Transwell Culture Research Articles

Recruitment of bone marrow-derived cells to the periodontal ligament via the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 axis.

The periodontal ligament (PDL) is a non-mineralized connective tissue that exists between the alveolar bone and root surface cementum and plays important roles in tooth function. The PDL harbors a remarkable reserve of multipotent stem cells, which maintain various types of cells. However, the sources of these stem cells, other than their developmental origin, are not well understood. To elucidate the recruitment of bone marrow (BM)-derived stem cells in the PDL, green fluorescent protein (GFP)-expressing BM-derived cells were transplanted into the femoral BM of immunodeficient rats, and the distribution and expression of stem cell markers in the PDL were analyzed in vivo. To evaluate the functional significance of BM-derived cells to the PDL, tooth replantation was performed and the expression of stromal cell-derived factor (SDF)-1, a critical chemotactic signal for mesenchymal stem cell recruitment, was analyzed. To confirm the SDF-1-dependency of BM-derived cell migration to the PDL, PDL-conditioned medium (CM) was prepared, and BM-derived cell migration was analyzed using a transwell culture system. Four weeks after cell transplantation, GFP-positive cells were detected in the PDL, and some of them were also positive for stem cell markers (i.e., CD29, SSEA4, and αSMA). Seven days after tooth replantation, the number of GFP- and SDF-1-positive cells significantly increased in PDL. Concurrently, the concentration of SDF-1 and the number of colony-forming units of fibroblasts in peripheral blood were increased. BM-derived cell migration increased in PDL-CM and was inhibited by an inhibitor of C-X-C chemokine receptor type 4 (CXCR4), an SDF-1 receptor. These results indicate that stem cells and their progeny in PDL are not only derived from their developmental origin but are also supplied from the BM via the blood as the need arises. Moreover, this BM-derived cell recruitment appears to be regulated, at least partially, by the SDF-1/CXCR4 axis.

Concentration-dependent roles of DMT1 and ZIP14 in cadmium absorption in Caco-2 cells.

Intestinal absorption of cadmium (Cd) is considered to be mediated mainly by the ferrous iron transporter DMT1, or the calcium transporter CaT1. The roles of zinc transporters such as ZIP8 and ZIP14 remain unclear, and the roles of these four transporters in the intestinal uptake of Cd under physiological conditions have not been compared. Here, we used a trans-well cell culture system to investigate the effects of the down-regulation of these four transporters on the uptake of Cd from the apical side of enterocytes. We used a Caco-2-kh cell line that can form tight junctions within a few days. The transfection of DMT1 siRNA significantly decreased the Cd uptake from the apical side at 5 μM, but not at 0.1 or 1 μM. The transfection of ZIP14 siRNA markedly decreased the Cd uptake at 0.1 and 1 μM, but not at 5 μM. The transfection of siRNA of CaT1 or ZIP8 did not alter the Cd uptake at any concentrations of Cd examined. These results suggest that DMT1 and ZIP14 play different roles in intestinal Cd absorption depending on the concentration of Cd.

Paris polyphylla Suppresses Proliferation and Vasculogenic Mimicry of Human Osteosarcoma Cells and Inhibits Tumor Growth In Vivo.

Paris polyphylla, a traditional antipyretic-detoxicate chinese medicinal herb, has been applied extensively in cancer treatments for nearly 2000 years. The purpose of the present study is to evaluate the potential anti-osteosarcoma effects of Paris polyphylla ethanol extract (PPEE) and to investigate its underlying mechanisms. The antiproliferation activity of PPEE was tested on 143B, MG-63, U-2 OS and hFOB1.19 cells using MTT assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by Hoechst 33342 staining and flow cytometry. The antimigratory, anti-invasive and antivasculogenic mimicry (VM) effects of PPEE were investigated by wound healing, Transwell and 3D culture assays. Mouse xenograft model was used to examine its anti-osteosarcoma efficacy in vivo. Hematologic profiles and hepatorenal functions were evaluated to assess the toxicity of PPEE.PPEE evidently suppressed cell proliferation of 143B, MG-63 and U-2 OS with IC50 values of 10-60[Formula: see text][Formula: see text]g/mL, but showed little cytotoxicity against normal osteoblastic cell. PPEE promoted apoptosis in 143B cell via caspase activation, increased Bax/Bcl-2 ratio and PARP cleavage. It also induced G2/M phase arrest associated with elevated phosphorylation of CDK1, Cdc25C, Chk2 and down-regulation of cyclin B1, CDK1, Cdc25C expression. Additionally, PPEE inhibited 143B cell migration, invasion and VM formation at noncytotoxic concentrations through decreasing the expression of FAK, Mig-7, MMP2 and MMP9. Finally, daily oral administration of PPEE for four weeks exhibits potent antitumor and anti-VM activity in 143B xenograft model with low toxicity. Taken together, these findings demonstrated PPEE possesses anti-osteosarcoma and anti-VM activity in vitro and in vivo, and therefore is a potential candidate for osteosarcoma treatment.

