Assuntos
Anemia Falciforme/patologia , Plaquetas/metabolismo , Eritrócitos/patologia , Neutrófilos/patologia , Anemia Falciforme/tratamento farmacológico , Antidrepanocíticos/uso terapêutico , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Eritrócitos/metabolismo , Humanos , Hidroxiureia/uso terapêutico , Neutrófilos/metabolismo , FenótipoRESUMO
The development of microfluidic chip-based cytometers has become an important area due to their advantages of compact size and low cost. Herein, we demonstrate a sheathless microfluidic cytometer which integrates a standing surface acoustic wave (SSAW)-based microdevice capable of 3D particle/cell focusing with a laser-induced fluorescence (LIF) detection system. Using SSAW, our microfluidic cytometer was able to continuously focus microparticles/cells at the pressure node inside a microchannel. Flow cytometry was successfully demonstrated using this system with a coefficient of variation (CV) of less than 10% at a throughput of ~1000 events s(-1) when calibration beads were used. We also demonstrated that fluorescently labeled human promyelocytic leukemia cells (HL-60) could be effectively focused and detected with our SSAW-based system. This SSAW-based microfluidic cytometer did not require any sheath flows or complex structures, and it allowed for simple operation over a wide range of sample flow rates. Moreover, with the gentle, bio-compatible nature of low-power surface acoustic waves, this technique is expected to be able to preserve the integrity of cells and other bioparticles.
Assuntos
Citometria de Fluxo/métodos , Técnicas Analíticas Microfluídicas/métodos , Células-Tronco Neoplásicas/citologia , Citometria de Fluxo/instrumentação , Corantes Fluorescentes/química , Células HL-60 , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Tamanho da Partícula , SomRESUMO
BACKGROUND: In murine embryonic stem cells, the onset of vascular endothelial growth factor receptor 2 (VEGFR-2) expression identifies endothelial precursors. Undifferentiated human embryonic stem cells express VEGFR-2, and VEGFR-2 expression persists on differentiation. The objective of our study was to identify a single population of endothelial precursors with common identifying features from both human and murine embryonic stem cells. METHODS AND RESULTS: We report that expression of the VEGF coreceptor neuropilin-1 (NRP-1) coincides with expression of Brachyury and VEGFR-2 and identifies endothelial precursors in murine and human embryonic stem cells before CD31 or CD34 expression. When sorted and differentiated, VEGFR-2(+)NRP-1(+) cells form endothelial-like colonies that express CD31 and CD34 7-fold more efficiently than NRP-1 cells. Finally, antagonism of both the VEGF and Semaphorin binding functions of NRP-1 impairs the differentiation of vascular precursors to endothelial cells. CONCLUSIONS: The onset of NRP-1 expression identifies endothelial precursors in murine and human stem cells. The findings define the origin of a single population of endothelial precursors from human and murine stem cells to endothelial cells. Additionally, the function of both the VEGF and Semaphorin binding activities of NRP-1 has important roles in the differentiation of stem cells to endothelial cells, providing novel insights into the role of NRP-1 in a model of vasculogenesis.
Assuntos
Antígenos CD34/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Neovascularização Fisiológica/fisiologia , Neuropilina-1/genética , Animais , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Meios de Cultura Livres de Soro/farmacologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Técnicas In Vitro , Camundongos , Neuropilina-1/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Semaforinas/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cellular inactivation through killer immunoglobulin-like receptors (KIRs) may allow neoplastic cells to evade host natural killer (NK) cell-mediated immunity. Recently, alloreactive NK cells were shown to mediate antileukemic effects against acute myelogenous leukemia (AML) after mismatched transplantation, when KIR ligand incompatibility existed in the direction of graft-versus-host disease (GVHD). Therefore, we investigated whether solid tumor cells would have similar enhanced susceptibility to allogeneic KIR-incompatible NK cells compared with their KIR-matched autologous or allogeneic counterparts. NK populations enriched and cloned from the blood of cancer patients or healthy donors homozygous for HLA-C alleles in group 1 (C-G1) or group 2 (C-G2) were tested in vitro for cytotoxicity against Epstein-Barr virus-transformed lymphoblastic cell lines (EBV-LCLs), renal cell carcinoma (RCC), and melanoma (MEL) cells with or without a matching KIR-inhibitory HLA-C ligand. Allogeneic NK cells were more cytotoxic to tumor targets mismatched for KIR ligands than their KIR ligand-matched counterparts. Bulk NK populations (CD3(-)/CD2(+)/CD56(+)) expanded 10(4)-fold from patients homozygous for C-G1 or C-G2 had enhanced cytotoxicity against KIR ligand-mismatched tumor cells but only minimal cytotoxicity against KIR ligand-matched targets. Further, NK cell lines from C-G1 or C-G2 homozygous cancer patients or healthy donors expanded but failed to kill autologous or KIR-matched MEL and RCC cells yet had significant cytotoxicity (more than 50% lysis at 20:1 effector-target [E/T] ratio) against allogeneic KIR-mismatched tumor lines. These data suggest immunotherapeutic strategies that use KIR-incompatible allogeneic NK cells might have superior antineoplastic effects against solid tumors compared with approaches using autologous NK cells.