructure in L-AgNPs/LL-treated C. albicans. Interestingly, L-AgNPs/LL showed less than 5% cytotoxicity when examined with either the primary bone marrow derived mesenchymal stem cell (BMSCs) or MCF-7 cell line at MIC values of L-AgNPs/LL. In conclusion, we identified L-AgNPs/LL as a potential biosynthesized-based drug for oral candidiasis in humans and animals.

LGR5/R-Spo1/Wnt3a axis promotes stemness and aggressive phenotype in hepatoblast-like hepatocellular carcinoma cell lines

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly defined stem cell marker in endoderm-derived organs such as the small intestine, colon and pancreas. Recently, LGR5 was demonstrated to be an important factor in liver regeneration and stem cell maintenance. Moreover, LGR5 expression is highly up-regulated in various cancers including hepatocellular carcinoma. Herein, we demonstrate that LGR5 expression is specifically observed in certain subset of HCC cell lines with “hepatoblast-like” appearance, characterized by the expression of liver fetal/progenitor markers. Notably, the activation of the canonical Wnt pathway significantly increases the expression of LGR5 in this subset of cell lines, whereas it does not cause any induction of LGR5 expression in mesenchymal like cell lines SNU-475 and SNU-449. Furthermore, we showed that treatment of the hepatoblast-like HCC cell lines HuH-7 and Hep3B with LGR5 ligand R-Spo1 significantly amplifies the induction of LGR5 expression, the phosphorylation of LRP6 and β-catenin resulting in enhanced TCF/LEF activity either alone or in combination with Wnt3a. Consistently, the silencing of the LGR5 gene attenuates the co-stimulatory effect of R-Spo1/Wnt3a on TCF/LEF activity while overexpression of LGR5 enhances it. On the contrary, overexpression of LGR5 does not change TCF/LEF activity induced by R-Spo1/Wnt3a in mesenchymal-like HCC line, SNU-449. Importantly, LGR5-overexpressing cells have increased expression of several Wnt target genes and stemness-related genes including EpCAM and CK19 upon R-Spo1/Wnt3a treatment. LGR5-overexpressing cells also have increased spheroid forming, migration and invasion abilities and stimulation with R-Spo1/Wnt3a augments these abilities of LGR5-overexpressing cells. In addition, ectopic overexpression of LGR5 significantly increases cell proliferation rate independent of R-Spo1/Wnt3a stimulation. Moreover, in vitro tubulogenesis assay demonstrates that treatment with R-Spo1/Wnt3a enhances the sprouting of capillary tubules in only LGR5-overexpressing cells. Finally, R-Spo1/Wnt3a significantly promotes dissemination of LGR5-overexpressing cells in vivo in a zebrafish xenograft model. Our study unravels a tumor-promoting role for LGR5 through activation of canonical Wnt/β-catenin signaling in the hepatoblast-like HCCs. In conclusion, our results suggest that LGR5/R-Spo1/Wnt3a generates an axis that mediates the acquisition of aggressive phenotype specifically in hepatoblast-like subset of HCCs and might represent a valuable target for treatment of HCC tumors with aberrant activation of Wnt/β-catenin pathway.

Abstract PO051: Fibroblast lineage tracing in metastatic and pre-metastatic microenvironments