Basophil Recruitment to Skin Lesions of Patients with Systemic Lupus Erythematosus Mediated by CCR1 and CCR2

Background/Aims: Basophils have been reported to infiltrate skin lesions in various skin diseases, but not in systemic lupus erythematosus (SLE). This study investigated basophil infiltration in SLE and its mechanism. Methods: Twenty newly diagnosed SLE patients and twenty healthy controls were enrolled. Nine SLE patients underwent skin biopsies. Flow cytometric analysis the phenotype of peripheral basophils and their migration rate toward RANTES and MCP-1 were analyzed with the transwell culture system, also the expression of these two chemokines in skin tissue were analyzed with immunohistochemistry. Results: Increased activation and decreased numbers of peripheral basophils were observed in SLE patients compared with controls. Basophil migration into skin lesions of SLE patients were observed, but not in normal skin tissue. This migration was related to the upregulation of chemokine receptors CCR1 and CCR2 on basophils. In vitro studies showed that migration rate toward RANTES and MCP-1 increased significantly in basophils from SLE patients compared with those from controls. Consistently, high levels of RANTES and MCP-1 expression were observed in skin lesions from SLE patients but not in normal skin tissue. Conclusion: Basophil recruitment to skin lesions of SLE patients mediated by CCR1 and CCR2, which may contribute to tissue damage in SLE.

Bone Marrow Mesenchymal Stem Cells Inhibit the Function of Dendritic Cells by Secreting Galectin-1.

This study aimed to investigate whether bone marrow-derived mesenchymal stem cells (BM-MSCs) can inhibit function of dendritic cells (DCs) by secreting Galectin-1 (Gal-1). BM-MSCs have been shown to inhibit the maturation and function of DCs, further inhibiting the activation and proliferation of T cells. However, the detailed mechanism remains unknown. In this current study, MSCs and DCs derived from mouse bone marrow were cocultured using Transwell culture plates under different in vitro conditions. The results showed that as the ratio of MSC to DC of the coculture system increased and the coculture time of the two cells prolonged, the concentrations of Gal-1, interleukin- (IL-) 10, and IL-12 in the supernatants were increased and the protein expression of Gal-1 on and within DCs was also enhanced. The phosphorylation of extracellular signal-regulated kinase (ERK) pathway in DCs was boosted, whereas p38 mitogen-activated protein kinase (MAPK) pathway phosphorylation was weakened. Meanwhile, the expression of costimulatory molecules on the surface of DCs was decreased, and the proliferative effect of DCs on allogeneic T cells was also decreased. Therefore, this present study indicated that Gal-1 secreted from MSCs upregulated expression of Gal-1 and stimulated formation of tolerance immunophenotype on DCs, where the underlying mechanism was the regulation of the MAPK signaling pathway in DCs, thereby inhibiting the function of DCs.

Periostin Supports Hematopoietic Stem/Progenitor Cells and Niche-Dependent Myeloblastoma Cells In Vitro

