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Dedifferentiation of vascular smooth muscle cells (VSMCs) from a functional phenotype to an inverse synthetic phenotype is a symptom of cardiovascular disorders, such as atherosclerosis and hypertension. The sympathetic nervous system (SNS) is an essential regulator of the differentiation of vascular smooth muscle cells (VSMCs). In addition, numerous studies suggest that SNS also stimulates VSMCs to retain their contractile phenotype. However, the molecular mechanisms for this stimulation have not been thoroughly studied. In this study, we used a novel in vitro co-culture method to evaluate the effective cellular interactions and stimulatory effects of sympathetic neurons on the differentiation of VSMCs. We co-cultured rat neural-like pheochromocytoma cells (PC12) and rat aortic VSMCs with this method. Expression of VSMCs contractile genes, including smooth muscle actin (acta2), myosin heavy chain (myh11), elastin (eln), and smoothelin (smtn), were determined by quantitative real-time-PCR analysis as an indicator of VSMCs differentiation. Fold changes for specific contractile genes in VSMCs grown in vitro for seven days in the presence (innervated) and absence (non-innervated) of sympathetic neurons were 3.5 for acta2, 6.5 for myh11, 4.19 for eln, and 4 for smtn (normalized to Tata Binding Protein (TBP)). As a result, these data suggest that sympathetic innervation promotes VSMCs' contractile gene expression and also maintains VSMCs' functional phenotype.

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Co-culturing is an essential method for unravelling the importance of cross talk and cellular interaction. This chapter describes the preparation of an indirect co-culture technique based on encapsulation of chondrocytes and mesenchymal stromal cells in polyurethane scaffolds and alginate beads, respectively. This way, both cell populations can communicate through paracrine effects in the absence of cell-cell contact. Due to the mechanical properties of polyurethane, this model can be employed in mechanobiology studies. The resulting engineered cultures can provide a more realistic environment, recreating the complex joints' microenvironment and physiology.

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Currently, the number of stem-cell based experimental therapies in neurological injuries and neurodegenerative disorders has been massively increasing. Despite the fact that we still have not obtained strong evidence of mesenchymal stem/stromal cells' neurogenic effectiveness in vivo, research may need to focus on more appropriate sources that result in more therapeutically promising cell populations. In this study, we used dedifferentiated fat cells (DFAT) that are proven to demonstrate more pluripotent abilities in comparison with standard adipose stromal cells (ASCs). We used the ceiling culture method to establish DFAT cells and to optimize culture conditions with the use of a physioxic environment (5% O2). We also performed neural differentiation tests and assessed the neurogenic and neuroprotective capability of both DFAT cells and ASCs. Our results show that DFAT cells may have a better ability to differentiate into oligodendrocytes, astrocytes, and neuron-like cells, both in culture supplemented with N21 and in co-culture with oxygen-glucose-deprived (OGD) hippocampal organotypic slice culture (OHC) in comparison with ASCs. Results also show that DFAT cells have a different secretory profile than ASCs after contact with injured tissue. In conclusion, DFAT cells constitute a distinct subpopulation and may be an alternative source in cell therapy for the treatment of nervous system disorders.

