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Miyoshi, Hirotoshi, Chiaki Sato, Yuichiro Shimizu e Misa Morita. "Expansion of mouse hematopoietic stem/progenitor cells in three-dimensional cocultures on growth-suppressed stromal cell layer". International Journal of Artificial Organs 42, n.º 7 (12 de fevereiro de 2019): 374–79. http://dx.doi.org/10.1177/0391398819827596.
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With the aim of establishing an effective method to expand hematopoietic stem/progenitor cells for application in hematopoietic stem cell transplantation, we performed ex vivo expansion of hematopoietic stem/progenitor cells derived from mouse fetal liver cells in three-dimensional cocultures with stromal cells. In these cocultures, stromal cells were first cultured within three-dimensional scaffolds to form stromal layers and then fetal liver cells containing hematopoietic cells were seeded on these scaffolds to expand the hematopoietic cells over the 2 weeks of coculture in a serum-containing medium without the addition of cytokines. Prior to coculture, stromal cell growth was suppressed by treatment with the DNA synthesis inhibitor mitomycin C, and its effect on hematopoietic stem/progenitor cell expansion was compared with that in control cocultures in which fetal liver cells were cocultured with three-dimensional freeze-thawed stromal cells. After coculture with mitomycin C-treated stromal cells, we achieved a several-fold expansion of the primitive hematopoietic cells (c-kit+hematopoietic progenitor cells >7.8-fold, and CD34+hematopoietic stem/progenitor cells >3.5-fold). Compared with control cocultures, expansion of hematopoietic stem/progenitor cells tended to be lower, although that of hematopoietic progenitor cells was comparable. Thus, our results suggest that three-dimensional freeze-thawed stromal cells have higher potential to expand hematopoietic stem/progenitor cells compared with mitomycin C-treated stromal cells.
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Hirschi, Karen K., Stephanie A. Rohovsky e Patricia A. D'Amore. "PDGF, TGF-β, and Heterotypic Cell–Cell Interactions Mediate Endothelial Cell–induced Recruitment of 10T1/2 Cells and Their Differentiation to a Smooth Muscle Fate". Journal of Cell Biology 141, n.º 3 (4 de maio de 1998): 805–14. http://dx.doi.org/10.1083/jcb.141.3.805.
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We aimed to determine if and how endothelial cells (EC) recruit precursors of smooth muscle cells and pericytes and induce their differentiation during vessel formation. Multipotent embryonic 10T1/2 cells were used as presumptive mural cell precursors. In an under-agarose coculture, EC induced migration of 10T1/2 cells via platelet-derived growth factor BB. 10T1/2 cells in coculture with EC changed from polygonal to spindle-shaped, reminiscent of smooth muscle cells in culture. Immunohistochemical and Western blot analyses were used to examine the expression of smooth muscle (SM)-specific markers in 10T1/2 cells cultured in the absence and presence of EC. SM-myosin, SM22α, and calponin proteins were undetectable in 10T1/2 cells cultured alone; however, expression of all three SM-specific proteins was significantly induced in 10T1/2 cells cocultured with EC. Treatment of 10T1/2 cells with TGF-β induced phenotypic changes and changes in SM markers similar to those seen in the cocultures. Neutralization of TGF-β in the cocultures blocked expression of the SM markers and the shape change. To assess the ability of 10T1/2 cells to contribute to the developing vessel wall in vivo, prelabeled 10T1/2 cells were grown in a collagen matrix and implanted subcutaneously into mice. The fluorescently marked cells became incorporated into the medial layer of developing vessels where they expressed SM markers. These in vitro and in vivo observations shed light on the cell–cell interactions that occur during vessel development, as well as in pathologies in which developmental processes are recapitulated.
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Gilead, L., O. Bibi e E. Razin. "Fibroblasts induce heparin synthesis in chondroitin sulfate E containing human bone marrow-derived mast cells". Blood 76, n.º 6 (15 de setembro de 1990): 1188–95. http://dx.doi.org/10.1182/blood.v76.6.1188.1188.
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Abstract Human bone marrow-derived mast cells (hBMMCs), differentiated in vitro in suspension culture and under the influence of human peripheral blood mononuclear cells conditioned medium (hCM), were tested for their response to recombinant human interleukin-3 (rhIL-3) and for their behavior in different microenvironments. The hBMMCs were incubated in the presence of rhIL-3 and the changes in their proliferation rate were determined. Recombinant hIL-3 induced a more than sixfold increase in 3H-thymidine uptake into the hBMMC DNA in a dose-dependent manner. Human CM used as a control for proliferation response induced a more than eightfold maximal proliferation rate increase. Rabbit anti-rhIL-3 completely inhibited hBMMC 3H-thymidine uptake induced by rhIL-3 and decreased the hCM-induced proliferation by approximately 50%. These hBMMCs were cocultured with four different mytomicin C-treated cell monolayers and assayed for phenotypic changes. After only 2 days in coculture with either embryonic mouse skin-derived fibroblasts (MESFs) or human skin-derived fibroblasts (HSFs), a marked increase in granule number and density was noted on staining with toluidine blue. Mast cells that initially stained alcian blue+/safranin- at day 0 of coculture became alcian blue+/safranin+ during the coculture period. Human BMMC proteoglycan synthesis shifted from approximately 85% chondroitin sulfate E to approximately 60% heparin within 14 to 19 days of coculture with the MESF monolayer and to approximately 50% heparin within 19 days of coculture with the HSF monolayer. None of the above- mentioned changes were noted in cocultures of hBMMCs with 3T3 cell line fibroblast monolayers or in cocultures with bovine vascular endothelium (BVE) cell monolayers. These results demonstrate microenvironmental effects exerted by the MESF and HSF monolayers on IL-3-dependent hBMMCs similar to those reported in the conversion of murine mast cell phenotype.
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Gilead, L., O. Bibi e E. Razin. "Fibroblasts induce heparin synthesis in chondroitin sulfate E containing human bone marrow-derived mast cells". Blood 76, n.º 6 (15 de setembro de 1990): 1188–95. http://dx.doi.org/10.1182/blood.v76.6.1188.bloodjournal7661188.
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Human bone marrow-derived mast cells (hBMMCs), differentiated in vitro in suspension culture and under the influence of human peripheral blood mononuclear cells conditioned medium (hCM), were tested for their response to recombinant human interleukin-3 (rhIL-3) and for their behavior in different microenvironments. The hBMMCs were incubated in the presence of rhIL-3 and the changes in their proliferation rate were determined. Recombinant hIL-3 induced a more than sixfold increase in 3H-thymidine uptake into the hBMMC DNA in a dose-dependent manner. Human CM used as a control for proliferation response induced a more than eightfold maximal proliferation rate increase. Rabbit anti-rhIL-3 completely inhibited hBMMC 3H-thymidine uptake induced by rhIL-3 and decreased the hCM-induced proliferation by approximately 50%. These hBMMCs were cocultured with four different mytomicin C-treated cell monolayers and assayed for phenotypic changes. After only 2 days in coculture with either embryonic mouse skin-derived fibroblasts (MESFs) or human skin-derived fibroblasts (HSFs), a marked increase in granule number and density was noted on staining with toluidine blue. Mast cells that initially stained alcian blue+/safranin- at day 0 of coculture became alcian blue+/safranin+ during the coculture period. Human BMMC proteoglycan synthesis shifted from approximately 85% chondroitin sulfate E to approximately 60% heparin within 14 to 19 days of coculture with the MESF monolayer and to approximately 50% heparin within 19 days of coculture with the HSF monolayer. None of the above- mentioned changes were noted in cocultures of hBMMCs with 3T3 cell line fibroblast monolayers or in cocultures with bovine vascular endothelium (BVE) cell monolayers. These results demonstrate microenvironmental effects exerted by the MESF and HSF monolayers on IL-3-dependent hBMMCs similar to those reported in the conversion of murine mast cell phenotype.
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Portnoy, Joshua, Tianli Pan, Charles A. Dinarello, John M. Shannon, Jay Y. Westcott, Lening Zhang e Robert J. Mason. "Alveolar type II cells inhibit fibroblast proliferation: role of IL-1α". American Journal of Physiology-Lung Cellular and Molecular Physiology 290, n.º 2 (fevereiro de 2006): L307—L316. http://dx.doi.org/10.1152/ajplung.00102.2005.
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Alveolar type II (ATII) cells inhibit fibroblast proliferation in coculture by releasing or secreting a factor(s) that stimulates fibroblast production of prostaglandin E2(PGE2). In the present study, we sought to determine the factors released from ATII cells that stimulate PGE2production in fibroblasts. Exogenous addition of rat IL-1α to cultured lung fibroblasts induced PGE2secretion in a dose-response manner. When fibroblasts were cocultured with rat ATII cells, IL-1α protein was detectable in ATII cells and in the coculture medium between days 8 and 12 of culture, correlating with the highest levels of PGE2. Furthermore, under coculture conditions, IL-1α gene expression increased in ATII cells (but not fibroblasts) compared with either cell cultured alone. In both mixed species (human fibroblasts-rat ATII cells) and same species cocultures (rat fibroblasts and ATII cells), PGE2secretion was inhibited by the presence of IL-1 receptor antagonist (IL-1Ra) or selective neutralizing antibody directed against rat IL-1α (but not IL-1β). Conditioned media from cocultures inhibited fibroblast proliferation, and this effect was abrogated by the addition of IL-1Ra. Addition of keratinocyte growth factor (KGF) resulted in an earlier increase in PGE2secretion and fibroblast inhibition ( day 8 of coculture). This effect was inhibited by indomethacin but was not altered by IL-1Ra. We conclude that in this coculture system, IL-1α secretion by ATII cells is one factor that stimulates PGE2production by lung fibroblasts, thereby inhibiting fibroblast proliferation. In addition, these studies demonstrate that KGF enhances ATII cell PGE2production through an IL-1α-independent pathway.
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Hyakumura, Tomoko, Stuart McDougall, Sue Finch, Karina Needham, Mirella Dottori e Bryony A. Nayagam. "Organotypic Cocultures of Human Pluripotent Stem Cell Derived-Neurons with Mammalian Inner Ear Hair Cells and Cochlear Nucleus Slices". Stem Cells International 2019 (20 de novembro de 2019): 1–14. http://dx.doi.org/10.1155/2019/8419493.
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Stem cells have been touted as a source of potential replacement neurons for inner ear degeneration for almost two decades now; yet to date, there are few studies describing the use of human pluripotent stem cells (hPSCs) for this purpose. If stem cell therapies are to be used clinically, it is critical to validate the usefulness of hPSC lines in vitro and in vivo. Here, we present the first quantitative evidence that differentiated hPSC-derived neurons that innervate both the inner ear hair cells and cochlear nucleus neurons in coculture, with significantly more new synaptic contacts formed on target cell types. Nascent contacts between stem cells and hair cells were immunopositive for both synapsin I and VGLUT1, closely resembling expression of these puncta in endogenous postnatal auditory neurons and control cocultures. When hPSCs were cocultured with cochlear nucleus brainstem slice, significantly greater numbers of VGLUT1 puncta were observed in comparison to slice alone. New VGLUT1 puncta in cocultures with cochlear nucleus slice were not significantly different in size, only in quantity. This experimentation describes new coculture models for assessing auditory regeneration using well-characterised hPSC-derived neurons and highlights useful methods to quantify the extent of innervation on different cell types in the inner ear and brainstem.
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Zotova, Anastasia, Anastasia Atemasova, Alexey Pichugin, Alexander Filatov e Dmitriy Mazurov. "Distinct Requirements for HIV-1 Accessory Proteins during Cell Coculture and Cell-Free Infection". Viruses 11, n.º 5 (26 de abril de 2019): 390. http://dx.doi.org/10.3390/v11050390.