Periostin (POSTN), the fasciclin family extracellular matrix protein also known as osteoblast-specific factor 2 (OSF-2), was previously reported to be required for optimal B lymphopoiesis in vitro. Now, our study first demonstrates the proof that POSTN might be a bona fide niche factor for both normal and malignant myelopoiesis, indicating that it is a niche molecule for hematopoietic stem cells and diverse hematopoietic precursor cells. The Mediator, composed of about 31 subunits, is a master transcriptional coregulator complex that is essential for global transcription governed by RNA polymerase II. Among the Mediator subunits, MED1 acts as a specific coactivator for activators that include nuclear receptors and GATA1. We previously reported that Med1−/− mouse embryonic fibroblasts (MEFs) have a decreased capability to support hematopoietic stem/progenitor cells (HSPCs) relative to wild-type MEFs in vitro, and that the attenuated expression of full-length osteopontin and FGF7 in Med1−/− MEFs is responsible for the observed phenotype. The microarray analyses, showing that the expression of POSTN was also suppressed in Med1−/− MEFs, prompted us to study the role for POSTN in support of both normal and malignant HSPCs in our in vitro niche model. When bone marrow (BM) cells were cocultured with mitomycin C-treated Med1+/+ MEFs, or OP-9 or MS-5 BM stromal cells, in the presence of anti-POSTN blocking antibody, the mitogenicity and growth of BM cells were attenuated. The number of long-term culture-initiating cells (LTC-ICs), i.e., number of both granulo-monocytic and erythroid colonies, was also decreased. When BM cells were cocultured with Med1-/- MEFs in the presence of recombinant POSTN, the mitogenicity and growth of BM cells and the number of LTC-ICs were restored. These results suggest that POSTN mediates mitogenicity of BM cells and HSPCs support. The MB-1 myeloblastoma cell line, originally established from a patient with myeloid crisis chronic myeloid leukemia, is a mesenchymal stromal cell-dependent cell line. These cells are unique in that they grow by forming cobblestone areas in the presence of niche cells but die of apoptosis when detached from stromal cells, thus faithfully conforming to a stochastic model of leukemic stem cells in vitro. Intriguingly, antibody-mediated blockage of stromal cells-derived POSTN markedly reduced the mitogenicity and growth, as well as the cobblestone formation, a leukemic stem cell feature, of MB-1 myeloblastoma cells. Therefore, it appears that niche cell-derived POSTN supports niche-dependent MB-1 myeloblastoma cells. While POSTN was expressed both in BM cells and variably in different BM stromal cells, expression in the latter cells was markedly increased by tactile interaction with hematopoietic cells. Specifically, POSTN was robustly induced 6 hours after BM stromal cells were cocultured with BM cells or MB-1 myeloblastoma cells, and the induction sustained for as long as 24 hours. However, POSTN expression was not enhanced when BM cells were cocultured but physically separated from MS-5 or OP-9 cells using transwell culture wells. Therefore, the major source of POSTN in the coculture appears to be the BM stromal cells associated with hematopoietic cells. The receptor for POSTN, integrin αvβ3, was expressed abundantly in BM stromal cells. Although β3 mRNA was especially prominent in both BM cells and MB-1 cells, in accordance with a previous report that integrin β3/CD61 marks HSPCs, western blot analysis showed that αv and β3 expression levels were below the detection level on BM cells. Hence, integrin αvβ3 is scarce on BM cells compared to BM stromal cells, although it does not exclude the possibility that functional integrin αvβ3 might be enriched on HSPCs as suggested previously. When an excess amount of exogenous POSTN was added to MS-5 or OP-9 BM stromal cells after 24-h serum starvation, FAK (the immediate target of integrin αvβ3) and MAP kinases ERK1/ERK2 (the intermediate hub of various intracellular signals) were robustly phosphorylated as early as 10 min, and the phosphorylation was sustained for over 60 min. Thus, POSTN effectively activates integrin αvβ3 and subsequent intracellular signaling in BM stromal cells. These results suggest that stromal cell POSTN supports both normal HSPCs and leukemia-initiating cells in vitro, at least in part, indirectly by acting on stromal cells in an autocrine or paracrine manner. DisclosuresNo relevant conflicts of interest to declare.

Overexpression of HIF-1α in Mesenchymal Stem Cells Contributes to Repairing Hypoxic-Ischemic Brain Damage in Rats

Introduction: Preclinical researches on the use of mesenchymal stem cells (MSCs) transplantation to treat hypoxic-ischemic (HI) brain damage have received some encouraging results. However, the insufficient migration of active cells to damaged tissues has limited their potential therapeutic effects. There is a good evidence that hypoxia inducible factor-1 alpha (HIF-1α) promotes the viability and migration of cells. This study was designed to investigate whether overexpression of HIF-1α in MSCs could be effective to enhance the therapeutic efficiency.Methods: MSCs were obtained from rat femoral and tibial bone marrow, and cells with HIF-1α overexpression were gained by recombinant lentiviral vector. The proliferation and migration of MSCs were assessed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and transwell culture system. Additionally, the therapeutic efficiency of MSCs after transplantion on HI brain damage in rats were investigated by Morris water maze test and histological examination to analyse the cognitive and pathological changes, and the recruitment in the hippocampus was also observed microscopically.Results: (1) As evidenced by phase contrast and fluorescence microscopy, more than 90% of the cells were GFP-positive when infected with the HIF-1α or GFP lentiviral, and the protein expression of HIF-1α in HIF-1α transduced MSCs was approximately 2-fold that in normal MSCs and GFP transduced MSCs (P<0.01). The results of MTT assay showed that overexpression of HIF-1α promoted the viability and proliferation of MSCs (P<0.01). (2) Meantime, we found MSCs were enhanced the migration both in vitro and in vivo experiments. According to transwell assay, the number of cells migrating to the lower compartment in HIF-1α overexpression group was more than other two groups (P<0.01). In vivo experiments in HI-induced rats, the number of MSCs in the hippocampus increased gradually in a time-dependent manner from day 7 to day 21 after transplantation. And a remarkably increased in the recruitment of HIF-1α transduced MSCs was observed in the hippocampus at every time point compared with GFP transduced MSCs (P<0.01). (3) HIF-1α overexpression also elevated the therapeutic efficiency of MSCs on behavioral and histological aspect in HI-induced brain damaged rats. Morris water maze test suggested that the rats suffered from cognitive dysfunction due to HI treatment. There were a significantly increased of time in the target quadrant in both MSCs transplanted groups compared with HI-vehicle group, but a more increase in the amount of time have been found in HIF-1α overexpressed MSCs group (P<0.01). In contrast with sham group, the pathological changes of the hippocampus in the HI-vehicle group revealed serious injury, and the cells were disordered and cavitation was visible by hematoxylin and eosin staining, but the damages were partly mitigated in HIF-1α overexpressed MSCs transplanted group as compared to other groups.Conclusion: Taken together, these data indicated that overexpression of HIF-1α could improve the therapeutic efficiency of MSCs, and transplantation of MSCs with HIF-1α overexpression may represent an attractive therapeutic option to treat HI brain damage in the future.Footnotes: This work was financially supported by National Natural Science Foundation of China (Gant No. 81270566, Gant No. 81500114). DisclosuresNo relevant conflicts of interest to declare.