Abstract Metastatic breast cancer is one of the leading causes of cancer-related mortality among women. However, the complex process of tumor cell dissemination and colonization of distant tissues is incompletely understood and largely incurable using existing therapies. Metastatic disease is not solely driven by tumor cell-intrinsic properties but is instead a consequence of dynamic interactions between cancer cells and other cell types in the tumor microenvironment (TME) including cancer-associated fibroblasts (CAFs). However, CAFs are known to display paradoxical functions across different cancer types, suggesting that heterogeneous CAF subpopulations play diverse or opposing roles in the TME. It was recently shown that periostin, a TGFβ-induced matricellular protein important for collagen cross-linking and extracellular matrix stiffening, can distinguish CAF subtypes in breast cancer. This specific CAF subpopulation is not well characterized, and their functional properties and contributions to disease progression have not been well-studied in vivo. Here, we have used in vivo genetic labelling of periostin+ subpopulations (Postn-Cre:ZSGreenl/s/l) and their progeny to assess their expansion and function during mammary tumor growth and metastasis. We find that ZSGreen-labelled periostin-expressing CAFs arise from mesenchymal-lineage cells and are more abundant in primary tumors, metastatic, and pre-metastatic sites of high-metastatic tumors versus their low-metastatic counterparts. We also find differential collagen organization in high- versus low-metastatic tumors: high-metastatic tumors display longer, aligned collagen fibers whereas low-metastatic tumors display shorter, disorganized fibers. Knocking down periostin in primary human breast CAFs inhibits tumor cell invasion through collagen in a CAF/tumor cell co-culture spheroid assay, indicating that periostin-expressing CAFs may remodel the extracellular matrix via collagen cross-linking to facilitate cell invasion. Finally, we show that periostin/ZSGreen+ cells in highly metastatic tumors are larger and more spindle-shaped, and that periostin knockdown in primary human breast CAFs reduces their size and ability to migrate, suggesting that periostin expression associates with CAF biomechanical properties such as spreading and motility. Our results indicate that highly-metastatic cancer cells mobilize periostin-expressing CAFs in the TME, which may promote collagen remodeling and collective migration of CAFs and cancer cells during tissue invasion. Citation Format: Jamie L. Null, Dae Joong Kim, Yu Zhang, James V. McCann, Thomas H. Barker, Andrew C. Dudley. Fibroblast lineage tracing in metastatic and pre-metastatic microenvironments [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO051.

Metabolism and chromatin: A dynamic duo that regulates development and ageing

Bone-marrow mesenchymal stem cell (BM-MSC) proliferation and lineage commitment are under the coordinated control of metabolism and epigenetics; the MSC niche contains low oxygen, which is an important determinant of the cellular metabolic state. In turn, metabolism drives stem cell fate decisions via alterations of the chromatin landscape. Due to the fundamental role of BM-MSCs in the development of adipose tissue, bones and cartilage, age-associated changes in metabolism and the epigenome perturb the balance between stem cell proliferation and differentiation leading to stem cell depletion, fat accumulation and bone-quality related diseases. Therefore, understanding the dynamics of the metabolism-chromatin interplay is crucial for maintaining the stem cell pool and delaying the development and progression of ageing. This review summarizes the current knowledge on the role of metabolism in stem cell identity and highlights the impact of the metabolic inputs on the epigenome, with regards to stemness and pluripotency.

The epithelial sodium channel has a role in breast cancer cell proliferation.

Breast cancer is the most common cancer affecting women worldwide with half a million associated deaths annually. Despite a huge global effort, the pathways of breast cancer progression are not fully elucidated. Ion channels have recently emerged as novel regulators of cancer cell proliferation and metastasis. The epithelial sodium channel, ENaC, made up of α, β and γ subunits is well known for its role in Na+ reabsorption in epithelia, but a number of novel roles for ENaC have been described, including potential roles in cancer. A role for ENaC in breast cancer, however, has yet to be described. Therefore, the effects of ENaC level and activity on breast cancer proliferation were investigated. Through the publicly available SCAN-B dataset associations between αENaC mRNA expression and breast cancer subtypes, proliferation markers and epithelial-mesenchymal transitionmarkers (EMT) were assessed. αENaC expression, through overexpression or siRNA-mediated knockdown, and activity, through the ENaC-specific inhibitor amiloride, were altered in MCF7, T47D, BT549, and MDAMB231 breast cancer cells. MTT and EdU cell proliferation assays were used to determine the effect of these manipulations on breast cancer cell proliferation. High αENaC mRNA expression was associated with less aggressive and less proliferative breast cancer subtypes and with reduced expression of proliferation markers. Decreased αENaC expression or activity, in the mesenchymal breast cancer cell lines BT549 and MDAMB231, increased breast cancer cell proliferation. Conversely, increased αENaC expression decreased breast cancer cell proliferation. αENaC expression is associated with a poor prognosis in breast cancer and is a novel regulator of breast cancer cell proliferation. Taken together, these results identify ENaC as a potential future therapeutic target.