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OBJECTIVE: To investigate the changes in the proliferation and migration ability of bone marrow mesenchymal stem cells (BMSCs) after indirect co-culturing with glioma C6 cells, and to examine the role of plasmacytoma variant translocation 1 gene ( PVT1), a long non-coding RNA (lncRNA), in these changes. METHODS: After separation, cultivation and identification of BMSCs, BMSCs of good growth condition were picked out and indirectly co-cultured with glioma C6 cells in Transwell chambers. These cells are henceforth referred to as the co-culture group. Normal BMSCs cultured separately were the control group. CCK-8 and soft agar colony formation assay were used to examine the proliferation ability of the two groups of cells. Flow cytometry was used to examine the cell cycle. Wound healing assay and Transwell assay were used to explore the migration ability of the cells. Quantitative real-time PCR (qRT-PCR) was used to examine the genetic expression level of PVT1 in the two groups. The above-mentioned tests were repeated after the co-cultured BMSCs were transfected with si- PVT1 (si- PVT1 group) and si-NC (si-NC group). In addition, qRT-PCR was done to evaluate the expression of CyclinD1, a cell cycle protein gene, and matrix metalloproteinases 2 and 9 ( MMP2 and MMP9), the migration-related genes in the si- PVT1 and si-NC transfected co-cultured BMSCs. RESULTS: The BMSCs used in the present study possess the capability of osteogeneic and adipogenic differentiation. Compared with the control group, the co-cultured BMSCs had smaller size, disorderly arrangement and the lack of intercellular contact inhibition. The proliferation and migration ability was significantly enhanced, the proportions of S and G 2 phase cells greatly increased and the expression level of PVT1 was significantly up-regulated ( P<0.05) in the co-cultured group in comparison with those of the control group. When compared with the si-NC group, the si- PVT1 group showed inhibited proliferation and migration ability of the co-cultured BMSCs; the percentage of G 1 phase cells increased, while that of S phase decreased; the expression of PVT1, CyclinD1, MMP2 and MMP9 mRNA also decreased ( P<0.05) in the si- PVT1 group. CONCLUSION: The enhanced proliferation and migration ability of BMSCs in the glioma C6 microenvironment may be associated with the up-regulated expression of PVT1 .

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INTRODUCTION: The present in vitro study was undertaken to learn about the effects of leukocytes on tenocytes in respect to complement regulation simulating an inflammatory scenario of the traumatized tissue. METHODS: Human hamstring tendon-derived tenocyte monolayers were co-cultured indirectly with human leukocytes (either Peripheral Blood Mononuclear Cells [PBMCs] or neutrophils) using a transwell system with/without (+ /wo) 10 ng/ml tumor necrosis factor α (TNFα) for 4 and 24 h. Tenocyte and leukocyte cell survival was assessed by live-dead assay. Tenocyte gene expression of TNFα, the anaphylatoxin receptor C5aR and the cytoprotective complement regulatory proteins (CRP) CD46, CD55 and CD59 was monitored using qPCR. TNFα was detected in the culture supernatants using ELISA. RESULTS: C5aR gene expression was significantly induced by TNFα after 4 h, but impaired in the presence of leukocytes + TNFα after 24 h. At 4 h, PBMCs activated by TNFα induced the CRP CD46 gene expression. However, CD55 was significantly suppressed after 24 h by neutrophils + /woTNFα. Leukocytes activated by TNFα decreased also significantly the gene expression of the more downstream acting CRP CD59 after 4 h. TNFα gene expression and ELISA analysis revealed an amplified TNFα expression/release in tenocyte co-cultures with PBMC + /woTNFα, probably contributing to complement regulation. CONCLUSION: TNFα might represent a crucial soluble mediator exerting diverse time-dependent effects on tenocyte complement regulation.

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This study aims to investigate the effect of substances secreted or metabolized by vascular endothelial cells on epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma cells under indirect co-culture condition. Human hepatocellular carcinoma cell line QGY-7703 was cultured in vitro, and then was co-cultured with conditioned medium of human umbilical vein endothelial cells (HUVEC). The morphological changes of QGY-7703 cells were observed by inverted phase contrast microscopy. The migration ability of QGY-7703 cells was analyzed by scratch-wound assays. The effect of conditioned medium on the expression and distribution of EMT related proteins was detected by Western blot and immunofluorescence assays, respectively. The results showed that the QGY-7703 cells gradually changed from polygonal to spindle shape, the migration ability promoted significantly, and both the expression and distribution of EMT related marker changed in a time-dependent manner after co-culturing. The results confirm that vascular endothelial cells can induce EMT in hepatocellular carcinoma cells under indirect co-culture condition.