Somatostatin signaling via SSTR1 contributes to the quiescence of colon cancer stem cells.

BackgroundNeuroendocrine cells (NECs) reside adjacent to colonic stem cells (SCs) in the crypt stem cell (SC) niche, but how NECs are involved in regulation of SCs is unclear. We investigated NECs expressing somatostatin (SST) and somatostatin receptor type 1 (SSTR1) because SST inhibits intestinal proliferation. Hypothesis: SSTR1 cells maintain SCs in a quiescent state, and aberrant SST signaling contributes to SC overpopulation in colorectal cancer (CRC).MethodsThe proportion of SCs to NECs cells was quantified, by flow cytometry, in CRC cell lines and primary normal/tumor tissues based on cellular ALDH and SSTR1 levels, respectively. Doubling time and sphere-formation was used to evaluate cell proliferation and stemness. CRC cell lines were treated with exogenous SST and SST inhibitor cyclosomatostatin (cycloSST) and analyzed for changes in SCs and growth rate. Paracrine signaling between NECs and SCs was ascertained using transwell cultures of ALDH+ and SSTR1+ cells.ResultsIn CRC cell lines, the proportion of ALDH+ cells inversely correlates with proportion of SSTR1+ cells and with rate of proliferation and sphere-formation. While primary normal tissue shows SST and SSTR1 expression, CRC shows only SSTR1 expression. Moreover, ALDH+ cells did not show SST or SSTR1 expression. Exogenous SST suppressed proliferation but not ALDH+ population size or viability. Inhibition of SSTR1 signaling, via cycloSST treatment, decreased cell proliferation, ALDH+ cell population size and sphere-formation. When co-cultured with SSTR1+ cells, sphere-formation and cell proliferation of ALDH+ cells was inhibited.ConclusionThat each CRC cell line has a unique ALDH+/SSTR1+ ratio which correlates with its growth dynamics, suggests feedback mechanisms exist between SCs and NECs that contribute to regulation of SCs. The growth suppression by both SST and cycloSST treatments suggests that SST signaling modulates this feedback mechanism. The ability of SSTR1+ cells to decrease sphere formation and proliferation of ALDH+ cells in transwell cultures indicates that the ALDH subpopulation is regulated by SSTR1 via a paracrine mechanism. Since ALDH+ cells lack SST and SSTR1 expression, we conjecture that SST signaling controls the rate of NEC maturation as SCs mature along the NEC lineage, which contributes to quiescence of SCs and inhibition of proliferation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2969-7) contains supplementary material, which is available to authorized users.

Mesenchymal stem cells attenuate hydrogen peroxide-induced oxidative stress and enhance neuroprotective effects in retinal ganglion cells

The apoptosis of retinal ganglion cells leads to visual impairment and blindness in ocular neurodegenerative diseases, especially in diabetic retinopathy (DR). Mounting evidence suggests that oxidative stress contributes to the pathogenesis of DR. In the present study, we investigated whether bone mesenchymal stem cells (BMSCs) have protective ability to relieve hydrogen peroxide (H2O2)-induced injury on retinal ganglion cells in vitro. An immortalized retinal ganglion cells, RGC-5 cells, were exposed to anindicated concentration of H2O2 for 24h. Cell viability was analyzed by CCK-8 assay to find out a certain concentration to build H2O2 oxidative damage model. Morphological changes in RGC-5 cells were observed under optical microscope, and cell apoptosis was detected with Hoechst fluorescence staining. Then, BMSCs were co-cultured with RGC-5 cells in a transwell culture system for 24 h and 48h. Flow cytometry was performed to qualify the apoptosis rate of RGC-5 cells. Conditioned medium was collected for evaluation the inflammatory cytokines by ELISA. The content of intracellular malondialdehyde (MDA) and superoxide dismutase (SOD) was assayed by thiobarbituric acid and xanthine oxidase method, respectively. qRT-PCR and ELISA were conducted for analysis of the expression changes in brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), respectively. After H2O2 exposure, the morphological varieties were observed as cytoplasm shrinking and paramorphia together with nuclear gathering. Meanwhile, the apoptotic cells had hyperfluorescence with Hoechst 33258 staining. Co-culture with BMSCs significantly inhibited retinal cell death. It was found that BMSCs reduced H2O2-induced inflammatory factors IL-1β and TNF-α, down-regulated intracellular oxidant factor MDA, up-regulated intracellular antioxidant factor SOD, and increased neurotrophins BDNF and CNTF expression. BMSCs may enhance protective effect of RGC-5 cells in H2O2-induced damage through improving antioxidant capacity, inhibiting pro-inflammatory cytokine secretion, and promoting neurotrophin expression.