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The role of accessory proteins during cell-to-cell transmission of HIV-1 has not been explicitly defined. In part, this is related to difficulties in measuring virus replication in cell cocultures with high accuracy, as cells coexist at different stages of infection and separation of effector cells from target cells is complicated. In this study, we used replication-dependent reporter vectors to determine requirements for Vif, Vpu, Vpr, or Nef during one cycle of HIV-1 cell coculture and cell-free infection in lymphoid and nonlymphoid cells. Comparative analysis of HIV-1 replication in two cell systems showed that, irrespective of transmission way, accessory proteins were generally less required for virus replication in 293T/CD4/X4 cells than in Jurkat-to-Raji/CD4 cell cocultures. This is consistent with a well-established fact that lymphoid cells express a broad spectrum of restriction factors, while nonlymphoid cells are rather limited in this regard. Remarkably, Vpu deletion reduced the level of cell-free infection, but enhanced the level of cell coculture infection and increased the fraction of multiply infected cells. Nef deficiency did not influence or moderately reduced HIV-1 infection in nonlymphoid and lymphoid cell cocultures, respectively, but strongly affected cell-free infection. Knockout of BST2—a Vpu antagonizing restriction factor—in Jurkat producer cells abolished the enhanced replication of HIV-1 ΔVpu in cell coculture and prevented the formation of viral clusters on cell surface. Thus, BST2-tethered viral particles mediated cell coculture infection more efficiently and at a higher level of multiplicity than diffusely distributed virions. In conclusion, our results demonstrate that the mode of transmission may determine the degree of accessory protein requirements during HIV-1 infection.
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Wang, Qishan, Bingxin Xu, Kaijian Fan, Jing Wu e Tingyu Wang. "CypB-CD147 Signaling Is Involved in Crosstalk between Cartilage and FLS in Collagen-Induced Arthritis". Mediators of Inflammation 2020 (29 de agosto de 2020): 1–12. http://dx.doi.org/10.1155/2020/6473858.
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To investigate the crosstalk between cartilage and fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA), we adopted an in vitro coculture system model of collagen-induced arthritis (CIA) cartilage and CIA FLS monolayer. CIA rat samples of the synovium and femur head were collected for isolation of FLS and coculture system. Cartilages were treated with vehicle (Ctrl group), 10 ng/mL interleukin- (IL-) 1α (IL-1α group), and 10 ng/mL IL-1α plus 10 μM dexamethasone (Dex group) for 3 days before coculture with FLS for further 2 days. After the coculture, FLS were collected to determine the influences of articular cartilage on synoviocytes. Whether the CypB-CD147 signaling pathway is involved in the interactions between cartilage and FLS is assayed. Results showed that IL-1α-stimulated CIA cartilage promoted the proliferation and reduced the apoptosis of FLS. Increased inflammatory cytokines and decreased p57 expression were found in cocultured FLS stimulated by IL-1α-challenged CIA cartilage. Upregulation of NF-κB and I-κB kinase β (IKK-β) and downregulation of the inhibitor of NF-κBα (I-κBα) protein were observed in cocultured FLS. After coculture, significant increases in the expression of cyclophilin B (CypB) and CD147 were observed in CIA cartilage and FLS, respectively. Furthermore, results of immunofluorescence staining showed that the anti-CD147 antibody significantly suppressed p65 nuclear translocation in cocultured FLS stimulated by IL-1α-challenged CIA cartilage. In conclusion, inflammatory effects in the cartilage-FLS coculture system are associated with the CypB-CD147 mediating NF-κB pathway which may further enhance the inflammation in RA.
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Loibl, Markus, Andreas Binder, Marietta Herrmann, Fabian Duttenhoefer, R. Geoff Richards, Michael Nerlich, Mauro Alini e Sophie Verrier. "Direct Cell-Cell Contact between Mesenchymal Stem Cells and Endothelial Progenitor Cells Induces a Pericyte-Like Phenotype In Vitro". BioMed Research International 2014 (2014): 1–10. http://dx.doi.org/10.1155/2014/395781.
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Tissue engineering techniques for the regeneration of large bone defects require sufficient vascularisation of the applied constructs to ensure a sufficient supply of oxygen and nutrients. In our previous work, prevascularised 3D scaffolds have been successfully established by coculture of bone marrow derived stem cells (MSCs) and endothelial progenitor cells (EPCs). We identified stabilising pericytes (PCs) as part of newly formed capillary-like structures. In the present study, we report preliminary data on the interactions between MSCs and EPCs, leading to the differentiation of pericyte-like cells. MSCs and EPCs were seeded in transwell cultures, direct cocultures, and single cultures. Cells were cultured for 10 days in IMDM 10% FCS or IMDM 5% FCS 5% platelet lysate medium. Gene expression of PC markers, CD146, NG2,αSMA, and PDGFR-β, was analysed using RT-PCR at days 0, 3, 7, and 10. The upregulation of CD146, NG2, andαSMA in MSCs in direct coculture with EPCs advocates the MSCs’ differentiation towards a pericyte-like phenotype in vitro. These results suggest that pericyte-like cells derive from MSCs and that cell-cell contact with EPCs is an important factor for this differentiation process. These findings emphasise the concept of coculture strategies to promote angiogenesis for cell-based tissue engineered bone grafts.
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Burger, Jan A., Maite P. Quiroga, Elena Hartmann, Andrea Bürkle, William G. Wierda, Michael J. Keating e Andreas Rosenwald. "High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation". Blood 113, n.º 13 (26 de março de 2009): 3050–58. http://dx.doi.org/10.1182/blood-2008-07-170415.
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Abstract In lymphatic tissues, chronic lymphocytic leukemia (CLL) cells are interspersed with CD68+ nurselike cells (NLCs), T cells, and other stromal cells that constitute the leukemia microenvironment. However, the mechanism regulating colocalization of CLL and these accessory cells are largely unknown. To dissect the molecular cross talk between CLL and NLCs, we profiled the gene expression of CD19-purified CLL cells before and after coculture with NLCs. NLC coculture induced high-level expression of B-cell maturation antigen and 2 chemoattractants (CCL3, CCL4) by CLL cells. CCL3/CCL4 induction in NLC cocultures correlated with ZAP-70 expression by CLL cells. High CCL3/CCL4 protein levels were found in CLL cocultures with NLCs, and CCL3/CCL4 induction was abrogated by R406, a Syk inhibitor, suggesting that NLCs induce these chemokines via B-cell receptor (BCR) activation. BCR triggering also caused robust CCL3/CCL4 protein secretion by CLL cells. High CCL3 and CCL4 plasma levels in CLL patients suggest that this pathway plays a role in vivo. These studies reveal a novel mechanism of cross talk between CLL cells and their microenvironment, namely, the secretion of 2 T-cell chemokines in response to NLC coculture and BCR stimulation. Through these chemokines, CLL cells can recruit accessory cells and thereby actively create a supportive microenvironment.
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Mhyre, Andrew J., A. Mario Marcondes, Emily Y. Spaulding e H. Joachim Deeg. "Stroma-dependent apoptosis in clonal hematopoietic precursors correlates with expression of PYCARD". Blood 113, n.º 3 (15 de janeiro de 2009): 649–58. http://dx.doi.org/10.1182/blood-2008-04-152686.
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Abstract The role of the marrow microenvironment in the pathophysiology of myelodysplastic syndromes (MDSs) remains controversial. Using stromal/hematopoietic cell cocultures, we investigated the effects of stroma-derived signals on apoptosis sensitivity in hematopoietic precursors. The leukemia-derived cell line KG1a is resistant to proapoptotic ligands. However, when cocultured with the human stromal cell line HS5 (derived from normal marrow) and exposed to tumor necrosis factor-α (TNF-α), KG1a cells showed caspase-3 activation and induction of apoptosis. Apoptosis was contact dependent. Identical results were obtained in coculture with primary stroma. Gene-expression profiling of KG1a cells identified coculture-induced up-regulation of various genes involved in apoptosis, including PYCARD. Suppression of PYCARD expression in KG1a by miRNA interfered with apoptosis. Knockdown of the TNF receptor 1 (TNFR1) or TNFR2 in HS5 cells had no effect. However, knockdown of R1 in KG1a cells prevented TNF-α–induced apoptosis, while apoptosis was still induced by TNF-α–related apoptosis-inducing ligand. Primary CD34+ cells from MDS marrow, when cocultured with HS5 and TNF-α, also underwent apoptosis. In contrast, no apoptosis was observed in CD34+ cells from the marrow of healthy donors. These data indicate that stroma may convey not only protective effects on hematopoietic cells, but, dependent upon the milieu, may also facilitate apoptosis.
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Stadlmann, Sylvia, Hans Feichtinger, Gregor Mikuz, Christian Marth, Alain Gustave Zeimet, Manfred Herold, Cornelius Knabbe e Felix Albert Offner. "Interactions of Human Peritoneal Mesothelial Cells With Serous Ovarian Cancer Cell Spheroids—Evidence for a Mechanical and Paracrine Barrier Function of the Peritoneal Mesothelium". International Journal of Gynecologic Cancer 24, n.º 2 (fevereiro de 2014): 192–200. http://dx.doi.org/10.1097/igc.0000000000000036.
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BackgroundOvarian carcinoma spreads by implantation of tumor cells onto the peritoneal mesothelium. We established a 3-dimensional coculture model to simulate the interactions of ovarian carcinoma cell aggregates with human peritoneal mesothelial cells (HPMC).MethodsMulticellular tumor spheroids (MCTS) of the human ovarian cancer cell line SK-OV-3 were directly inoculated onto either confluent HPMC monolayers or their submesothelial matrix or were cocultured with mesothelium without direct cellular contact.Results and DiscussionsInoculation of MCTS onto submesothelial matrix resulted in rapid attachment (within 30 minutes) of the tumor cell aggregates followed by rapid dissemination (within 12 hours) and growth of tumor cells. Intact mesothelium increased the time required for MCTS attachment (up to 180 minutes) and led to almost complete inhibition of tumor cell dissemination and to 47% tumor growth suppression. Bromodeoxyuridine incorporation into tumor cell nuclei was almost completely abolished in cocultured MCTS. Growth also was inhibited in MCTS treated with supernatants of HPMC. Analysis of coculture supernatants revealed that HPMC-derived transforming growth factor β (TGF-β) was almost completely bound by MCTS. Addition of a function-blocking anti–TGF-β antibody (30 μg/mL) to the cocultures abrogated the growth inhibitory effect of the mesothelium by 50%.ConclusionsThe present model provides a dynamic system to study the complex interactions of ovarian carcinoma cells with HPMC over extended periods and suggests that the mesothelium constitutes a mechanical and partly TGF-β–mediated paracrine barrier to the progression of ovarian cancer.
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Wang, Ying, Boping Yang, Pan Hu, Shentao Lu, Li Lei e Lubin Liu. "The Role of Gap Junctions in the Generation of Smooth Muscle Cells from Bone Marrow Mesenchymal Stem Cells". Disease Markers 2022 (12 de agosto de 2022): 1–9. http://dx.doi.org/10.1155/2022/1491327.
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Background. Studies have shown that stem cell transplantation can improve smooth muscle cell (SMC) regeneration and remodelling. Gap junctions can enhance the cytoprotective effects of neighbouring cells. We investigated the effect of gap junctions on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into SMCs. Materials and Methods. Rat BMSCs and SMCs were obtained from the bone marrow and bladder of Sprague-Dawley rats, respectively. Flow cytometry and multilineage differentiation were performed to assess the characteristics of these cells. BMSCs and SMCs were incubated together in cocultures in the presence and absence of heptanol, an uncoupler of gap junctions. Cocultures were divided into three groups consisting of a contact coculture, noncontact coculture, and contact coculture plus heptanol groups. The expression of BMSC-specific markers and the effect of gap junctions on the differentiation of BMSCs were evaluated by performing real-time reverse transcription-polymerase chain reaction, immunofluorescence analysis, and western blotting after cocultures. Results. CD90 and CD44 were markedly expressed, and CD31 and CD45 were weakly or not expressed in BMSCs. The cells also showed good osteogenic and adipogenic differentiation ability. Compared with the noncontact coculture group, the SMC markers such as α-SMA, calponin, and connexin43 increased in the contact coculture group. The effect of contact in the coculture group was significantly weakened by heptanol. Conclusions. The results suggested that gap junctions play an important role in the generation of SMCs from BMSCs. The formation of SMCs can potentially be used to repair the sphincter muscle of patients with stress urinary incontinence.
