Project description:To facilitate the research on the interaction between mammary basal cells and M2-like macrophages, we established a co-culture system of mammary basal cells and M2-like macrophages in vitro, and performed microarray of M2-like macrophage co-cultured basal cells comparing with control, for investigating the changes of downstream signals after co-culturing with M2-like macrophages in the basal cells.
Project description:Single end 3' RNA sequencing of GFP-transduced germ cell tumor cell lines (TCam-2, 2102EP, JAR, GCT72) co-cultured in 3D with mCherry-transduced or deepred-stained microenvironmental cells (HUVEC, JURKAT, THP-1-M2, HVHF2) for 72 h before cell sorting both populations using flow cytometry.
Project description:The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Using iTRAQ-based quantitative assessments, we found that the primary proteins involved in carbohydrate and fatty acid metabolism, adipogenesis and the electron transport chain were highly expressed in 3D cell culture system when compared to those of 2D-cultured cells. Furthermore, it was also shown that the expression levels of proteins associated with metabolic pathways, carbon metabolism and glycolysis/gluconeogenesis were up-regulated, whereas proteins implicated in both DNA replication and the cell cycle were expressed at lower levels compared to those of the 2D mono-cultured cells. Based on these results, the 3D adipocyte model can help elucidate the mechanisms underpinning metabolic syndromes and aid the development of new medical treatments for metabolic disorders.
Project description:We showed that co-culture with TAMs triggered Bmi1 expression in gastrointestinal cancer cell lines. miRNAs have been found to target various oncogenes and tumor suppressors. We therefore hypothesized that the regulation of Bmi1 expression in gastrointestinal cancer cells may be mediated by miRNAs using miRNA microarray analysis. THP-1 cells were seeded in the transwell inserts (3540, Corning) for 6-well plates (1 M-CM-^W 106 cells/well). For preparation of M1-polarized THP-1 macrophages, 320 nM phorbol myristate acetate (PMA) was added to THP-1 cells for 6 h, followed by PMA plus 20 ng/ml interferon (IFN)-M-NM-3 and 100 ng/ml lipopolysaccharide for the following 18 h. For preparation of M2-polarized THP-1 macrophages, 320 nM PMA was added to THP-1 cells for 6 h, followed by PMA plus 20 ng/ml interleukin (IL)-4/IL-13 for the following 18 h. After three washes to remove cytokines, M1- or M2-polarized THP-1 macrophages were co-cultured in upper inserts with AGS cells in 6-well plates (1 M-CM-^W 105 cells/well) without direct contact, in each medium without 10% FBS as described above. After 24 h of co-culture, the upper inserts containing macrophages were discarded. AGS cells were collected and analyzed to identify downregulated microRNA in a gastric cancer cell line co-cultured with M1- or M2-polarized macrophage.
Project description:A genomic expression comparison was done among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neural spheres (in vivo surrogate), with the goal of assessing the feasibility of establishing the meaning of 3D and associated physiological relevance at the molecular level Neural progenitor cells were cultured on 2D surfaces, in 3D scaffolds and as 3D neural spheres. Chemical cues are controlled by coating. Only spacial properties of the culture systems were compared.
Project description:Hair follicle growth inhibitory effect of dihydrotestosterone (DHT)-treated two-dimensional (2D)- and 3D-cultured DPCs were evaluated. 2D- and 3D-cultured DPC proliferation was inhibited after co-culturing with outer root sheath (ORS) cells under DHT treatment. Moreover, gene expression levels of β‐catenin and neural cell adhesion molecule (NCAM) were significantly decreased and those of cleaved caspase-3 significantly increased in 2D- and 3D-cultured DPCs with increasing DHT concentrations. ORS cell proliferation also significantly increased after co-culturing in the control-3D model compared with the control-2D model. ki67 downregulation and cleaved caspase-3 upregulation in DHT-2D and 3D groups significantly inhibited ORS cell proliferation. Sequencing showed an increase in the expression of genes related to extracellular matrix synthesis in the 3D model group. Additionally, the top 10 hub genes were identified, and the expression of nine chemokine-related genes in DHT-treated DPCs was found significantly increased. We also identified the interactions between transcription factor (TF)–genes and microRNAs with hub genes and the TF–microRNA coregulatory network. Overall, the findings indicate that 3D-cultured DPCs are more representative of in vivo conditions than are 2D-cultured DPCs, and contribute to our understanding of the molecular mechanisms underlying androgen-induced alopecia.
Project description:Adult stem cells have the ability to self-renew and to generate specialized cells. Self-renewal is dependent on extrinsic niche factors but few of those signals have been identified. We show that adult mammary glands contain a Wnt-responsive cell population that is enriched for stem cells. In cell culture experiments, exposure to purified Wnt protein clonally expands mammary stem cells for many generations and maintains their ability to generate functional glands in transplantation assays. We propose here that Wnt3A treated mammary stem cells retain their stemness through the regulation of its downstream target genes. We used microarrays to detail the global gene expression pattern underlying mammary stem cells response towards Wnt3A treatment and identified distinct classes of genes during this process Mammary glands from 8- to 12-week-old virgin female mice were isolated and single-cell suspension was obtained. Mammary stem cell enriched population (Lin-, CD24+, CD29hi) cells were isolated using BD FACSAria. The purity of sorted population was routinely checked and ensured to be more than 95%.Total RNA from 2nd colonies passage cultured in the presence of vehicle and Wnt3A was extracted with PicoPure (Arcturs) in accordance with the manufacturerM-bM-^@M-^Ys protocol. RNA concentration was determined with NanoDrop ND-1000, and quality was determined using the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies). Affymetrix microarray analysis, fragmentation of RNA, labelling, hybridization to Mouse Genome 430 2.0 microarrays, and scanning were performed in accordance with the manufacturerM-bM-^@M-^Ys protocol (Affymetrix). Total 4 samples representing two sets of replicates were analyzed.
Project description:MSCs in vivo have a markedly different three-dimensioanal (3D) niche compared to the traditional two-dimensional (2D) culture in vitro. We used microarrays to detail the global difference of gene expression between MSCs cultured on 3D and 2D matrixes