Novel approach to study the cardiovascular effects and mechanism of action of urban particulate matter using lung epithelial-endothelial tetra-culture system

In vitro models have become increasingly sophisticated, and their usefulness in supporting toxicity testing is well established. The present study was designed to establish a novel in vitro model that mimics the cellular network surrounding airways and pulmonary blood vessels, to study the cardiovascular toxic effects of particulate matter (PM). Transwell culture method was used to develop a novel tetra-culture system consisting of tri-cultures (one lung epithelial and two immune cell lines) in the apical chamber and endothelial cells in the basolateral chamber. Tri-cultures were exposed to standard reference material (SRM) 1648a, an urban PM. SRM 1648a did not show cytotoxic effects; however, it increased IL-6 level in apical and basolateral chambers. The cells in the basolateral chamber showed increased monocyte adhesion. Furthermore, exposure of tri-cultured cells to SRM 1648a in the apical chamber induced ICAM-1 expression in endothelial cells in the basolateral chamber by activating the IL-6/STAT3 pathway. In conclusion, a tetra-culture system was established to facilitate the identification of cellular adhesion molecule expression induced by the interaction between pulmonary epithelial and endothelial cells. The tetra-culture system will contribute to elucidation of the relationships between inhalable PM and cardiovascular diseases.

Mitochondrial transfer of mesenchymal stem cells effectively protects corneal epithelial cells from mitochondrial damage.

Recent studies have demonstrated that mesenchymal stem cells (MSCs) can donate mitochondria to airway epithelial cells and rescue mitochondrial damage in lung injury. We sought to determine whether MSCs could donate mitochondria and protect against oxidative stress-induced mitochondrial dysfunction in the cornea. Co-culturing of MSCs and corneal epithelial cells (CECs) indicated that the efficiency of mitochondrial transfer from MSCs to CECs was enhanced by Rotenone (Rot)-induced oxidative stress. The efficient mitochondrial transfer was associated with increased formation of tunneling nanotubes (TNTs) between MSCs and CECs, tubular connections that allowed direct intercellular communication. Separation of MSCs and CECs by a transwell culture system revealed no mitochiondrial transfer from MSCs to CECs and mitochondrial function was impaired when CECs were exposed to Rot challenge. CECs with or without mitochondrial transfer from MSCs displayed a distinct survival capacity and mitochondrial oxygen consumption rate. Mechanistically, increased filopodia outgrowth in CECs for TNT formation was associated with oxidative inflammation-activated NFκB/TNFαip2 signaling pathways that could be attenuated by reactive oxygen species scavenger N-acetylcysteine (NAC) treatment. Furthermore, MSCs grown on a decellularized porcine corneal scaffold were transplanted onto an alkali-injured eye in a rabbit model. Enhanced corneal wound healing was evident following healthy MSC scaffold transplantation. And transferred mitochondria was detected in corneal epithelium. In conclusion, mitochondrial transfer from MSCs provides novel protection for the cornea against oxidative stress-induced mitochondrial damage. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.

Ectoderm mesenchymal stem cells promote differentiation and maturation of oligodendrocyte precursor cells

Many neurological diseases are closely associated with demyelination caused by pathological changes of oligodendrocytes. Although intrinsic remyelination occurs after injury, the regeneration efficiency of myelinating oligodendrocytes remains to be improved. Herein, we reported an initiative finding of employing a valuable cell source, namely neural crest-derived ectoderm mesenchymal stem cells (EMSCs), for promoting oligodendrocyte differentiation and maturation by co-culturing oligodendrocyte precursor cells (OPCs) with the EMSCs. The results demonstrated that the OPCs/EMSCs co-culture could remarkably increase the number and length of oligodendrocyte processes in comparison with the mono-cultured OPCs and non-contact OPCs/EMSCs transwell culture. Furthermore, the inhibition experiments revealed that the EMSCs-produced soluble factor Sonic hedgehog, gap junction protein connexin 43 and extracellular matrix molecule laminin accounted for the promoted OPC differentiation since inhibiting the function of anyone of the three proteins led to substantial retraction of processes and detachment of oligodendrocytes. Altogether, OPCs/EMSCs co-culture system could be a paradigmatic approach for promoting differentiation and maturation of oligodendrocytes, and EMSCs will be a promising cell source for the treatment of neurological diseases caused by oligodendrocyte death and demyelination.