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Bam, Rakesh, Sathisha Upparahalli Venkateshaiah, Xin Li, Sharmin Khan, Wen Ling, Randal S. Shelton, Joshua Epstein, Bart Barlogie e Shmuel Yaccoby. "Healthy Donor Whole Bone Marrow Cells Preconditioned With Myeloma Patient Serum Support Long-Term Survival Of Primary Myeloma and Reveal Altered Microenvironmental Pathways". Blood 122, n.º 21 (15 de novembro de 2013): 3118. http://dx.doi.org/10.1182/blood.v122.21.3118.3118.
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Abstract Primary human myeloma (MM) cells do not survive in culture while current in vitro and in vivo systems for growing these cells are limited to coculture with specific bone marrow (BM) cell type or growth in immunodeficient animal model. The aim of the study was to determine long-term survival and interaction of primary MM plasma cells with a healthy adult human BM that include immune cells capable of functional activation. This system is different from the autologous BM culture that is already affected by the disease. Whole BM cells from healthy donors were cultured in αMEM medium supplemented with 10% FBS and 10% serum pooled from MM patients. Following 7-9 days the cultures were composed of adherent and nonadherent cellular compartments. The nonadherent compartment contained typical BM hematopoietic cells such as monocytes, B and T lymphocytes and NK and normal plasma cells as assessed by flow cytometry, while the adherent compartment contained cells that morphologically resemble macrophages, osteoclasts, megakaryocytes and fibroblast-like cells. At this culture stage, CD138-selected MM cells from 20 patients were added to the BM cultures (4:1 BM:MM cell ratio) and survival and growth of MM cells were determined after 7 days by assessing proportion of CD45low/intermediate/CD38high MM plasma cells among total number of cells. MM and BM cell viability was constantly high (>90%) in cocultures. Subsets of primary MM plasma cells, regardless of molecular risk or subtype, were survived and detected in all cases while in 14 of 20 experiments, number of MM plasma cells was increased by 58±12% (p<0.0005, n=14). MM cell proliferation following long-term coculture was evident by the loss of cell membrane PKH26 dye or by BudR uptake in dividing cocultured MM cells. Growth of primary MM was superior in cocultures supplemented with patient serum compared to healthy donor serum. In additional study, we stably infected IL6- or stroma-dependent MM lines, or two primary MM cell cases capable of passaging in SCID-hu mice with EGFP/luciferase construct and demonstrated increased MM cell growth in all experiments in coculture using bioluminescence analysis (statistical significance range: p<0.04 to p<0.0003). Growth of OPM2 MM line was also enhanced in coculture compared to culture alone. The coculture conditions protected OPM2 cells from dexamethasone but not bortezomib while proportion of MM cell killing by lenalidomide was enhanced compared to culture of OPM2 cells alone. To assess the effect of MM cells on BM cells in coculture, global gene expression profile was performed on BM cells cultured alone or plasma cell-depleted BM after coculture with MM cells from 4 patients. Among the top underexpressed genes we identified immunoglobulin genes related to polyclonal plasma cells, extracellular factors associated with osteoblastogenesis (e.g. MGP, IGFBP2), WNT signaling (e.g. SOX4, LRP1, LRP6) and TGFb bioavailability (e.g. FBN1, LTBP1). Top upregulated genes include immuneregulatory factors such as PROK2, LRG1, OLFM4 and IL16, and cellular markers (e.g. ARG1 expressed by MDSCs). This culture system demonstrates the ability of primary MM cells to interact with and to survive in coculture with healthy adult BM that was first cultivated by patients' serum and is appropriate for studying MM-microenvironment interaction, characterization of MM cell subpopulations capable of long term survival and targeted therapy. Disclosures: No relevant conflicts of interest to declare.
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Powell, Richard J., Jaya Bhargava, Marc D. Basson e Bauer E. Sumpio. "Coculture conditions alter endothelial modulation of TGF-β1 activation and smooth muscle growth morphology". American Journal of Physiology-Heart and Circulatory Physiology 274, n.º 2 (1 de fevereiro de 1998): H642—H649. http://dx.doi.org/10.1152/ajpheart.1998.274.2.h642.
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We examined whether endothelial cells (ECs) inhibit smooth muscle cell (SMC) transforming growth factor-β1 (TGF-β1) activation in bilayer coculture. Western analysis showed that SMCs cocultured with ECs as a bilayer had lower amounts of active TGF-β1 protein compared with SMCs cultured alone and SMCs cocultured with ECs as a monolayer. EC inhibition of TGF-β1 activation could be blocked with plasminogen activator inhibitor-1 (PAI-1) antibody. Similarly, SMC hill-and-valley growth, a marker for TGF-β1 activity, was present in SMCs cultured alone and SMCs cocultured with ECs as a monolayer but was absent in SMCs cocultured as a bilayer. SMCs cocultured with ECs as a bilayer migrated at a greater rate than SMCs cultured either alone or cocultured as a monolayer. The EC effect on SMC migration was inhibited by the addition of 5 ng/ml TGF-β1. ECs had no effect on SMC RNA levels of TGF-β1. PAI-1 levels were increased in ECs and ECs cocultured with SMCs compared with SMCs cultured alone. ECs inhibit TGF-β1 activation in bilayer coculture. This appears to be mediated through an increase in EC PAI-1 release. Alterations in coculture conditions, in particular the degree of EC-SMC cell contact, have profound effects on this process.
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Chen, Weibo, Junhua Wu, Hua Shi, Zhongxia Wang, Guang Zhang, Yin Cao, Chunping Jiang e Yitao Ding. "Hepatic Stellate Cell Coculture Enables Sorafenib Resistance in Huh7 Cells through HGF/c-Met/Akt and Jak2/Stat3 Pathways". BioMed Research International 2014 (2014): 1–10. http://dx.doi.org/10.1155/2014/764981.
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Purpose. Tumor microenvironment confers drug resistance to kinase inhibitors by increasing RKT ligand levels that result in the activation of cell-survival signaling including PI3K and MAPK signals. We assessed whether HSC-LX2 coculture conferred sorafenib resistance in Huh7 and revealed the mechanism underlying the drug resistance.Experimental Design. The effect of LX2 on sorafenib resistance was determined by coculture system with Huh7 cells. The rescue function of LX2 supernatants was assessed by MTT assay and fluorescence microscopy. The underlying mechanism was tested by administration of pathway inhibitors and manifested by Western blotting.Results. LX2 coculture significantly induced sorafenib resistance in Huh7 by activating p-Akt that led to reactivation of p-ERK. LX2 secreted HGF into the culture medium that triggered drug resistance, and exogenous HGF could also induce sorafenib resistance. The inhibition of p-Akt blocked sorafenib resistance caused by LX2 coculture. Increased phosphorylation of Jak2 and Stat3 was also detected in LX2 cocultured Huh7 cells. The Jak inhibitor tofacitinib reversed sorafenib resistance by blocking Jak2 and Stat3 activation. The combined administration of sorafenib and p-Stat3 inhibitor S3I-201 augmented induced apoptosis even in the presence of sorafenib resistance.Conclusions. HSC-LX2 coculture induced sorafenib resistance in Huh7 through multiple pathways: HGF/c-Met/Akt pathway and Jak2/Stat3 pathway. A combined administration of sorafenib and S3I-201 was able to augment sorafenib-induced apoptosis even in the presence of LX2 coculture.
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Chiu, Jeng-Jiann, Li-Jing Chen, Chih-I. Lee, Pei-Ling Lee, Ding-Yu Lee, Min-Chien Tsai, Chia-Wen Lin, Shunichi Usami e Shu Chien. "Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress". Blood 110, n.º 2 (15 de julho de 2007): 519–28. http://dx.doi.org/10.1182/blood-2006-08-040097.
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Abstract E-selectin is a major adhesion molecule expressed by endothelial cells (ECs), which are exposed to shear stress and neighboring smooth muscle cells (SMCs). We investigated the mechanisms underlying the modulation of EC E-selectin expression by SMCs and shear stress. SMC coculture induced rapid and sustained increases in expression of E-selectin and phosphorylation of interleukin-1 (IL-1) receptor-associated kinase glycoprotein-130, as well as the downstream mitogen-activated protein kinases (MAPKs) and Akt. By using specific inhibitors, dominant-negative mutants, and small interfering RNA, we demonstrated that activations of c-Jun-NH2-terminal kinase (JNK) and p38 of the MAPK pathways are critical for the coculture-induced E-selectin expression. Gel shifting and chromatin immunoprecipitation assays showed that SMC coculture increased the nuclear factor-κB (NF-κB)–promoter binding activity in ECs; inhibition of NF-κB activation by p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced E-selectin promoter activity. Protein arrays and blocking assays using neutralizing antibodies demonstrated that IL-1β and IL-6 produced by EC/SMC cocultures are major contributors to the coculture induction of EC signaling and E-selectin expression. Preshearing of ECs at 12 dynes/cm2 inhibited the coculture-induced EC signaling and E-selectin expression. Our findings have elucidated the molecular mechanisms underlying the SMC induction of EC E-selectin expression and the shear stress protection against this SMC induction.
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Lee, K. H., T. Kinashi, K. Tohyama, K. Tashiro, N. Funato, K. Hama e T. Honjo. "Different stromal cell lines support lineage-selective differentiation of the multipotential bone marrow stem cell clone LyD9." Journal of Experimental Medicine 173, n.º 5 (1 de maio de 1991): 1257–66. http://dx.doi.org/10.1084/jem.173.5.1257.
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An interleukin 3-dependent multipotential stem cell clone, LyD9, has been shown to generate mature B lymphocytes, macrophages, and neutrophils by coculture with primary bone marrow stromal cells. We report here that coculture with the cloned stromal cell lines PA6 and ST2 can support differentiation of LyD9 cells predominantly into granulocyte/macrophage colony-stimulating factor (GM-CSF)- and granulocyte (G)-CSF-responsive cells, respectively. However, these stromal cell lines were unable to support lymphopoiesis of LyD9 cells. The GM-CSF-dependent line, L-GM, which was derived from LyD9 cells cocultured with PA6 stromal cells, could differentiate into macrophages and granulocytes in the presence of GM-CSF. The L-GM line can further differentiate predominantly into neutrophils by coculture with ST2 stromal cells. The G-CSF-dependent line, L-G, which was derived from LyD9 cells cocultured with ST2 stromal cells, differentiated into neutrophils in response to G-CSF. Although the stromal cell-supported differentiation of LyD9 cells required the direct contact between LyD9 and stromal cells, a small fraction of LyD9 cells that were pretreated with 5-azacytidine could differentiate into neutrophils and macrophages without direct contact with stromal cells. These results indicate that different stromal cell lines support lineage-selective differentiation of the LyD9 stem cell and that 5-azacytidine treatment can bypass the requirement of direct contact with stromal cells, albeit with a lower frequency.
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White, S. R., A. Garland, B. Gitter, I. Rodger, L. E. Alger, J. Necheles, A. R. Nawrocki e J. Solway. "Proliferation of guinea pig tracheal epithelial cells in coculture with rat dorsal root ganglion neural cells". American Journal of Physiology-Lung Cellular and Molecular Physiology 268, n.º 6 (1 de junho de 1995): L957—L965. http://dx.doi.org/10.1152/ajplung.1995.268.6.l957.