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This study aims to investigate the effect of substances secreted or metabolized by vascular endothelial cells on epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma cells under indirect co-culture condition. Human hepatocellular carcinoma cell line QGY-7703 was cultured , and then was co-cultured with conditioned medium of human umbilical vein endothelial cells (HUVEC). The morphological changes of QGY-7703 cells were observed by inverted phase contrast microscopy. The migration ability of QGY-7703 cells was analyzed by scratch-wound assays. The effect of conditioned medium on the expression and distribution of EMT related proteins was detected by Western blot and immunofluorescence assays, respectively. The results showed that the QGY-7703 cells gradually changed from polygonal to spindle shape, the migration ability promoted significantly, and both the expression and distribution of EMT related marker changed in a time-dependent manner after co-culturing. The results confirm that vascular endothelial cells can induce EMT in hepatocellular carcinoma cells under indirect co-culture condition.

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OBJECTIVE: Stem cell therapy, specifically, pre-induction of mesenchymal stem cells toward male germ-like cells may be useful in patients with azoospermia. The aim of this study was to evaluate in vitro differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs) into male germ-like cells by indirect co-culture with testicular cells in the presence of bone morphogenetic protein 4 (BMP4). METHODS: Experimental groups included: control (mouse BMSCs), treatment group-1 (BMSCs treated with BMP4), treatment group-2 (indirect co-culture of BMSCs with mouse testicular cells in the presence of BMP4) and treatment group-3 (indirect co-culture of BMSCs with testicular cells). BMSCs-derived male germ-like cells were evaluated by the expression of Dazl, and Stra8 using RT-qPCR. RESULTS: Stra8 gene expression was significantly increased in the treatment group-2 and Dazl gene was significantly increased in the treatment group-1 compared to other groups. In conclusion, indirect co-culturing of BMSCs with testicular cells and BMP4 leads to the differentiation of BMSCs into male germ-like cells which express specific male germ-like genes. Testicular cells released factors that contributed to the differentiation of BMSCs into male germ progenitor cells. CONCLUSION: This study suggests that mesenchymal stem cells may be differentiated into male germ-like cells and therefore, may be a novel treatment option for men with azoospermia.

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The interaction of peripheral nerves with different cells of the skin is a relevant aspect of many physiological processes including nociception, temperature control, and wound healing. Here we describe a protocol for the setup of an indirect co-culture system of peripheral nerve cells and sweat gland-derived stem cells, which can be used to quantify neurite outgrowth.

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Mesenchymal stem cells (MSCs) hold great promise as attractive vehicles to deliver therapeutic agents against cancer, while the cross-talk between MSCs and cancer cells remains controversial. Here in an indirect co-culture system we observed that MSCs induced the malignancy transformation of low malignancy cancer cells HT29 and MCF7, whereas MSCs were reprogrammed by high malignancy cancer cells HCT116 and MDA-MB-231 without exerting an obvious influence on them. We further demonstrated that the RNA-binding protein polypyrimidine tract-binding protein 1 (PTBP1) was suppressed in low malignancy cancer cells co-cultured with MSCs. Moreover, shRNA mediated silencing of PTBP1 could promote the invasiveness of HT29 cells while over-expression of PTBP1 attenuate the MSC-induced invasion of HT29 cells. Our results suggested that differential effects of MSCs on the invasion of cancer cells partially corresponded to PTBP1 expression in cancer cells and the maintenance of biological characteristics in MSCs, which insight could provide a theoretical basis for evaluating the safety of MSC application and PTBP1 targeting in cancer treatment.

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In biological systems, extracellular vesicles including exosomes have recently been revealed to play a significant role in the communication between various cells, and the number of papers on this subject has dramatically increased. In current conventional exosome studies, the standard research method is to use liquid biopsies to analyze extracts of various disease exosomes. However, exosomes are only one of many key players in natural cellular interactions. Reproducing the phenomena occurring in vivo and investigating the interactions are required in order to examine their role fully. For exosome research, an alternative to the liquid biopsy method for observing natural interactions is the co-culturing technique. It does not require an exosome extraction procedure, and while the technique has been used in many studies thus far, its application to exosome research has been limited. However, the use of co-culturing technologies is necessary to examine the essential interactions of exosomes. An overview of exosome research methodologies and co-culturing systems is thus provided here.