Gene expression profiling of CD8+ T cells induced by ovarian cancer cells suggests a possible mechanism for CD8+ Treg cell production

The aim of this study was to investigate a possible mechanism of CD8+ regulatory T-cell (Treg) production in an ovarian cancer (OC) microenvironment. Agilent microarray was used to detect changes in gene expression between CD8+ T cells cultured with and without the SKOV3 ovarian adenocarcinoma cell line. QRT-PCR was performed to determine glycolysis gene expression in CD8+ T cells from a transwell culturing system and OC patients. We also detected protein levels of glycolysis-related genes using Western blot analysis. Comparing gene expression profiles revealed significant differences in expression levels of 1420 genes, of which 246 were up-regulated and 1174 were down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis indicated that biological processes altered in CD8+ Treg are particularly associated with energy metabolism. CD8+ Treg cells induced by co-culture with SKOV3 had lower glycolysis gene expression compared to CD8+ T cells cultured alone. Glycolysis gene expression was also decreased in the CD8+ T cells of OC patients. These findings provide a comprehensive bioinformatics analysis of DEGs in CD8+ T cells cultured with and without SKOV3 and suggests that metabolic processes may be a possible mechanism for CD8+ Treg induction.

Geometric correction factor for transepithelial electrical resistance measurements in transwell and microfluidic cell cultures

Transepithelial electrical resistance (TEER) measurements are regularly used in in vitro models to quantitatively evaluate the cell barrier function. Although it would be expected that TEER values obtained with the same cell type and experimental setup were comparable, values reported in the literature show a large dispersion for unclear reasons. This work highlights a possible error in a widely used formula to calculate the TEER, in which it may be erroneously assumed that the entire cell culture area contributes equally to the measurement. In this study, we have numerically calculated this error in some cell cultures previously reported. In particular, we evidence that some TEER measurements resulted in errors when measuring low TEERs, especially when using Transwell inserts 12 mm in diameter or microfluidic systems that have small chamber heights. To correct this error, we propose the use of a geometric correction factor (GCF) for calculating the TEER. In addition, we describe a simple method to determine the GCF of a particular measurement system, so that it can be applied retrospectively. We have also experimentally validated an interdigitated electrodes (IDE) configuration where the entire cell culture area contributes equally to the measurement, and it also implements minimal electrode coverage so that the cells can be visualized alongside TEER analysis.

Downregulation of nucleophosmin expression inhibited proliferation and induced apoptosis in salivary gland adenoid cystic carcinoma.

This study aimed to explore the relationship between nucleophosmin (NPM1) and patient clinical characteristics. Moreover, we investigated the effect of NPM1 in tumor proliferation and apoptosis of salivary gland adenoid cystic carcinoma (SACC). NPM1 expression was examined in 74 specimens of SACC and 31 non-cancerous epithelium adjacent to carcinoma (NCEAC) by immunohistochemistry (IHC). RNA interference technology was used to silence NPM1 expression in SACC cells. We used transwell culture assay, cell counting kit-8 tests, and colony formation assay to test the proliferation, cisplatin resistance, migration, and invasiveness of SACC cells. The nuclear and cytoplasmic expression of NPM1 in SACC tissue was overexpressed and was tightly linked to perineural invasion and lymph node metastasis. The downregulation of NPM1 inhibited proliferation and induced apoptosis in SACC cells. Knockdown of NPM1 expression had no effect on chemoresistance migration, or invasiveness. NPM1 may play an important role in tumor progress in SACC and is a potential biomarker for SACC.

In vitro response of macrophages to ceramic scaffolds used for bone regeneration.

Macrophages, the primary cells of the inflammatory response, are major regulators of healing, and mediate both bone fracture healing and the inflammatory response to implanted biomaterials. However, their phenotypic contributions to biomaterial-mediated bone repair are incompletely understood. Therefore, we used gene expression and protein secretion analysis to investigate the interactions in vitro between primary human monocyte-derived macrophages and ceramic scaffolds that have been shown to have varying degrees of success in promoting bone regeneration in vivo Specifically, baghdadite (Ca3ZrSi2O9) and strontium-hardystonite-gahnite (Sr-Ca2ZnSi2O7-ZnAl2O4) scaffolds were chosen as two materials that enhanced bone regeneration in vivo in large defects under load compared with clinically used tricalcium phosphate-hydroxyapatite (TCP-HA). Principal component analysis revealed that the scaffolds differentially regulated macrophage phenotype. Temporal changes in gene expression included shifts in markers of pro-inflammatory M1, anti-inflammatory M2a and pro-remodelling M2c macrophage phenotypes. Of note, TCP-HA scaffolds promoted upregulation of many M1-related genes and downregulation of many M2a- and M2c-related genes. Effects of the scaffolds on macrophages were attributed primarily to direct cell-scaffold interactions because of only minor changes observed in transwell culture. Ultimately, elucidating macrophage-biomaterial interactions will facilitate the design of immunomodulatory biomaterials for bone repair.