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Neuropeptides secreted by sensory afferent nerves in airways may modulate growth of airway epithelial cells. To determine whether airway sensory C-fiber nerves secrete neuropeptides that stimulate airway epithelial cell proliferation, we measured S-phase traversal in guinea pig tracheal epithelial (GPTE) cells after coculture with rat dorsal root ganglion (DRG) cells. GPTE cells were grown in subconfluent culture on collagen-coated filters for 2 days. DRG cells were harvested from newborn rat pups and grown in primary culture for 7-10 days in separate wells. GPTE and DRG cells then were cocultured for 48 h, and 10 mM bromodeoxyuridine (BrdU), a thymidine analogue, was added in the final 24 h. Control GPTE cells were grown under similar conditions but without DRG cells. Coculture with DRG cells stimulated GPTE cell traversal of S phase. BrdU labeling in cocultured GPTE cells was 42.8 +/- 5.8 compared with 18.1 +/- 7.2% in control GPTE cells (P < 0.001, n = 6). Coculture in the presence of either the neurokinin (NK)1 receptor antagonists LY-297911 or CP-99,994, the NK2 receptor antagonist SR-48,968, or the calcitonin gene-related peptide (CGRP) receptor antagonist hCGRP-(8-37) (10(-7) M of each) during coculture attenuated proliferation of GPTE cells. Treatment with all three antagonists together during coculture decreased BrdU labeling to 2.4 +/- 0.9% of labeled cells vs. 8.5 +/- 0.5% of labeled cells during coculture without antagonists (n = 4, P < 0.02). DRG cells in coculture secreted substantial concentrations of CGRP [71.0 +/- 11.3 (+/- SE) pmol/ml], substance P (1.26 +/- 0.35 pmol/ml), and neurokinin A (0.45 +/- 0.10 pmol/ml) (n = 19 for each).(ABSTRACT TRUNCATED AT 250 WORDS)
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Dreier, Rita, Shona Wallace, Susanne Fuchs, Peter Bruckner e Susanne Grässel. "Paracrine interactions of chondrocytes and macrophages in cartilage degradation: articular chondrocytes provide factors that activate macrophage-derived pro-gelatinase B (pro-MMP-9)". Journal of Cell Science 114, n.º 21 (1 de novembro de 2001): 3813–22. http://dx.doi.org/10.1242/jcs.114.21.3813.
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Cells of the monocyte/macrophage lineage are involved in the development of inflammatory joint diseases such as rheumatoid arthritis. This disease is characterized by cartilage degradation and synovial membrane inflammation with a progressive loss of joint function. The pathological processes are still not well understood. Therefore it would be interesting to develop a suitable experimental in vitro model system for defined studies of monocyte/macrophage and chondrocyte interactions at the molecular level. For that purpose we cocultured chondrocytes from adult human articular cartilage with human monocytes and macrophages for defined periods of time in agarose without addition of serum. We performed zymographic and western blot analysis of culture medium, completed by quantitative RT-PCR of each chondrocyte, monocyte and macrophage RNA, respectively. The reliability of the newly established coculture systems is confirmed by causing a clear decrease of intact aggrecan in the coculture medium plus concurrent appearance of additional smaller fragments and a reduction of chondrocyte aggrecan and collagen II gene expression in the presence of monocytes. In culture medium from cocultures we detected active forms of the matrix metalloproteinases MMP-1, MMP-3 and MMP-9 accompanied by induction of gene expression of MMP-1, membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in chondrocytes. No gene expression of MMP-9 was detectable in chondrocytes, the enzyme was solely expressed in monocytes and macrophages and was downregulated in the presence of chondrocytes. Our results suggest that MMP-9 protein in coculture medium originated from monocytes and macrophages but activation required chondrocyte-derived factors. Because addition of plasmin, a partial activator of pro-MMP-3 and pro-MMP-1, enhanced the activation of pro-MMP-9 and pro-MMP-1 in cocultures but not in monocultured macrophages, and the presence of MMP-3 inhibitor II prevented pro-MMP-9 activation, we assumed a stepwise activation process of pro-MMP-9 that is dependent on the presence of at least MMP-3 and possibly also MMP-1.
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Kim, Sung Min, Yoo Ri Ko, Hye Seon Park e Se Jin Jang. "Abstract 218: Deciphering tumoroid-CAF interactions through a spatially segregated coculture model". Cancer Research 84, n.º 6_Supplement (22 de março de 2024): 218. http://dx.doi.org/10.1158/1538-7445.am2024-218.
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Abstract In the context of lung cancers, cancer-associated fibroblast (CAF) plays a pivotal role as a key component of the tumor microenvironment (TME), affecting tumor growth, invasion, metastasis, immune modulation, and drug resistance. Despite the recognized impact of CAFs, the precise interaction between the CAF and tumor cell remains elusive. Previous studies have reported that CAFs stimulate tumor cell growth both in vitro and in vivo. However, our tumoroid models reveal an intriguing phenomenon where CAFs inhibit tumoroids growth within Matrigel. This led us to hypothesize that paracrine signaling might be the primary mode of interaction between CAFs and tumor cells. To unravel the intricacies of this interaction, we developed an innovative coculture method. Using hydrophobic barrier, we segregated the coculture of nine sets of CAFs and tumoroids within a single well, preventing CAFs from infiltrating the tumoroid culture area. This setup enabled interaction solely through paracrine signaling. Surprisingly, cocultured tumoroids exhibited no significant differences compared to individually cultured tumoroids. Conversely, cocultured CAFs displayed a remarkable increase in growth compared to their individually cultured counterparts. This suggests that tumoroids influence CAF growth, while CAFs do not significantly impact tumoroid growth. Furthermore, in three coculture sets, tumor cells within the Matrigel migrated towards the CAF culture area, indicating that CAFs induce tumor cell metastasis. Immunofluorescence staining confirmed this metastatic phenomenon. To identify the paracrine factors influencing CAFs and tumoroids, we employed RNA-sequencing, single-cell RNA-sequencing, and proteomic analysis of culture soup. Proteomic analysis revealed a substantial increase in proteins and signaling pathways related to the extracellular matrix in cocultures compared to single cultures. Our findings suggest that tumor cells recruit CAFs, promoting CAF growth to establish and fortify their TME, ultimately leading to drug resistance, metastasis, and immune modulation Citation Format: Sung Min Kim, Yoo Ri Ko, Hye Seon Park, Se Jin Jang. Deciphering tumoroid-CAF interactions through a spatially segregated coculture model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 218.
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Uchida, K., e B. J. Ballermann. "Sustained activation of PGE2 synthesis in mesangial cells cocultured with glomerular endothelial cells". American Journal of Physiology-Cell Physiology 263, n.º 1 (1 de julho de 1992): C200—C209. http://dx.doi.org/10.1152/ajpcell.1992.263.1.c200.
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Glomerular endothelial cells synthesize and release endothelin-1 (ET-1), and mesangial cells, normally closely apposed to endothelial cells in vivo, respond to ET-1 with contraction, proliferation, and prostaglandin E2 (PGE2) release. This study sought to determine whether chronic coculture of mesangial cells with glomerular endothelial cells alters mesangial cell PGE2 synthesis. Mesangial cells cocultured with endothelial cells were found to release PGE2 at rates much greater than those observed in mesangial cells not cocultured with endothelial cells. This effect persisted for at least 24 h after the mesangial cells were removed from coculture with endothelial cells. The increase in basal mesangial cell PGE2 synthesis was dependent on endothelial cell-derived ET-1. Despite the increase in basal PGE2 synthesis after coculture with endothelial cells, acute ET-1-stimulated PGE2 release was markedly blunted in mesangial cells that had been cocultured with endothelial cells when compared with mesangial cells in solo-culture. This lack of responsiveness was specific for ET-1 and resulted from a profound downregulation of mesangial cell endothelin receptors. Thus coculture with endothelial cells produces two apparently opposing and ET-1-dependent effects in mesangial cells, namely a sustained increase in basal PGE2 synthesis by the cells and a loss of responsiveness to further stimulation with ET-1. It is postulated that the induction of sustained PGE2 synthesis may also occur in vivo if endothelin release from endothelial cells is stimulated and may explain, in part, the extraordinary sensitivity of some patients with glomerular disease to cyclooxygenase inhibitors.
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Ishikawa, Shohei, Kazutoshi Iijima, Kohei Sasaki, Masaaki Kawabe e Hidenori Otsuka. "Improvement of Hepatic Functions by Spheroids Coculture with Fibroblasts in 3D Silica Nonwoven Fabrics". Journal of Nanoscience and Nanotechnology 19, n.º 6 (1 de junho de 2019): 3326–33. http://dx.doi.org/10.1166/jnn.2019.16103.
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In order to realize organ-on-a-chip as an effective tool for regenerative medicine and drug development, tissue-mimic cell culture methods which promote liver-specific function for long period have been developed. We have previously demonstrated that coculture of hepatocyte spheroids on fibroblasts using micropatterned substrate improved the hepatic functions due to the heterotypic cell–cell interactions and paracrine signaling from each other. In addition, hepatocyte function cultured as monolayer was also promoted in separately coculture with fibroblasts cultured as monolayer, and it is more improved in separately coculture with fibroblasts in 3D silica nonwoven fabrics. In this study, separately coculture of hepatocyte spheroids with fibroblasts cultured on 3D silica nonwoven fabrics was estimated for further improvement of hepatocyte functions. The hepatic function cocultured with fibroblast was more promoted than mono spheroids culture. The functional enhancement was significantly most improved in separately coculture with fibroblast in 3D silica nonwoven fabrics. Thus, these results were suggested that 3D culture of fibroblasts in 3D silica nonwoven fabrics increased the heterotypic secretion of paracrine factors, and it is essential for improved hepatic performance.
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Hielscher, Abigail C., Connie Qiu e Sharon Gerecht. "Breast cancer cell-derived matrix supports vascular morphogenesis". American Journal of Physiology-Cell Physiology 302, n.º 8 (15 de abril de 2012): C1243—C1256. http://dx.doi.org/10.1152/ajpcell.00011.2012.
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The extracellular matrix (ECM), important for maintaining tissue homeostasis, is abnormally expressed in mammary tumors and additionally plays a crucial role in angiogenesis. We hypothesize that breast cancer cells (BCCs) deposit ECM that supports unique patterns of vascular morphogenesis of endothelial cells (ECs). Evaluation of ECM expression revealed that a nontumorigenic cell line (MCF10A), a tumorigenic cell line (MCF7), and a metastatic cell line (MDA-MB-231) express collagens I and IV, fibronectin, and laminin, with tenascin-C limited to MCF10A and MCF7. The amount of ECM deposited by BCCs was found to be higher in MCF10A compared with MCF7 and MDA231, with all ECM differing in their gross structure but similar in mean fiber diameter. Nonetheless, deposition of ECM from BCC lines was overall difficult to detect and insufficient to support capillary-like structure (CLS) formation of ECs. Therefore, a coculture approach was undertaken in which individual BCC lines were cocultured with fibroblasts. Variation in abundance of deposited ECM, deposition of ECM proteins, such as absent collagen I deposition from MDA231-fibroblast cocultures, and fibril organization was found. Deposited ECM from fibroblasts and each coculture supported rapid CLS formation of ECs. Evaluation of capillary properties revealed that CLS grown on ECM deposited from MDA231-fibroblast cocultures possessed significantly larger lumen diameters, occupied the greatest percentage of area, expressed the highest levels of von Willebrand factor, and expressed the greatest amount of E-selectin, which was upregulated independent of exposure to TNF-α. To our knowledge, this is the first study to report tumor cell ECM-mediated differences in vascular capillary features, and thus offers the framework for future investigations interrogating the role of the tumor ECM in supporting vascular morphogenesis.
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Schmelzer, Eva, Vitale Miceli, Cinzia Maria Chinnici, Alessandro Bertani e Jörg C. Gerlach. "Effects of Mesenchymal Stem Cell Coculture on Human Lung Small Airway Epithelial Cells". BioMed Research International 2020 (30 de março de 2020): 1–8. http://dx.doi.org/10.1155/2020/9847579.
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Mesenchymal stem cells (MSCs) and their secreted extracellular vesicles have been used effectively in different lung disease animal models and clinical trials. Their specific beneficial effects, the potential differences between MSCs derived from different organs, and interactions between MSC products and target cells still need to be studied further. Therefore, we investigated the effects of secreted products of human MSCs derived from the bone marrow and adipose tissue on human lung small airway epithelial (AE) cells in vitro. AE cells were cocultured with MSCs in inserts that allowed the free exchange of medium but did not allow direct cell-to-cell contact. We examined the effects on AE cell viability, proliferation, cell numbers, expression of AE cell-specific genes, and CD54 (intercellular adhesion molecule 1 (ICAM1)) surface positivity, as well as the secretion/uptake of growth factors relevant for AE cell. We found that coculture increased the viability of AE cells. The majority of AE cells expressed CD54 on their surface, but the percentage of cells being positive for CD54 did not increase in coculture. However, ICAM1 gene expression was increased in coculture. Also, we observed increased gene expression of mucin (MUC1), a lung-enriched cell surface glycoprotein. These observed effects were the same between bone marrow and adipose tissue MSCs. However, MSCs derived from adipose tissue reduced angiopoietin concentrations in coculture, whereas those from the bone marrow did not. Conclusively, MSCs influenced AE cells positively by increasing their viability and affecting gene expression, with some effects being specific for the tissue origin of MSCs.