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Fibroblasts express androgen receptor (AR) in the normal prostate and during prostate cancer development. We have reported that loss of AR expression in prostate cancer-associated fibroblasts is a poor prognostic indicator. Here we report outcomes of direct and indirect co-cultures of immortalised AR-positive (PShTert-AR) or AR-negative (PShTert) myofibroblasts with prostate cancer cells. In the initial co-cultures the AR-negative PC3 cell line was used so AR expression and signalling were restricted to the myofibroblasts. In both direct and indirect co-culture with PShTert-AR myofibroblasts, paracrine signalling to the PC3 cells slowed proliferation and induced apoptosis. In contrast, PC3 cells proliferated with PShTert myofibroblasts irrespective of the co-culture method. In direct co-culture PC3 cells induced apoptosis in and destroyed PShTerts by direct signalling. Similar results were seen in direct co-cultures with AR-negative DU145 and AR-positive LNCaP and C4-2B prostate cancer cell lines. The AR ligand 5α-dihydrotestosterone (DHT) inhibited the proliferation of the PShTert-AR myofibroblasts, thereby reducing the extent of their inhibitory effect on cancer cell growth. These results suggest loss of stromal AR would favour prostate cancer cell growth in vivo, providing an explanation for the clinical observation that reduced stromal AR is associated with a poorer outcome.

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The therapy of patients suffering the psychiatric disorder schizophrenia requires the usage of antipsychotic drugs that are classified into two different groups, the first-generation (FGAs) and the second-generation antipsychotics (SGAs). This study compares the effects of the two FGAs haloperidol and flupentixol with those of the SGA olanzapine on synapse formation and synaptic activity of embryonic rat hippocampal neurons. To this end, the development of perineuronal nets (PNNs), the formation of synapses and the resulting spontaneous network activity under control and treatment conditions were studied using an indirect co-culture system of neurons and astrocytes in completely defined media. The number and extent of PNNs that consist of extracellular matrix superstructures surrounding synapses was not altered in hippocampal neurons by exposure to antipsychotic drugs. In contrast treatment of hippocampal neurons with haloperidol led to a slight decrease whereas olanzapine induced a significant increase of the number of structural synapses after 13days. This differential effect concerning synapse numbers was also reflected in the spontaneous activity of neuronal networks, as monitored on multielectrode arrays (MEAs). In that context, application of haloperidol reduced while olanzapine significantly enhanced network activity. Unexpectedly, flupentixol that is regarded as an FGA caused similar effects than the SGA olanzapine in that it augmented synapse number as well as network activity. Our pilot study provides a proof of concept that the neuron-astrocyte co-culture model can be used to investigate the impact of antipsychotics on pivotal parameters of neuronal cell biology. Thereby, it may support the comparative analysis of antipsychotics applied in the therapy of schizophrenia.

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BACKGROUND AND AIM: Cerebral ischemia is characterized by both acute and delayed neuronal injuries. Neuro-protection is a major issue that should be properly addressed from a pharmacological point of view, and cell-based treatment approaches are of interest due to their potential pleiotropic effects. Endothelial progenitor cells have the advantage of being mobilized from the bone marrow into the circulation, but have been less studied than other stem cells, such as mesenchymal stem cells. Therefore, the comparison between human endothelial progenitor cells (hEPC) and human mesenchymal progenitor cells (hMSC) in terms of efficacy in rescuing neurons from cell death after transitory ischemia is the aim of the current study, in the effort to address further directions. MATERIALS AND METHODS: In vitro model of oxygen-glucose deprivation (OGD) on a primary culture of rodent cortical neurons was set up with different durations of exposure: 1, 2 and 3hrs with assessment of neuron survival. The 2hrs OGD was chosen for the subsequent experiments. After 2hrs OGD neurons were either placed in indirect co-culture with hMSC or hEPC or cultured in hMSC or hEPC conditioned medium and cell viability was evaluated by MTT assay. RESULTS: At day 2 after 2hrs OGD exposure, mean neuronal survival was 47.9±24.2%. In contrast, after treatment with hEPC and hMSC indirect co-culture was 74.1±27.3%; and 69.4±18.8%, respectively. In contrast, treatment with conditioned medium did not provide any advantage in terms of survival to OGD neurons CONCLUSION: The study shows the efficacy of hEPC in indirect co-culture to rescue neurons from cell death after OGD, comparable to that of hMSC. hEPC deserve further studies given their potential interest for ischemia.