The rise in expression and activity of 11β-HSD1 in human mesenchymal progenitor cells induces adipogenesis through increased local cortisol synthesis

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) plays an important role in pre-receptor glucocorticoid metabolism. This enzyme is expressed in bone, increases with age, and catalyzes the conversion of the inactive glucocorticoid cortisone into the active glucocorticoid cortisol and vice versa. Here we hypothesized that the physiological activity of 11β-HSD1 to produce cortisol in human mesenchymal progenitor cells (hMSC) is principally sufficient to shift the differentiation potential in the direction of adipogenic. We thus investigated differentiating hMSCs and the mesenchymal stem cell line SCP-1 cultured under osteogenic conditions and stimulated with supra-physiological cortisone levels. The release of active cortisol into the medium was monitored and the influence on cell differentiation analyzed. We revealed an increase in 11β-HSD1 expression followed by increased reductive activity of the enzyme, thereby inducing a more adipogenic phenotype of the cell models via cortisol with negative effects on osteogenesis. Through inhibition experiments with the specific inhibitor 10 j, we proved the enzyme specificity for cortisol synthesis and adipogenic differentiation. Increased expression of 11β-HSD1 followed by higher cortisol levels might thus explain bone marrow adiposity followed by reduced bone quality and stability in old age or in situations of supra-physiological glucocorticoid exposure.

Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT.

Glypican-5 (GPC5) is a heparan sulfate proteoglycan (HSPG) localized to the plasma membrane. We previously reported that in the human mesenchymal stem cell line UE6E7T-3, GPC5 is overexpressed in association with transformation and promotes cell proliferation by acting as a co-receptor for Sonic hedgehog signaling. In this study, we found using immunofluorescence microscopy that in transformed cells (U3DT), GPC5 localized not only at primary cilia on the cell surface, but also at the leading edge of migrating cells, at the intercellular bridge and blebs during cytokinesis, and in extracellular vesicles. In each subcellular region, GPC5 colocalized with fibroblast growth factor receptor (FGFR) and the small GTPases Rab11 and ARF6, indicating that GPC5 is delivered to these regions by Rab11-associated recycling endosomes. These colocalizations suggest that GPC5 plays an important role in FGF2 stimulation of cell migration, which was abrogated by knockdown of GPC5. Our findings indicate that GPC5 plays a role in regulation of U3DT cell migration and provides several insights into the functions of GPC5 that could be elucidated by future studies.

Heterogeneity of radiation response in mesenchymal subtype glioblastoma: molecular profiling and reactive oxygen species generation.

Radiotherapy-induced tumor death remains critical in the successful first-line management of glioblastoma, whereas resistance to radiation serves as a major factor in disease progression. Mesenchymal shift has been identified as a driver in GBM recurrence, with gene expression associated with enhanced repair of macromolecular damage caused by radiation. Using distinct mesenchymal subtype GBM cells lines, radiation response was assessed by clonogenic assay and orthotopic mouse tumor model. RNA-sequencing was performed in the setting of increasing radiation dosing while real-time assessment of ROS generation was achieved by the measurement of hydroxyl spin trap adducts via electron paramagnetic resonance. Radiation-induced cell death determined by clonogenic assay was significantly different at low dose (4-8Gy) between the resistant U3035 cells and the sensitive U3020 cells. Similar trends were present in the in vivo NSG mouse model following radiation dosing on post-implantation day 7-10, with the rate of reduction in tumor bioluminescence reversing between the U3020 and U3035 cells after the third dose of radiation. Changes in gene expression following radiation determined by RNA-sequencing indicate both U3035 and U3020 cells demonstrate a shift toward more mesenchymal profiles, with concurrent shift away from pro-neural subtype gene expression in the U3020 cells that appeared to develop resistance to radiation in vivo. Persistence of ROS generated following radiation was greater in U3020 cells shown to be more sensitive to radiation. Despite the same molecular classification, distinct GBM cell lines can demonstrate differential response to radiation and potential for mesenchymal shift associated with radiation resistance.

Bioprinting of Magnetically Deformable Scaffolds

Mechanical stimulation of cells embedded in scaffolds is known to increase the cellular performance toward osteogenic or chondrogenic differentiation and tissue development. Three-dimensional bioplotting of magnetically deformable scaffolds enables the spatially defined distribution of magnetically inducible scaffold regions. In this study, a magnetic bioink based on alginate (alg, 3%) and methylcellulose (MC, 9%) with incorporated magnetite microparticles (25% w/w) was developed and characterized. The size and shape of particles were monitored via scanning electron microscopy and X-ray micro-computed tomography. Shear-thinning properties of the algMC ink were maintained after the addition of different concentrations of magnetite microparticles to the ink. Its viscosity proportionally increased with the added amount of magnetite, and so did the level of saturation magnetization as determined via vibrating sample magnetometry. The printability and shape fidelity of various shapes were evaluated, so that the final composition of algMC + 25% w/w magnetite was chosen. With application of this ink, cytocompatibility was proven in indirect cell culture and bioplotting experiments using a human mesenchymal stem cell line. Toward the deformation of cell-laden scaffolds to support cell differentiation in the future, radiography allowed the real-time monitoring of magnetically induced deformation of scaffolds of different pore architectures and scaffold orientations inside the magnetic field. Varying the strand distance and scaffold design will allow fine-tuning the degree of deformation in stimulatory experiments.

