RESUMO
Bone marrow stromal cells taken from EGFP transgenic mice were sorted by magnetic beads with surface markers for Sca-1 and Thy-1. The cells were then co-cultured on organotypic hippocampal slice or with neuronal cell feeder in dish. On hippocampus, both Sca-1 and Thy-1 positive cells showed 4- 8 folds higher potential to show neuron-like morphology than negative cells. In dish, negative cells fewly survived but each positive cells survived and showed neuron-like differentiation. In both culture condition, retinoic acid supplement accelerate differentiation. Differentiated Sca-1 and Thy-1 positive cells were immunohistochemically GFAP- and NeuN-negative but nestin-, neurofilament- and NSE-positive. Neuron-like differentiation of bone marrow cells can be enhanced by selection using cell surface proteins.
Assuntos
Antígenos Ly/fisiologia , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Proteínas de Membrana/fisiologia , Neurônios/citologia , Antígenos Thy-1/fisiologia , Animais , Antígenos Ly/biossíntese , Antígenos Ly/genética , Células da Medula Óssea/metabolismo , Células Cultivadas , Técnicas de Cocultura , Genes Reporter , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Neurônios/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Antígenos Thy-1/biossíntese , Antígenos Thy-1/genéticaRESUMO
The development of tumor vasculature is thought to occur through two complementary processes: sprouting angiogenesis from preexisting blood vessels of the host, and vasculogenesis, which involves the spontaneous development of vessels through specific recruitment, differentiation, and vascular incorporation of circulating endothelial cells (EC), endothelial progenitor cells (EPC), or potentially bone marrow-derived cells. Recent reports, however, have challenged the belief that bone marrow-derived cells contribute to tumor neovascularization, claiming an exclusive role for sprouting angiogenesis in tumor blood vessel development. In the present study, we explored the recruitment behavior of bone marrow-derived lin(-)c-kit(+)Sca-1+ stem cells to subcutaneously implanted Lewis lung carcinoma in a syngeneic bone marrow transplantation model. We observed that although lin(-)c-kit(+)Sca-1+ and their derived cells demonstrate significant recruitment to carcinomas in vivo, they do not appear to functionally contribute to tumor neovascularization. Furthermore, our results support the hypothesis that new vessel formation in carcinomas occurs primarily through endothelialization from adjacent and preexisting vasculature.
Assuntos
Antígenos Ly/genética , Antígenos Ly/fisiologia , Células da Medula Óssea/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Neovascularização Patológica , Oxiquinolina/análogos & derivados , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/fisiologia , Animais , Medula Óssea/patologia , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Carcinoma , Carcinoma Pulmonar de Lewis/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Células Endoteliais/metabolismo , Endotélio Vascular/patologia , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Transplante de Neoplasias , Compostos Organometálicos/química , Oxiquinolina/química , Células-Tronco/citologia , Distribuição TecidualRESUMO
NK cells can migrate into sites of inflammatory responses or malignancies in response to chemokines. Target killing by rodent NK cells is restricted by opposing signals from inhibitory and activating Ly49 receptors. The rat NK leukemic cell line RNK16 constitutively expresses functional receptors for the inflammatory chemokine CXC chemokine ligand (CXCL)10 (CXCR3) and the homeostatic chemokine CXCL12 (CXCR4). RNK-16 cells transfected with either the activating Ly49D receptor or the inhibitory Ly49A receptor were used to examine the effects of NK receptor ligation on CXCL10- and CXCL12-mediated chemotaxis. Ligation of Ly49A, either with Abs or its MHC class I ligand H2-D(d), led to a decrease in chemotactic responses to either CXCL10 or CXCL12. In contrast, Ly49D ligation with Abs or H2-D(d) led to an increase in migration toward CXCL10, but a decrease in chemotaxis toward CXCL12. Ly49-dependent effects on RNK-16 chemotaxis were not the result of surface modulation of CXCR3 or CXCR4 as demonstrated by flow cytometry. A mutation of the Src homology phosphatase-1 binding motif in Ly49A completely abrogated Ly49-dependent effects on both CXCL10 and CXCL12 chemotaxis, suggesting a role for Src homology phosphatase-1 in Ly49A/chemokine receptor cross-talk. Ly49D-transfected cells were pretreated with the Syk kinase inhibitor Piceatannol before ligation, which abrogated the previously observed changes in migration toward CXCL10 and CXCL12. Piceatannol also abrogated Ly49A-dependent inhibition of chemotaxis toward CXCL10, but not CXCL12. Collectively, these data suggest that Ly49 receptors can influence NK cell chemotaxis within sites of inflammation or tumor growth upon interaction with target cells.