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Chao, C. C., S. Hu, T. W. Molitor, E. G. Shaskan e P. K. Peterson. "Activated microglia mediate neuronal cell injury via a nitric oxide mechanism." Journal of Immunology 149, n.º 8 (15 de outubro de 1992): 2736–41. http://dx.doi.org/10.4049/jimmunol.149.8.2736.
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Abstract Activated microglial have been proposed to play a pathogenetic role in immune-mediated neurodegenerative diseases. To test this hypothesis, purified murine neonatal microglial were cocultured with neuronal cells derived from fetal brain. Activation with IFN-gamma and LPS of these cocultures brought about a sharp decrease in uptake of gamma-amino butyric acid and a marked reduction in neuronal cell survival. These effects varied with the density of microglia, the concentrations of the activation signals (IFN-gamma and LPS), and the duration of coculture. Inasmuch as addition of NG-monomethyl-L-arginine blocked these effects, a L-arginine-dependent neurocytotoxic mechanism was implicated. Abundant nitrite, a metabolite of the free radical nitric oxide (NO) derived from L-arginine, was detected in activated microglial/neuronal cell cocultures and in purified microglial cell cultures but not in purified astrocyte or neuronal cell cultures, suggesting that microglial were the principal source of the NO. These findings support the hypothesis that microglia are the source of a neurocytotoxic-free radical, and shed light on an additional mechanism of immune-mediated brain injury.
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Schmidt, Elena, Maike Haase, Elke Ziegler, Günter Emons e Carsten Gründker. "Kisspeptin-10 Inhibits Stromal-Derived Factor 1–Induced Invasion of Human Endometrial Cancer Cells". International Journal of Gynecologic Cancer 24, n.º 2 (fevereiro de 2014): 210–17. http://dx.doi.org/10.1097/igc.0000000000000050.
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ObjectivesThe cross talk between metastatic cancer cells and target sites is critical for the development and progression of metastases. Disruption of this interaction will allow to design mechanism-based effective and specific therapeutic interventions for metastases. We have established a coculture system of cells derived from different tumor entities and MG63 human osteoblastlike cells to analyze tumor cell invasion. Recently, we have shown that breast cancer cell invasion was dramatically increased when cocultured with MG63 cells.Using this model, we have now analyzed whether stromal-derived factor 1 (SDF-1) is responsible for human endometrial cancer cell invasion and whether kisspeptin-10 (KP-10) treatment affects SDF-1–induced invasion of endometrial cancer cells in vitro.MethodsInvasion was quantified by assessment of endometrial cancer cell migration rate through an artificial basement membrane in a modified Boyden chamber during coculture with MG63 cells or after treatment with SDF-1α, SDF-1β, or the combination of both SDF-1 isoforms. In addition, the role of SDF-1 in invasion of endometrial cancer cells was analyzed by blocking SDF-1 secretion during coculture with MG64 cells. Furthermore, the effects of KP-10 treatment on MG63 coculture-driven and SDF-1–induced invasion were analyzed.ResultsEndometrial cancer cell invasion was significantly increased when cocultured with MG63 cells. Treatment with KP-10 reduced the ability to invade a reconstituted basement membrane and to migrate in response to the cellular stimulus. This effect was significant in a dose window of 10−13 to 10−11 mol/L. During coculture, SDF-1 protein expression of MG63 cells was significantly increased. The MG63 coculture-induced increase of endometrial cancer cell invasion could be blocked by anti–SDF-1 antibodies. Treatment of endometrial cancer cells in monoculture (without MG63) with SDF-1α, SDF-1β, or the combination of both isoforms resulted in a significant increase of endometrial cancer cell invasion. The SDF-1–induced increase of endometrial cancer cell invasion was significantly reduced after treatment with KP-10.ConclusionsOur findings suggest that SDF-1 plays a major role in endometrial cancer invasion. Stromal-derived factor 1–induced invasion can be inhibited by KP-10 treatment.
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Lim, Ivor Jiun, Toan-Thang Phan, Boon-Huat Bay, Robert Qi, Hung Huynh, Walter Tiang-Lee Tan, Seng-Teik Lee e Michael Thornton Longaker. "Fibroblasts cocultured with keloid keratinocytes: normal fibroblasts secrete collagen in a keloidlike manner". American Journal of Physiology-Cell Physiology 283, n.º 1 (1 de julho de 2002): C212—C222. http://dx.doi.org/10.1152/ajpcell.00555.2001.
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Keloid scars represent a pathological response to cutaneous injury, reflecting a new set point between synthesis and degradation biased toward extracellular matrix (ECM) collagen accumulation. Using a serum-free two-chamber coculture model, we recently demonstrated a significant increase in normal fibroblast proliferation when cocultured with keloid-derived keratinocytes. We hypothesized that similar keratinocyte-fibroblast interactions might influence fibroblast collagen production and examined conditioned media and cell lysate from coculture for collagen I and III production by Western blot, allied with Northern analysis for procollagen I and III mRNA. Normal fibroblasts cocultured with keloid keratinocytes produced increased soluble collagen I and III with a corresponding increase in procollagen I and III mRNA transcript levels. This was associated with decreased insoluble collagen from cell lysate. When keloid fibroblasts were cocultured with keloid keratinocytes, both soluble and insoluble collagen were increased with associated procollagen III mRNA upregulation. Transmission electron microscopy of normal fibroblasts cocultured with keloid keratinocytes showed an ECM appearance similar to in vivo keloid tissue, an appearance not seen when normal fibroblasts were cocultured with normal keratinocytes.
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Ellington, Joanna E., Juan Samper, Allison Jones, Sylvia A. Oliver, Katherine Burnett e Ray W. Wright. "Effects of bovine serum albumin on function of cryopreserved stallion spermatozoa during medium culture and uterine tube epithelial cell coculture". American Journal of Veterinary Research 60, n.º 3 (1 de março de 1999): 363–67. http://dx.doi.org/10.2460/ajvr.1999.60.03.363.
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Abstract Objective To compare function of cultured cryopreserved stallion spermatozoa in a modified Tyrode’s medium (TM), with or without bovine serum albumin (BSA), or in uterine tube (oviduct) epithelial cell (OEC) coculture in TM, with or without BSA. Sample Population Cryopreserved spermatozoa from 6 proven stallions and OEC from bovine reproductive tracts in follicular phase. Procedure Thawed spermatozoa were cultured in TM, with or without BSA, or cocultured with OEC monolayers in TM, with or without BSA. Percentages of capacitated and acrosome-reacted spermatozoa were measured at 5 hours for TM cultures. Spermatozoal survival and motility characteristics were observed over time for all culture methods. Number of spermatozoa attaching to OEC were compared for cocultures. Results Use of TM without BSA altered spermatozoal function in cell-free medium culture and OEC coculture. A higher percentage of spermatozoa were acrosome reacted in TM with BSA, although percentages of capacitated spermatozoa did not differ. Spermatozoa survived longer and maintained superior motion in TM culture without BSA and in OEC cocultures. More spermatozoa were able to attach to OEC in TM without BSA. Conclusions Incubation of cryopreserved spermatozoa in media with BSA resulted in rapid decrease in percentage of intact, motile spermatozoa and limited their ability to interact with OEC. Clinical Relevance Current culture media used for assisted reproduction techniques in horses do not provide functionally capacitated spermatozoa. Removal of BSA from such media improves spermatozoal quality and survival. (Am J Vet Res 1999;60: 363–367)
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Nygaard, Svein J. T., Hans K. R. Haugland, Ole Didrik Laerum, Morten Lund-Johansen, Rolf Bjerkvig e Ole-Björn Tysnes. "Dynamic determination of human glioma invasion in vitro". Journal of Neurosurgery 89, n.º 3 (setembro de 1998): 441–47. http://dx.doi.org/10.3171/jns.1998.89.3.0441.
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Object. The goal of this study was to evaluate whether there is any relationship between survival of patients with brain tumor and tumor proliferation or tumor invasion in vitro. Methods. Samples of freshly resected brain tumors from 14 patients with glioblastoma multiforme (GBM) were directly grown as three-dimensional multicellular spheroids. The tumor spheroids were cocultured with fetal rat brain cell aggregates (BCAs), used to represent an organotypical normal brain tissue model. Before the coculture, the tumor spheroids and the BCAs were stained with two different carbocyanine dyes, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and 3,3′-dioctadecycloxacarbocyanine perchlorate (DiO), respectively. During the coculture, confocal laser scanning microscopy allowed a sequential analysis of tumor cell invasion by visualizing dynamic aspects of the invasive process. Single cocultures were examined at three different time points (24, 48, and 96 hours). During the observation period there was a change in the structural morphology of the cocultures, with a progressive decrease in BCA volume. Furthermore, the scanning confocal micrographs revealed a bidirectional movement of tumor cells and normal cells into brain and tumor tissue, respectively. It is also shown that there is a considerable variation in the rate of BCA destruction in cocultures of glioma spheroids generated directly from biopsy specimens. This variation is seen both between spheroids generated from the same biopsy as well as between spheroids that are grown from different biopsy specimens. Cell proliferation measured by Ki-67 immunohistochemical analysis of biopsy samples obtained in the same patients revealed a correlation between tumor cell proliferation and tissue destruction of the BCAs, as determined by a reduction in BCA volume (p = 0.0338). No correlation was found when survival was related to the same parameters (p > 0.05). Conclusions. The present work provides a model for quick and efficient assessment of dynamic interactions between tumor and normal brain tissue shortly after surgery.
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Gairhe, Salina, Natalie N. Bauer, Sarah A. Gebb e Ivan F. McMurtry. "Serotonin passes through myoendothelial gap junctions to promote pulmonary arterial smooth muscle cell differentiation". American Journal of Physiology-Lung Cellular and Molecular Physiology 303, n.º 9 (1 de novembro de 2012): L767—L777. http://dx.doi.org/10.1152/ajplung.00183.2012.
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Myoendothelial gap junctional signaling mediates pulmonary arterial endothelial cell (PAEC)-induced activation of latent TGF-β and differentiation of cocultured pulmonary arterial smooth muscle cells (PASMCs), but the nature of the signal passing from PAECs to PASMCs through the gap junctions is unknown. Because PAECs but not PASMCs synthesize serotonin, and serotonin can pass through gap junctions, we hypothesized that the monoamine is the intercellular signal. We aimed to determine whether PAEC-derived serotonin mediates PAEC-induced myoendothelial gap junction-dependent activation of TGF-β signaling and differentiation of PASMCs. Rat PAECs and PASMCs were monocultured or cocultured with (touch) or without (no-touch) direct cell-cell contact. In all cases, tryptophan hydroxylase 1 (Tph1) transcripts were expressed predominantly in PAECs. Serotonin was detected by immunostaining in both PAECs and PASMCs in PAEC/PASMC touch coculture but was not found in PASMCs in either PAEC/PASMC no-touch coculture or in PASMC/PASMC touch coculture. Furthermore, inhibition of gap junctions but not of the serotonin transporter in PAEC/PASMC touch coculture prevented serotonin transfer from PAECs to PASMCs. Inhibition of serotonin synthesis pharmacologically or by small interfering RNAs to Tph1 in PAECs inhibited the PAEC-induced activation of TGF-β signaling and differentiation of PASMCs. We concluded that serotonin synthesized by PAECs is transferred through myoendothelial gap junctions into PASMCs, where it activates TGF-β signaling and induces a more differentiated phenotype. This finding suggests a novel role of gap junction-mediated intercellular serotonin signaling in regulation of PASMC phenotype.
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Kim, Minwook, David R. Steinberg, Jason A. Burdick e Robert L. Mauck. "Extracellular vesicles mediate improved functional outcomes in engineered cartilage produced from MSC/chondrocyte cocultures". Proceedings of the National Academy of Sciences 116, n.º 5 (15 de janeiro de 2019): 1569–78. http://dx.doi.org/10.1073/pnas.1815447116.