Microfluidics meets metabolomics to reveal the impact of Campylobacter jejuni infection on biochemical pathways.

Microfluidic devices that are currently being used in pharmaceutical research also have a significant potential for utilization in investigating exposure to infectious agents. We have established a microfluidic device cultured with Caco-2 cells, and utilized metabolomics to investigate the biochemical responses to the bacterial pathogen Campylobacter jejuni. In the microfluidic devices, Caco-2 cells polarize at day 5, are uniform, have defined brush borders and tight junctions, and form a mucus layer. Metabolomics analysis of cell culture media collected from both Caco-2 cell culture systems demonstrated a more metabolic homogenous biochemical profile in the media collected from microfluidic devices, compared with media collected from transwells. GeneGo pathway mapping indicated that aminoacyl-tRNA biosynthesis was perturbed by fluid flow, suggesting that fluid dynamics and shear stress impacts the cells translational quality control. Both microfluidic device and transwell culturing systems were used to investigate the impact of Campylobacter jejuni infection on biochemical processes. Caco-2 cells cultured in either system were infected at day 5 with C. jejuni 81-176 for 48h. Metabolomics analysis clearly differentiated C. jejuni 81-176 infected and non-infected medias collected from the microfluidic devices, and demonstrated that C. jejuni 81-176 infection in microfluidic devices impacts branched-chain amino acid metabolism, glycolysis, and gluconeogenesis. In contrast, no distinction was seen in the biochemical profiles of infected versus non-infected media collected from cells cultured in transwells. Microfluidic culturing conditions demonstrated a more metabolically homogenous cell population, and present the opportunity for studying host-pathogen interactions for extended periods of time.

Inhibition of the Rac1-WAVE2-Arp2/3 signaling pathway promotes radiosensitivity via downregulation of cofilin-1 in U251 human glioma cells.

The Ras-related C3 botulinum toxin substrate1 (Rac1)-WASP-family verprolin-homologous protein-2 (WAVE2)-actin-related protein 2/3 (Arp2/3) signaling pathway has been identified to be involved in cell migration and invasion in various types of cancer cell. Cofilin‑1 (CFL‑1), which is regulated by the Rac1‑WAVE2‑Arp2/3 signaling pathway, may promote radioresistance in glioma. Therefore, the present study aimed to investigate the potential role of the Rac1‑WAVE2‑Arp2/3 signaling pathway in radioresistance in U251 human glioma cells and elucidate its affect on CFL‑1 expression. Western blot analysis was performed to evaluate the protein expression of CFL‑1. In the present study, Rac1 was inhibited by NSC23766, WAVE2 was inhibited by transfection with short hairpin (sh)RNA‑WAVE2 using Lipofectamine™2000 and Arp2/3 was inhibited by CK‑666. Cell viability was measured using the 3‑(4,5‑dimethylthiazol‑2‑yl)-2,5‑diphenyltetrazolium bromide assay, the cell migration ability was examined by a wound‑healing assay, and the cell invasion ability was assessed using a Transwell culture chamber system. The results showed that inhibition of the Rac1‑WAVE2‑Arp2/3 signaling pathway using NSC23766, shRNA‑WAVE2 or CK‑666 reduced the cell viability, migration and invasion abilities in U251 human glioma cells, concordant with a reduced expression of CFL‑1. Furthermore, the expression of CFL‑1 was significantly increased in radioresistant U251 glioma cells when compared with normal U251 human glioma cells. These findings indicate that inhibition of the Rac1‑WAVE2‑Arp2/3 signaling pathway may promote radiosensitivity, which may partially result from the downregulation of CFL‑1 in U251 human glioma cells.