Deletion of phospholipase D1 decreases bone mass and increases fat mass via modulation of Runx2, β-catenin-osteoprotegerin, PPAR-γ and C/EBPα signaling axis

In osteoporosis, mesenchymal stem cells (MSCs) prefer to differentiate into adipocytes at the expense of osteoblasts. Although the balance between adipogenesis and osteogenesis has been closely examined, the mechanism of commitment determination switch is unknown. Here we demonstrate that phospholipase D1 (PLD1) plays a key switch in determining the balance between bone and fat mass. Ablation of Pld1 reduced bone mass but increased fat in mice. Mechanistically, Pld1/− MSCs inhibited osteoblast differentiaion with diminished Runx2 expression, while osteoclast differentiation was accelerated in Pld1−/− bone marrow-derived macrophages. Pld1−/− osteoblasts showed decreased expression of osteogenic makers. Increased number and resorption activity of osteoclasts in Pld1−/− mice were corroborated with upregulation of osteoclastogenic markers. Moreover, Pld1−/− osteoblasts reduced β-catenin mediated-osteoprotegerin (OPG) with increased RANKL/OPG ratio which resulted in accelerated osteoclast differentiation. Thus, low bone mass with upregulated osteoclasts could be due to the contribution of both osteoblasts and osteoclasts during bone remodeling. Moreover, ablation of Pld1 further increased bone loss in ovariectomized mice, suggesting that PLD1 is a negative regulator of osteoclastogenesis. Furthermore, loss of PLD1 increased adipogenesis, body fat mass, and hepatic steatosis along with upregulation of PPAR-γ and C/EBPα. Interestingly, adipocyte-specific Pld1 transgenic mice rescued the compromised phenotypes of fat mass and adipogenesis in Pld1 knockout mice. Collectively, PLD1 regulated the bifurcating pathways of mesenchymal cell lineage into increased osteogenesis and decreased adipogenesis, which uncovered a previously unrecognized role of PLD1 in homeostasis between bone and fat mass.

Anti-Obesity Effects ofArtemisia annuaExtract in Zucker Fatty Rats and High-Fat Diet Sprague Dawley Rats through Upregulation of Uncoupling Protein 1

BackgroundObesity is a widespread disease and is caused mainly by excessive adipocyte differentiation and fat accumulation. Peroxisome proliferation-activated receptor γ (PPARγ) and CCAAT/enhancer-binding proteins (C/EBP) are major components for regulating adipocyte differentiation. Uncoupling protein 1 (UCP1) is a transmembrane protein that can convert white fat to brown adipose tissue. Artemisia annua L. has long been used in East Asia as an herbal drug for anti-oxidant, anti-bacterial, and anti-obesity purposes.MethodsWe investigated the effects of water extracts of A. annua (WEAA) in C3H10T1/2, a mesenchymal stem cell line, by measuring the level of intracellular fat accumulation and the expression of genes associated with adipocyte differentiation. We also evaluated anti-obesity effects of WEAA in Zucker rats, a genetic model for the study of obesity, and in Sprague Dawley rats with high-fat diet (HFD)-induced obesity.ResultsIn this study, WEAA reduced the expression levels of PPARγ and C/EBPα in C3H10T1/2 cells, as well as the expression of enzymes that regulate fatty acid metabolism. In the Zucker fatty rat model and the HFD-induced obesity rat model, WEAA significantly decreased adipogenic differentiation and white fat accumulation between the scapulae, in contrast to the brown fat that remained unchanged between the groups. A. annua suppressed the expression of the adipocyte differentiation-promoting genes, while increasing the expression of UCP1.ConclusionThese results indicated that WEAA could reduce adipocyte differentiation and fat accumulation in in vitro and in vivo model systems, resulting in suppression of obesity and the occurrence of fatty liver due to a HFD.