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Several recent studies have demonstrated that coculture of chondrocytes (CHs) with bone marrow-derived mesenchymal stem cells (MSCs) improves their chondrogenesis. This implies that intercellular communication dictates fate decisions in recipient cells and/or reprograms their metabolic state to support a differentiated function. While this coculture phenomenon is compelling, the differential chondroinductivity of zonal CHs on MSC cocultures, the nature of the molecular cargo, and their transport mechanisms remains undetermined. Here, we demonstrate that juvenile CHs in coculture with adult MSCs promote functional differentiation and improved matrix production. We further demonstrate that close proximity between the two cell types is a prerequisite for this response and that the outcome of this interaction improves viability, chondrogenesis, matrix formation, and homeostasis in the recipient MSCs. Furthermore, we visualized the transfer of intracellular contents from CHs to nearby MSCs and showed that inhibition of extracellular vesicle (EV) transfer blocks the synergistic effect of coculture, identifying EVs as the primary mode of communication in these cocultures. These findings will forward the development of therapeutic agents and more effective delivery systems to promote cartilage repair.
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Ern, Christina, Vera Krump-Konvalinkova, Denitsa Docheva, Stefanie Schindler, Oliver Rossmann, Wolfgang Böcker, Wolf Mutschler e Matthias Schieker. "Interactions of Human Endothelial and Multipotent Mesenchymal Stem Cells in Cocultures". Open Biomedical Engineering Journal 4, n.º 1 (11 de outubro de 2010): 190–98. http://dx.doi.org/10.2174/1874120701004010190.
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Current strategies for tissue engineering of bone rely on the implantation of scaffolds, colonized with human mesenchymal stem cells (hMSC), into a recipient. A major limitation is the lack of blood vessels. One approach to enhance the scaffold vascularisation is to supply the scaffolds with endothelial cells (EC). The main goal of this study was to establish a coculture system of hMSC and EC for the purposes of bone tissue engineering. Therefore, the cell behaviour, proliferation and differentiation capacity in various cell culture media as well as cell interactions in the cocultures were evaluated. The differentiation capacity of hMSC along osteogenic, chondrogenic, and adipogenic lineage was impaired in EC medium while in a mixed EC and hMSC media, hMSC maintained osteogenic differentiation. In order to identify and trace EC in the cocultures, EC were transduced with eGFP. Using time-lapse imaging, we observed that hMSC and EC actively migrated towards cells of their own type and formed separate clusters in long term cocultures. The scarcity of hMSC and EC contacts in the cocultures suggest the influence of growth factor-mediated cell interactions and points to the necessity of further optimization of the coculture conditions.
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KLINEFELTER, GARY R., NAOMI L. ROBERTS e JUAN D. SUAREZ. "Direct Effects of Ethane Dimethanesulphonate on Epididymal Function in Adult Rats". Journal of Andrology 13, n.º 5 (10 de setembro de 1992): 409–21. http://dx.doi.org/10.1002/j.1939-4640.1992.tb03334.x.
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Abstract: It was recently demonstrated that the Leydig cell toxicant ethane dimethanesulphonate (EDS) produces multiple effects on the epididymis after a single in vivo exposure. To determine whether any of the perturbations were mediated by a direct action of the compound, we used a novel system for the coculture of epididymal epithelial cells and sperm from the caput epididymidis. This system maintains the morphologic integrity and cell polarity of the epididymal epithelial cells before and during coculture, and the sperm recovered after coculture have intact plasma and acrosomal membranes. In addition, several functions required for epididymal sperm maturation are expressed, including the secretion of protein by the epididymal epithelium, the association of secreted protein with the plasma membrane of cocultured sperm, and the acquisition of progressive motility by cocultured sperm. In vitro exposure of epididymal epithelial cells and sperm to EDS results in a significant decline in protein secretion by the epithelial cells during coculture, and in particular, a dose‐dependent decline in a 36‐ to 38‐kd protein (PI 4.0 to 4.5) and a 34‐ to 36‐kd protein (PI 4.5 to 5.0). Moreover, these and other proteins are not recovered from the sperm membrane of cocultured sperm after EDS treatment. Finally, EDS results in a dose‐dependent decline in the percentage of both motile and progressively motile sperm recovered after coculture compared with that of sperm from untreated co‐cultures. These effects on sperm motility were not observed when sperm were pretreated with EDS and subsequently co‐cultured with untreated epithelial cells. We conclude that EDS alters epididymal sperm maturation by acting directly on the epididymal epithelium to mediate changes in sperm membrane protein, and that this may subsequently alter the development of the progressive motility of sperm.
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Bam, Rakesh, Ricky D. Edmondson, Caleb K. Stein, Xin Li, Wen Ling, Sharmin Khan, Randal S. Shelton, Bart Barlogie e Shmuel Yaccoby. "Sustained Growth of Primary Myeloma Cells in Coculture with Whole Donor Bone Marrow Is Associated with Induced Secretion of the Microenvironmental Mediator of Cytokinesis, Hemicentin-1". Blood 124, n.º 21 (6 de dezembro de 2014): 3403. http://dx.doi.org/10.1182/blood.v124.21.3403.3403.
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Abstract Primary human myeloma (MM) cells do not survive in culture while current systems for growing these cells are limited to coculture with specific bone marrow (BM) cell type or growth in immunodeficient animals. The aim of the study was to establish a coculture system for studying long-term growth of primary MM and their interaction with whole BM microenvironment, namely normal bone marrow (NBM) system . Whole BM cells from healthy donors (n=20) were cultured in medium supplemented with serum (10% v/v) pooled from MM patients for 7 days followed by coculture with CD138-selected primary MM cells (4:1 NBM:MM ratio) or MM cell lines (10:1 NBM:MM ratio) for ≥7 days. This NBM system is composed of adherent and non-adherent compartments. Adherent cells were mainly macrophages and mesenchymal stem cells (MSCs) whereas non-adherent cells contained typical hematopoietic cells including CD19+, CD3+, CD11b+ and CD33+ cells. Growth of MM cells was determined by CD45/CD38 flow cytometry and by bioluminescence of luciferase-expressing MM cells. MM cells or subset of MM cells from all patients (n=60) survived and grew in this system regardless of molecular risk or subtype, and MM growth was comparable to coculture with the supportive osteoclasts or MSCs. Adherent and non-adherent compartments supported MM cells which required patient’s serum for optimal growth. In 14 of 20 experiments, number of MM plasma cells, quantified by flow cytometry or bioluminescence analysis was increased by 58±12% (p<0.0005) in the NBM system and cell proliferation was evident by the loss of cell membrane PKH26 dye or by BudR uptake in dividing cocultured MM cells. Growth of OPM2, H929 and ARP1 lines was also stimulated in the NBM system which protected these cells from dexamethasone (1-2.5µM) but not bortezomib (0.01-5nM), while the effect of lenalidomide varied (0.1-5µM). For identifying secreted proteins that may mediate MM growth in the NBM system, supernatant were collected from serum-free culture of NBM, MM cells and NBM/MM coculture (18 hrs, n=3). Proteomics analysis performed on supernatant samples identified 1843 proteins. The clinical markers B2M and LDHA were present at high levels and were significantly higher by 2-2.4 folds in NBM/MM coculture compared to cultured NBM (p<0.04). Further filtration revealed 89 proteins that were significantly changed upon NBM/MM coculture but minimally detected in the MM cells culture: 14 were significantly lower and 75 were higher in NBM/MM cocultures compared to cultured NBM. These factors include mediators of extracellular matrix, immunity, and inflammation. A microenvironmental secreted factor that was not detected in the supernatant from MM cells or NBM but was secreted in cocultures was hemicentin-1 (HMCN1), a unique extracellular matrix protein directly involved in cytokinesis (Xu and Vogel, Curr Biol 2011) but has yet not been implicated in MM. Hemicentin-1 gene expression was detected in cultured NBM and MSCs but not in primary MM cells, MM lines or CD11b+ NBM cells. Induction of hemicentin-1 expression in MSCs after coculture with MM cells was validated by immunohistochemistry. Hemicentin-1 expression is higher in random bone biopsies from newly diagnosed MM patients (n=406) compared to donor biopsies (n=25, p<0.008) and highest in MM focal lesion biopsies (n=49, q<0.0005 vs. paired random bone biopsies). Higher baseline HMCN1 expression in biopsies was associated with inferior overall survival in TT3b clinical trial (p<0.027). The NBM system demonstrates the ability of primary MM plasma cells to interact with and to survive in coculture with healthy allogeneic adult BM through secretion of factors involved in immune evasion and extracellular matrix modification. Ongoing work is underway to unravel the role of hemicentin-1 in MM growth. Disclosures No relevant conflicts of interest to declare.
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Peterson, P. K., G. Gekker, C. C. Chao, R. Schut, J. Verhoef, C. K. Edelman, A. Erice e H. H. Balfour. "Cocaine amplifies HIV-1 replication in cytomegalovirus-stimulated peripheral blood mononuclear cell cocultures." Journal of Immunology 149, n.º 2 (15 de julho de 1992): 676–80. http://dx.doi.org/10.4049/jimmunol.149.2.676.
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Abstract Cocaine and CMV each have been suggested to promote the progression of HIV-1 infection. In the present study, the interaction of cocaine and CMV was investigated in a PBMC coculture assay in which release of HIV-1 p24 Ag into coculture supernatants was used as an index of HIV-1 replication. CMV was an effective activation signal for HIV-1 replication when PBMC from CMV-seropositive donors were used in the coculture assay, and cocaine markedly increased replication of HIV-1 in these cocultures. The synergistic activity of cocaine was reduced by neutralizing antibodies to TNF-alpha and by pentoxifylline, an inhibitor of TNF-alpha mRNA production. Also, antibodies to transforming growth factor-beta (TGF-beta) eliminated the amplifying effect of cocaine on HIV-1 replication, whereas antibodies to IL-6 were inactive. The potentiating effect of cocaine could be reproduced by addition of rTNF-alpha or rTGF-beta to the cocultures of CMV-activated PBMC, although TGF-beta was substantially more potent than TNF-alpha. The possibility that TNF-alpha may act indirectly through stimulation of TGF-beta was suggested by the finding of reduced TGF-beta levels in culture supernatants of PBMC that were treated with CMV and cocaine in the presence of antibodies to TNF-alpha. Thus, cocaine amplifies HIV-1 replication in cocultures containing CMV-activated PBMC via a mechanism that appears to involve both TNF-alpha and TGF-beta. The results of this study support the possibility that cocaine and CMV could enhance HIV-1 replication and, thus, aggravate HIV-1-related disease.
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Hogaboam, Cory M., Nicholas W. Lukacs, Stephen W. Chensue, Robert M. Strieter e Steven L. Kunkel. "Monocyte Chemoattractant Protein-1 Synthesis by Murine Lung Fibroblasts Modulates CD4+ T Cell Activation". Journal of Immunology 160, n.º 9 (1 de maio de 1998): 4606–14. http://dx.doi.org/10.4049/jimmunol.160.9.4606.
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Abstract This study addressed the role of endogenous monocyte chemoattractant protein-1 (MCP-1) in Ag-stimulated lymphokine synthesis and proliferation by CD4+ T cells during their coculture with purified lung fibroblasts or splenic macrophages. Initial experiments showed that fibroblasts exposed to IL-4, TNF α, or IL-4 and TNF-α (all at 10 ng/ml) for 24 h released five- to eightfold more MCP-1 than similarly treated splenic macrophages. In 72-h coculture experiments, the synthesis of IL-4 by OVA-activated CD4+ T cells added to lung fibroblasts or splenic macrophages was significantly inhibited when endogenous MCP-1 was neutralized using polyclonal anti-MCP-1 antiserum. In these same cocultures, IFN-γ levels were significantly enhanced. Similarly, IFN-γ levels were significantly enhanced in 72-h cocultures of a purified peptide derivative-activated CD4+ Th1 clone and lung fibroblasts or splenic macrophages following immunoneutralization of MCP-1. In separate experiments, the selective inhibition of MCP-1 synthesis by lung fibroblasts and splenic macrophages using an MCP-1 antisense oligonucleotide significantly enhanced the proliferation of CD4+ T cells during a 96-h coculture. Taken together, these data suggest that MCP-1 exerts an immunomodulatory effect on CD4+ T cell-derived IL-4 and IFN-γ release and CD4+ T cell proliferation during cell-to-cell interactions.