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The aim of the present study was to investigate the effect of small molecules: Reversine and 5-azacytidine (5-AC), in an indirect co-culture condition with the cardiac fibroblasts as well as non co-culture condition, in order to explore the effect of such molecules in the process of differentiation of the ovine bone-marrow mesenchymal stem cells (BM-MSCs) towards cardiomyocytes. Surface antigens of the isolated cells were analysed using flow-cytometry. In addition, following to three passages cells were examined for their differentiation capacity into osteocytes and adipose cells, in order to ensure the mesenchymal origin of the stem cells. Six types of treatments were carried out in the present investigation, such that, in the first treatment BM-MSCs were cultured for 28 days as control group; the second treatment was composed of culturing ovine fetal cardiac fibroblasts on inserts, aiming to use these inserts for culturing plates which were seeded with BM-MSCs (Chamber group). As the third treatment, BM-MSCs were supplemented with 10-µM 5-AC and incubated for 48 h. The fourth treatment was composed of supplementing BM-MSCs with the 600-nM reversine, incubated for 48 h, and subsequently the incubation was further extended for another 48 h in the presence of 5-AC. The fifth treatment was composed of supplementing the chamber group with 10-µM 5-AC and incubation for 48 h, and the last or the sixth treatment was such that chamber group was supplemented with 600-nM reversine and an incubation period of 48 h. Following to the incubation, medium was replaced with 10-µM 5-AC and further incubated for another round of 48 h. In all treatments, following to addition of the small molecules incubations were carried out for 28 days; same as controls. Expression of cardiac alpha-actinin was analysed by immunocytochemistry. BM-MSCs have shown to express CD44 and CD166 along with a weak expression of the CD90, CD34, in addition to CD45. Multilineage differentiation has indicated that BM-MSCs could differentiate into adipose and osteocytes cells as well. In the treatment 4 it was observed that FGF signalling involved genes and all cardiac-related genes (ANP, MYH6 and Troponin I) were significantly expressed, except connexin 43 compared to other treatments. All treatments received small molecules, either alone or as a co-culture were seen to express sarcomeric alpha-actinin. This finding was partially supported by immunocytochemistry. These results validate that reversine and 5-AC have an effect on ovine BM-MSC differentiation into cardiomyocytes. Copyright © 2016 John Wiley & Sons, Ltd.

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Oral epithelial-mesenchymal interactions play a key role in tooth development and assist differentiation of dental pulp. Many epithelial and mesenchymal factors in the microenvironment influence dental pulp stem cells to differentiate and regenerate. To investigate the interaction between oral cells during differentiation, we designed a microfluidic device system for indirect co-culture. The system has several advantages, such as consumption of low reagent volume, high-throughput treatment of reagents, and faster mineralization analysis. In this study, stem cells from human exfoliated deciduous teeth were treated with media cultured with human gingival fibroblasts or periodontal ligament stem cells. When human exfoliated deciduous teeth was incubated in media cultured in human gingival fibroblasts and human periodontal ligament stem cells under the concentration gradient constructed by the microfluidic system, no remarkable change in human exfoliated deciduous teeth mineralization efficiency was detected. However, osteoblast gene expression levels in human exfoliated deciduous teeth incubated with human gingival fibroblasts media decreased compared to those in human exfoliated deciduous teeth treated with human periodontal ligament stem cells media, suggesting that indirect co-culture of human exfoliated deciduous with human gingival fibroblasts may inhibit osteogenic cytodifferentiation. This microfluidic culture device allows a co-culture system set-up for sequential treatment with co-culture media and differentiation additives and facilitated the mineralization assay in a micro-culture scale.