PD38-10 BLADDER CANCER EXOSOMES FROM HIGH-GRADE MUSCLE INVASIVE BLADDER CANCER CONTAIN LONG NON-CODING RNA AND MESSENGER RNA

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2016PD38-10 BLADDER CANCER EXOSOMES FROM HIGH-GRADE MUSCLE INVASIVE BLADDER CANCER CONTAIN LONG NON-CODING RNA AND MESSENGER RNA Claudia Berrondo, Jonathan Flax, Victor Kucherov, Aisha Siebert, Thomas Osinski, Alex Rosenberg, Christopher Fucile, and Carla Beckham Claudia BerrondoClaudia Berrondo More articles by this author , Jonathan FlaxJonathan Flax More articles by this author , Victor KucherovVictor Kucherov More articles by this author , Aisha SiebertAisha Siebert More articles by this author , Thomas OsinskiThomas Osinski More articles by this author , Alex RosenbergAlex Rosenberg More articles by this author , Christopher FucileChristopher Fucile More articles by this author , and Carla BeckhamCarla Beckham More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1490AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Many long non-coding RNAs (lncRNA) play critical roles in tumor biology. The lncRNA HOX Antisense Intergenic RNA (HOTAIR) is overexpressed in many solid tumors and regulates genes involved in epithelial-to-mesenchyme transition (EMT). There is a need to incorporate lncRNA into biomarker and therapeutic target discovery algorithms. Exosomes may be a good source for biomarkers for UBC because they can be isolated from urine and their contents are protected from degradation. To determine the role of HOTAIR in UBC progression and to identify novel mRNA and lncRNA in urinary exosomes of patients with high-grade muscle invasive (HGMI) UBC. METHODS HOTAIR was knocked down using lentiviral shRNA. Migration was measured with a wound-scratch assay. Invasion was measured with trans-well assay and 3D culture system. Tumor, distal normal tissue and urine from patients with HGMI UBC and healthy volunteers (HVs) was obtained with IRB approval. mRNA and lncRNA levels were measured by qRT-PCR. Exosomes were isolated by ultracentrifugation and confirmed with electron microscopy, nanoparticle tracing and Western blot. RNA sequencing was completed using Illumina HiSeq2500 platform with 20,000,000 pair-ended reads and 125 base pair length. RESULTS UBC cell lines express elevated levels of tumor-associated mRNA and lncRNA (including HOTAIR). The knockdown of HOTAIR in UBC cell lines reduces migration and invasion and correlates with the loss of expression of EMT factors. Tumor from patients with HGMI UBC express elevated levels of tumor-associated mRNA and lncRNA (including HOTAIR). Exosomes from UBC cell lines and urinary exosomes from patients with HGMI UBC have elevated levels of tumor-associated mRNA and lncRNA (including HOTAIR). RNA sequencing revealed elevated levels of 22 novel mRNA and 4 novel lncRNA in the urinary exosomes of patients with HGMI UBC compared to HVs. qRT-PCR confirmed the differential enrichment of mRNA PDX1, TEDDM1, ZBTB4 and lncRNA HYMA1, LOC100506688 and OXT2-AS1. CONCLUSIONS UBC cell lines, tumors from patients with HGMI UBC and the exosomes they produce have elevated expression of tumor-associated mRNA and lncRNA (including HOTAIR). HOTAIR is overexpressed in UBC cell lines and UBC patient tumors and is important in UBC progression in vitro through its effects on EMT factor expression, cell migration and invasion. Urinary exosomes from patients with HGMI UBC contain elevated levels of novel mRNA and lncRNA. Taken together, these data suggest that urinary exosomes may contain mRNA and lncRNA that can serve as biomarkers for UBC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e928 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Claudia Berrondo More articles by this author Jonathan Flax More articles by this author Victor Kucherov More articles by this author Aisha Siebert More articles by this author Thomas Osinski More articles by this author Alex Rosenberg More articles by this author Christopher Fucile More articles by this author Carla Beckham More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of mesenchymal stem cell-derived cytokines on the functional properties of endothelial progenitor cells

Human mesenchymal stem cell (hMSC) is a potential source for cell therapy due to its property to promote tissue repair. Although, it has been known that hMSCs promote tissue repair via angiogenic cytokines, the interaction between hMSC-derived cytokines and the endothelial progenitor cells (EPCs), which play an important role in tissue neovascularization, is poorly characterized. We investigate the effect of cytokine released from different sources of hMSCs including bone marrow and gestational tissues on the EPC functions in vitro. The migration, extracellular matrix invasion and vessel formation of EPCs were studied in the presence or absence of cytokines released from various sources of hMSCs using transwell culture system. The migration of EPCs was highest when co-culture with secretory factors from placenta-derived hMSCs (PL-hMSCs) compared to those co-culture with other sources of hMSCs. For invasion and vessel formation, secretory factors from bone marrow-derived hMSCs (BM-hMSCs) could produce the maximal enhancement compared to other sources. We further identified the secreted cytokines and found that the migratory-enhancing cytokine from PL-hMSCs was PDGF-BB while the enhancing cytokine from BM-hMSCs on invasion was IGF-1. For vessel formation, the cytokines released from BM-hMSCs were IGF1 and SDF-1. In conclusion, hMSCs can release angiogenic cytokines which increase the migration, invasion and vessel forming capacity of EPCs. We can then use hMSCs as a source of angiogenic cytokines to induce neovascularization in injured/ischemic tissues.