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Weber, MC, e ML Tykocinski. "Bone marrow stromal cell blockade of human leukemic cell differentiation". Blood 83, n.º 8 (15 de abril de 1994): 2221–29. http://dx.doi.org/10.1182/blood.v83.8.2221.bloodjournal8382221.
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Bone marrow (BM) stromal cell inhibition of leukemic cell differentiation was studied in cellular coculture experiments. In coculture, a significant percentage of cells from the human myeloid leukemic cell lines HL-60, PLB-985, and K562 adhere to fibroblastic KM- 102 BM stromal cells. A sensitive two-color immunofluorescence assay was developed to monitor stromal cellular effects upon leukemic cell differentiation. After chemical induction with 1 alpha,25- dihydroxyvitamin D3, strongly adherent HL-60 and PLB-985 cells were inhibited from differentiating into more mature monocytic cells, as measured by the monocytic surface marker CD14. In contrast, loosely adherent and nonadherent HL-60 and PLB-985 leukemic cells in the same cocultures, as well as both adherent and nonadherent K562 cells induced with phorbol ester, were not blocked in their capacity to differentiate. Scanning electron microscopy and intercellular dye transfer experiments correlated intimate stromal cell/leukemic cell interaction and intercellular communication with the blockade of leukemic cell differentiation. These studies indicate that there is significant variability among leukemic lines with respect to the nature of their adhesion to stromal cells. Moreover, the data implicate gap- junction formation as a potentially significant event in stromal cell- mediated leukemic cell regulation.
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Chung, Daesuk, e Sanjoy K. Das. "Mouse Primary Uterine Cell Coculture System Revisited: Ovarian Hormones Mimic the Aspects of in Vivo Uterine Cell Proliferation". Endocrinology 152, n.º 8 (21 de junho de 2011): 3246–58. http://dx.doi.org/10.1210/en.2011-0223.
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Previously, the uterine epithelial-stromal coculture system had limited success mimicking in vivo ovarian hormone-dependent cell-specific proliferation. Here, we established a mouse primary uterine coculture system, in which cells collected in pseudopregnancy specifically on d 4 are conducive to supporting hormone-induced cell-specific proliferation. When two cell types are placed in coculture without direct contact via cell culture inserts (nonadjacent), as opposed to with contact (adjacent), epithelial cells exhibit significant proliferation by estradiol-17β (E2), whereas progesterone in combination with E2 caused inhibition of epithelial cell proliferation and a major shift in proliferation from epithelial to stromal cells. Epithelial cell integrity, with respect to E-cadherin expression, persisted in nonadjacent, but not adjacent, conditions. In subsequent studies of nonadjacent cocultures, localization of estrogen receptor (ER)α and progesterone receptor (PR), but not ERβ, appeared to be abundant, presumably indicating that specific ER or PR coregulator expression might be responsible for this difference. Consistently, an agonist of ERα, but not ERβ, was supportive of proliferation, and antagonists of ER or PR totally eliminated cell-specific proliferation by hormones. RT-PCR analyses also revealed that hormone-responsive genes primarily exhibit appropriate regulation. Finally, suppression of immunoglobulin heavy chain binding protein, a critical regulator of ERα signaling, in epithelial and/or stromal cells caused dramatic inhibition of E2-dependent epithelial cell proliferation, suggesting that a molecular perturbation approach is applicable to mimic in vivo uterine control. In conclusion, our established coculture system may serve as a useful alternative model to explore in vivo aspects of cell proliferation via communication between the epithelial and stromal compartments under the direction of ovarian hormones.
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Chen, Zhefeng, Kai Shen, Ziyang Zheng, Jinchun Zhou, Shujie Zhao, Huanghe Song, Jiuxiang Liu, Xuan Zhao, Feng Liu e Qiang Zuo. "Kindlin-2 Promotes Chondrogenesis and Ameliorates IL-1beta-Induced Inflammation in Chondrocytes Cocultured with BMSCs in the Direct Contact Coculture System". Oxidative Medicine and Cellular Longevity 2022 (12 de abril de 2022): 1–13. http://dx.doi.org/10.1155/2022/3156245.
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The osteoarthritis caused by trauma or inflammation is associated with severe patient morbidity and economic burden. Accumulating studies are focusing on the repair of articular cartilage defects by constructing tissue-engineered cartilage. Recent evidence suggests that optimizing the source and quality of seed cells is one of the key points of cartilage tissue engineering. In this study, we demonstrated that Kindlin-2 and its activated PI3K/AKT signaling played an essential role in promoting extracellular matrix (ECM) secretion and ameliorating IL-1beta-induced inflammation in chondrocytes cocultured with bone marrow stem cells (BMSCs). In vivo experiments revealed that coculture significantly promoted hyaline cartilage regeneration. In vitro studies further uncovered that chondrocytes cocultured with BMSCs in the direct contact coculture system upregulated Kindlin-2 expression and subsequently activated the PI3K/AKT signaling pathway, which not only increases Sox9 and Col2 expression but also restores mitochondrial membrane potential and reduces ROS levels and apoptosis under inflammatory conditions. Overall, our findings indicated that direct contact BMSC-chondrocyte coculture system could promote chondrogenesis, and identified Kindlin-2 represents a key regulator in this process.
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Goff, J. P., A. S. Kirshenbaum, J. P. Albert, S. W. Kessler e D. D. Metcalfe. "Human mast cells but not human basophils coculture with mouse 3T3 fibroblasts". Proceedings, annual meeting, Electron Microscopy Society of America 50, n.º 1 (agosto de 1992): 604–5. http://dx.doi.org/10.1017/s0424820100123428.
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Human mast cells and basophils have been shown to arise from CD34+ pluripotent progenitor cells in the presence of rhIL-3 (1). Two different culture systems using CD34+ cells, the agarose interphase culture and 3T3 fibroblast/CD34 + coculture, give rise to mast cells and basophils but with markedly different results. (1,2). By 3 weeks in interphase agarose cultures, CD34+ cells give rise to approximately 25-45% basophils and 1-5% tryptase positive mast cells. These mast cells have homogeneous granule patterns that resemble immature mast cells. With the addition of stem cell factor (rhSCF) (3) to rhIL-3, total cell number increases and mast cell maturation is promoted without altering the percentages of mast cells and basophils (4). These mast cells have tryptase-positive granules, with characteristic patterns seen in mature mast cells. In contrast, CD34+ cells cocultured with mouse 3T3 fibroblasts give rise by 6 weeks to approximately 50% tryptase positive mast cells which adher to the monolayer and possess granule scroll patterns. No basophils are ever identified by six weeks in cocultures.
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Juntunen, Miia, Sanna Hagman, Anaick Moisan, Susanna Narkilahti e Susanna Miettinen. "In Vitro Oxygen-Glucose Deprivation-Induced Stroke Models with Human Neuroblastoma Cell- and Induced Pluripotent Stem Cell-Derived Neurons". Stem Cells International 2020 (29 de outubro de 2020): 1–13. http://dx.doi.org/10.1155/2020/8841026.
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Stroke is a devastating neurological disorder and one of the leading causes of mortality and disability. To understand the cellular and molecular mechanisms of stroke and to develop novel therapeutic approaches, two different in vitro human cell-based stroke models were established using oxygen-glucose deprivation (OGD) conditions. In addition, the effect of adipose stem cells (ASCs) on OGD-induced injury was studied. In the present study, SH-SY5Y human neuroblastoma cells and human induced pluripotent stem cells (hiPSCs) were differentiated into neurons, cultured under OGD conditions (1% O2) for 24 h, and subjected to a reperfusion period for 24 or 72 h. After OGD, ASCs were cocultured with neurons on inserts for 24 or 72 h to study the neuroprotective potential of ASCs. The effect of OGD and ASC coculture on the viability, apoptosis, and proliferation of and axonal damage to neuronal cells was studied. The results showed that OGD conditions induced cytotoxicity and apoptosis of SH-SY5Y- and hiPSC-derived neurons, although more severe damage was detected in SH-SY5Y-derived neurons than in hiPSC-derived neurons. Coculture with ASCs was protective for neurons, as the number of dead ASC-cocultured neurons was lower than that of control cells, and coculture increased the proliferation of both cell types. To conclude, we developed in vitro human cell-based stroke models in SH-SY5Y- and hiPSC-derived neurons. This was the first time hiPSCs were used to model stroke in vitro. Since OGD had different effects on the studied cell types, this study highlights the importance of using several cell types in in vitro studies to confirm the outcomes of the study. Here, ASCs exerted a neuroprotective effect by increasing the proliferation and decreasing the death of SH-SY5Y- and hiPSC-derived neurons after OGD.
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Janusz, M. J., e M. Hare. "Cartilage degradation by cocultures of transformed macrophage and fibroblast cell lines. A model of metalloproteinase-mediated connective tissue degradation." Journal of Immunology 150, n.º 5 (1 de março de 1993): 1922–31. http://dx.doi.org/10.4049/jimmunol.150.5.1922.
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Abstract A number of human and mouse macrophage and fibroblast cell lines were examined for their ability to degrade cartilage proteoglycan in an attempt to establish a cell culture model of cartilage degradation. The mouse transformed macrophage cell line J774A.1 alone or in combination with the mouse transformed fibroblast cell line 10ME HD A.5R.1 were the only cell lines capable of extensively degradating cartilage proteoglycan. Incubation of the macrophage cell line J774A.1 on heat-killed cartilage disks resulted in the release of 36% +/- 8 (mean +/- SEM, n = 5) of the radiolabeled cartilage proteoglycan. The fibroblast cell line 10ME HD A.5R.1 alone did not degrade cartilage. However, cocultures of J774A.1 macrophages and 10ME HD A.5R.1 fibroblasts incubated on cartilage discs resulted in the release of 69% +/- 6 (mean +/- SEM, n = 5) of radiolabeled proteoglycan. There was little degradation of cartilage by macrophage/fibroblast cocultures during the first 3 days of culture. Cartilage degradation increased with each subsequent day in culture from 7% +/- 2 on day 4 to 68% +/- 3 (n = 3) by day 7. Supernatants from the macrophage/fibroblast cocultures were incubated with cartilage discs in the presence of general class-specific proteinase inhibitors. The metalloproteinase inhibitors 1,10 phenanthroline, EDTA, and recombinant tissue inhibitor of metalloproteinase were the only inhibitors that significantly blocked cartilage degradation by coculture supernatant. The cartilage degrading metalloproteinase in the macrophage/fibroblast coculture supernatant eluted as a broad peak on Sephacryl S-200HR with an estimated molecular mass between 22 and 55 kDa. These studies suggest that the macrophage/fibroblast coculture model of cartilage degradation may be a useful experimental system for the study of metalloproteinase-mediated connective tissue degradation.
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Dayton, E. T., J. P. Caulfield, A. Hein, K. F. Austen e R. L. Stevens. "Regulation of the growth rate of mouse fibroblasts by IL-3-activated mouse bone marrow-derived mast cells." Journal of Immunology 142, n.º 12 (15 de junho de 1989): 4307–13. http://dx.doi.org/10.4049/jimmunol.142.12.4307.