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Oral epithelial-mesenchymal interactions play a key role in tooth development and assist differentiation of dental pulp. Many epithelial and mesenchymal factors in the microenvironment influence dental pulp stem cells to differentiate and regenerate. To investigate the interaction between oral cells during differentiation, we designed a microfluidic device system for indirect co-culture. The system has several advantages, such as consumption of low reagent volume, high-throughput treatment of reagents, and faster mineralization analysis. In this study, stem cells from human exfoliated deciduous teeth were treated with media cultured with human gingival fibroblasts or periodontal ligament stem cells. When human exfoliated deciduous teeth was incubated in media cultured in human gingival fibroblasts and human periodontal ligament stem cells under the concentration gradient constructed by the microfluidic system, no remarkable change in human exfoliated deciduous teeth mineralization efficiency was detected. However, osteoblast gene expression levels in human exfoliated deciduous teeth incubated with human gingival fibroblasts media decreased compared to those in human exfoliated deciduous teeth treated with human periodontal ligament stem cells media, suggesting that indirect co-culture of human exfoliated deciduous with human gingival fibroblasts may inhibit osteogenic cytodifferentiation. This microfluidic culture device allows a co-culture system set-up for sequential treatment with co-culture media and differentiation additives and facilitated the mineralization assay in a micro-culture scale.

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ABSTRACT:Objective To investigate the effects of phenytoin (PHT)on the secretion of vascular endothelial growth factor (VEGF)and stem cell factor (SCF)based on the establishment of indirect co-culture system of rat bone marrow mesenchymal stem cells (BMSCs)and vascular endothelial cells (VECs).Methods Indirect co-culture model of rat BMSCs and VECs was established.Experimental groups:indirect co-culture groups (PHT concentrations were 0,20 and 40 μg/mL);the control group:BMSCs culture group and VECs culture group (PHT concentrations were 0,20 and 40μg/mL).The contents of VEGF and SCF in the culture supernatant were measured using double antibody sandwich ABC-ELISA method on cultivation days 2,4,6.Results ELISA assay of the rBMSCs and rVECs in indirect co-culture supernatants,collected on culture days 2,4 and 6 showed that:① VEGF:On culture day 2,VEGF level in the co-culture groups was significantly higher than those in BMSCs group (P SCF increased as the incubation time increased;but as the incubation time increased, PHT concentration of 40 μg/mL made SCF content decrease.Each group did not significantly differ (P > 0.05 ).Conclusion PHT promotes the secretion of VEGF and may reduce the secretion of SCF.

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Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use.

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The metastatic potential of non-small cell lung cancer (NSCLC) has been shown to be associated with interactions with the tumor microenvironment, which primarily comprises of cancer-associated fibroblasts (CAFs). Heterotypic cell-cell interactions occur via released signaling molecules and direct physical contact. To investigate the differential contribution of direct cell-cell contact and paracrine signaling factors to NSCLC metastasis, we performed two types of co-cultures: direct co-cultures of the NSCLC cell line H358 with primary cultures of CAFs from patients with resected NSCLC; and indirect co-cultures across a separable membrane. We showed that CAFs more potently induce epithelial-to-mesenchymal transition (EMT) in NSCLC H358 cells through direct contacts than through indirect interactions, as indicated by an elongated and disseminated appearance. Immunocytochemical experiments show that EMT accompanies the expression of mesenchymal cytoskeletal proteins, including vimentin. However, H358 cells proliferate more slowly in direct co-culture than in indirect co-culture. Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed that H358 cells in direct contact with CAFs up-regulate the expression of the pan-mesenchymal markers α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), transforming growth factor-ß (TGFß) signaling effector SMAD family number-3 (SMAD3), and hedgehog signaling effector GLI family zinc finger-1 (GLI1), compared with the indirect co-culture system. Furthermore, we found that the direct GLI1 transcription targets snail family zinc finger-1 (SNAI1) and SNAI2 are up-regulated, suggesting that the hedgehog signaling pathway is active in direct co-culture. A scratch wound assay showed that direct contact co-culture increases the motility of H358 cells. In conclusion, these findings provide evidence that paracrine factors and direct physical contact between NSCLC cells and CAFs might control the metastatic potential of NSCLC through the hedgehog signaling pathway.

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