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Abstract When mouse bone marrow-derived mast cells (BMMC) are cocultured with a confluent layer of mouse 3T3 fibroblasts in the presence of WEHI-3-conditioned medium, the mast cells undergo a phenotypic change toward that of a connective tissue mast cell, and the fibroblasts increase their synthesis of globopentaosylceramide. We now demonstrate that fibroblasts lose their contact inhibition and multiply such that by the 2nd and the 4th wk of coculture there are, respectively, approximately four-fold and six-fold more fibroblasts than in the cultures that are not exposed to BMMC. This in vitro increase in the number of fibroblasts is dependent on the number of mast cells (over the range of 6 x 10(4) to 1 x 10(6) BMMC/culture) initially seeded with the fibroblasts and on the concentration of WEHI-3-conditioned medium present during the coculture. That the fibroblasts also multiply in BMMC/fibroblast cocultures exposed to synthetic IL-3 or to purified IL-3 indicates that IL-3 is a component in WEHI-3-conditioned medium that induces mast cells to produce the fibroblast growth factor. The number of fibroblasts does not increase if fibroblasts are exposed to lysates of BMMC, or to BMMC-derived conditioned medium, or if the two cell types are separated from one another during the coculture with a 3-microns filter or a 0.4-microns filter. Thus, IL-3-activated BMMC must be in proximity to fibroblasts to induce them to multiply. Because of their increased numbers per culture dish, total fibroblasts that were cocultured with mast cells synthesized approximately two-fold more 35S-labeled proteoglycans, incorporated approximately 3-fold more [3H] proline into collagenase-sensitive proteins, and had substantially more alpha 2(I) collagen mRNA than fibroblasts that were maintained in the absence of mast cells. These is vitro studies reveal a sequence by which IL-3-activated mast cells may play a role in the induction of fibrosis.
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Parfenova, H., T. H. Eidson e C. W. Leffler. "Upregulation of COX-2 in cerebral microvascular endothelial cells by smooth muscle cell signals". American Journal of Physiology-Cell Physiology 273, n.º 1 (1 de julho de 1997): C277—C288. http://dx.doi.org/10.1152/ajpcell.1997.273.1.c277.
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Cyclooxygenase (COX) isoform expression, intracellular localization, and function in endothelial cells from the newborn pig cerebral microvessels were investigated using COX-1- and COX-2-specific antibodies and the COX-2 inhibitor NS-398. Cerebral microvessels, microvascular endothelium, and cultured endothelial cells constitutively express both COX-1 and COX-2. NS-398 inhibits 70-90% of endothelial prostanoid production. Endothelial cells grown in noncontact coculture with smooth muscle cells for 24-48 h demonstrate a stable induction of COX-2 protein and an NS-398-sensitive increase in prostanoid synthesis. The induction of endothelial COX in mixed cell coculture is accompanied by intracellular redistribution of COX-2. In cocultured endothelial cells, COX-2 is observed in the nucleus, nuclear envelope, and cytoplasm, compared with the mainly intranuclear localization of COX-2 in cells cultured separately. No changes were observed in COX-1 protein, localized in endothelial cell cytoplasm and the nuclear envelope. These results indicate that smooth muscle cells may modify endothelial function by upregulating COX-2, which is a major functional COX isoform in cerebral microvascular endothelial cells.
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Nishiyama-Naruke, Anita, e Rui Curi. "Phosphatidylcholine participates in the interaction between macrophages and lymphocytes". American Journal of Physiology-Cell Physiology 278, n.º 3 (1 de março de 2000): C554—C560. http://dx.doi.org/10.1152/ajpcell.2000.278.3.c554.
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The role of phosphatidylcholine molecules as mediator for the control of lymphocyte proliferation by macrophages was investigated. Phosphatidylcholine added to the culture medium inhibited the concanavalin A-stimulated lymphocyte proliferation in a concentration-dependent manner. The potency of this effect was dependent on the presence of arachidonic acid in the phosphatidylcholine molecules. The phosphatidylcholine transfer from macrophages to lymphocytes was then investigated. Macrophages incorporated phosphatidylcholine at a much higher rate than lymphocytes and exported phosphatidylcholine to the culture medium. When cocultured, a significant amount of phosphatidylcholine incorporated by macrophages was transferred to lymphocytes. To examine the possible physiological importance of the transfer process, the lymphocyte proliferation was measured in coculture conditions. Macrophages were treated with phosphatidylcholine and washed, and then these cells were cocultured with concanavalin A-stimulated lymphocytes. The effect observed in coculture was an inhibition of lymphocyte proliferation, which was also dependent on the molecular species of the phosphatidylcholine. Therefore, phosphatidylcholine may act as a mediator of the macrophage effect on lymphocyte proliferation.
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Gupta, Pankaj, Bruce R. Blazar, Kalpna Gupta e Catherine M. Verfaillie. "Human CD34+ Bone Marrow Cells Regulate Stromal Production of Interleukin-6 and Granulocyte Colony-Stimulating Factor and Increase the Colony-Stimulating Activity of Stroma". Blood 91, n.º 10 (15 de maio de 1998): 3724–33. http://dx.doi.org/10.1182/blood.v91.10.3724.
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Abstract Cytokines produced by stromal cells induce the proliferation and differentiation of hematopoietic cells in the marrow microenvironment. We hypothesized that cross-talk between hematopoietic cells at different stages of differentiation and stromal cells influences stromal cytokine production and is responsible for maintaining steady-state hematopoiesis and responding to stress situations. We show that coculture of primitive CD34+ cells in contact with or separated by a transwell membrane from irradiated human bone marrow stromal layers induces a fourfold to fivefold increase in interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) levels in the stromal supernatant (SN) during the first week. Levels of both cytokines decreased to baseline after coculture of CD34+cells for 3 to 5 weeks. Coculture of more mature CD15+/CD14− myeloid precursors induced only a transient 1.5- to 2-fold increase in IL-6 and G-CSF at 48 hours. Neither CD34+ nor CD15+/CD14−cells produced IL-6, G-CSF, IL-1β, or tumor necrosis factor α. When CD34+ cells were cultured in methylcellulose medium supplemented with cytokines at concentrations found in stromal SN or supplemented with stromal SN, a fourfold to fivefold increase in colony formation was seen over cultures supplemented with erythropoietin (EPO) only. When cultures were supplemented with the increased concentrations of IL-6 and G-CSF detected in cocultures of stroma and CD34+ cells or when CD34+ cells were cocultured in methylcellulose medium in a transwell above a stromal layer, a further increase in the number and size of colonies was seen. The colony-forming unit–granulocyte-macrophage–stimulating activity of stromal SN was neutralized by antibodies against G-CSF or IL-6. These studies indicate that primitive CD34+ progenitors provide a soluble positive feedback signal to induce cytokine production by stromal cells and that the observed increase in cytokine levels is biologically relevant.
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Gupta, Pankaj, Bruce R. Blazar, Kalpna Gupta e Catherine M. Verfaillie. "Human CD34+ Bone Marrow Cells Regulate Stromal Production of Interleukin-6 and Granulocyte Colony-Stimulating Factor and Increase the Colony-Stimulating Activity of Stroma". Blood 91, n.º 10 (15 de maio de 1998): 3724–33. http://dx.doi.org/10.1182/blood.v91.10.3724.3724_3724_3733.
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Cytokines produced by stromal cells induce the proliferation and differentiation of hematopoietic cells in the marrow microenvironment. We hypothesized that cross-talk between hematopoietic cells at different stages of differentiation and stromal cells influences stromal cytokine production and is responsible for maintaining steady-state hematopoiesis and responding to stress situations. We show that coculture of primitive CD34+ cells in contact with or separated by a transwell membrane from irradiated human bone marrow stromal layers induces a fourfold to fivefold increase in interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) levels in the stromal supernatant (SN) during the first week. Levels of both cytokines decreased to baseline after coculture of CD34+cells for 3 to 5 weeks. Coculture of more mature CD15+/CD14− myeloid precursors induced only a transient 1.5- to 2-fold increase in IL-6 and G-CSF at 48 hours. Neither CD34+ nor CD15+/CD14−cells produced IL-6, G-CSF, IL-1β, or tumor necrosis factor α. When CD34+ cells were cultured in methylcellulose medium supplemented with cytokines at concentrations found in stromal SN or supplemented with stromal SN, a fourfold to fivefold increase in colony formation was seen over cultures supplemented with erythropoietin (EPO) only. When cultures were supplemented with the increased concentrations of IL-6 and G-CSF detected in cocultures of stroma and CD34+ cells or when CD34+ cells were cocultured in methylcellulose medium in a transwell above a stromal layer, a further increase in the number and size of colonies was seen. The colony-forming unit–granulocyte-macrophage–stimulating activity of stromal SN was neutralized by antibodies against G-CSF or IL-6. These studies indicate that primitive CD34+ progenitors provide a soluble positive feedback signal to induce cytokine production by stromal cells and that the observed increase in cytokine levels is biologically relevant.
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Weber, MC, e ML Tykocinski. "Bone marrow stromal cell blockade of human leukemic cell differentiation". Blood 83, n.º 8 (15 de abril de 1994): 2221–29. http://dx.doi.org/10.1182/blood.v83.8.2221.2221.
Texto completo da fonteResumo:
Abstract Bone marrow (BM) stromal cell inhibition of leukemic cell differentiation was studied in cellular coculture experiments. In coculture, a significant percentage of cells from the human myeloid leukemic cell lines HL-60, PLB-985, and K562 adhere to fibroblastic KM- 102 BM stromal cells. A sensitive two-color immunofluorescence assay was developed to monitor stromal cellular effects upon leukemic cell differentiation. After chemical induction with 1 alpha,25- dihydroxyvitamin D3, strongly adherent HL-60 and PLB-985 cells were inhibited from differentiating into more mature monocytic cells, as measured by the monocytic surface marker CD14. In contrast, loosely adherent and nonadherent HL-60 and PLB-985 leukemic cells in the same cocultures, as well as both adherent and nonadherent K562 cells induced with phorbol ester, were not blocked in their capacity to differentiate. Scanning electron microscopy and intercellular dye transfer experiments correlated intimate stromal cell/leukemic cell interaction and intercellular communication with the blockade of leukemic cell differentiation. These studies indicate that there is significant variability among leukemic lines with respect to the nature of their adhesion to stromal cells. Moreover, the data implicate gap- junction formation as a potentially significant event in stromal cell- mediated leukemic cell regulation.
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Doukas, J., e J. S. Pober. "Lymphocyte-mediated activation of cultured endothelial cells (EC). CD4+ T cells inhibit EC class II MHC expression despite secreting IFN-gamma and increasing EC class I MHC and intercellular adhesion molecule-1 expression." Journal of Immunology 145, n.º 4 (15 de agosto de 1990): 1088–98. http://dx.doi.org/10.4049/jimmunol.145.4.1088.
Texto completo da fonteResumo:
Abstract Endothelial cells (EC) were cocultured with allogeneic PBL, CD4+ T cells, or CD8+ T cells, and the degrees of EC activation induced examined by determining patterns of endothelial class I and class II MHC and intercellular adhesion molecule-1 (ICAM-1) expression. Coculture with PBL or CD8+ T cells uniformly increases class I MHC and ICAM-1 expression on all EC within a culture, but induces class II MHC expression on only a subpopulation(s) of EC. This heterogeneous EC response to coculture contrasts with the uniform class II expression on all EC induced by IFN-gamma in replicate wells. CD4+ T cells, when compared to equal numbers of unfractionated PBL or CD8+ T cells, are more effective at increasing class I MHC and ICAM-1 but are unable to induce class II MHC expression. The failure of CD4+ T cells to induce EC class II MHC Ag is not due to insufficient activation of the T cells, as PHA-activated CD4+ T cells also do not induce significant class II expression. In addition, conditioned media (CM) from CD4+ T cell/EC contain greater levels of immunoreactive IFN-gamma than do CM from PBL/EC cocultures. Rather, CD4+ T cells appear to actively inhibit the induction of EC class II Ag but not class I or ICAM-1 by IFN-gamma. Inhibition occurs at the time of induction, as CD4+ T cells are not capable of down-regulating previously induced class II Ag. CM from CD4+/EC (but not PBL/EC) cocultures also inhibits IFN-gamma induction of EC class II MHC expression. The inhibitory activity is generated during CD4+ T cell-EC cell contact, and is enhanced by PHA. The inhibitory activity(ies) of the CD4+/EC-CM is as yet unidentified, and is only minimally reversible by cocktails of neutralizing antibodies directed against TNF-alpha, TNF-beta (lymphotoxin), IFN-alpha and IFN-beta. In conclusion, CD4+ and CD8+ T cells are each effective activators of EC, but the patterns of activation produced by these subsets are quite distinct, largely due to generation of a soluble inhibitor(s) of class II MHC induction during coculture of CD4+ T cells with EC.