RESUMEN
Recombinant retroviruses are most commonly used in hematopoietic stem cell gene therapy trials, but gene transfer efficiency is still inadequate with the present vectors. One approach for overcoming this problem is to develop methods of selecting and enriching the successfully transduced cells. We investigated the feasibility of using the green fluorescent protein (GFP) gene as a selectable marker of hematopoietic cells. When M1 murine leukemia cells were electroporated with GFP expression vectors, a red-shifted mutant (S65T) GFP showed several-fold greater fluorescence than the wild-type GFP and generated readily detectable green light under control of SRalpha or CAG promoter. We then inserted an SRalpha-S65T GFP cassette into the MSCV retrovirus vector and established virus producer cells. Infection of primary murine bone marrow cells resulted in a distinct population with green fluorescence, which was separated by fluorescence-activated cell sorting. The fractionated bright cells gave rise to fluorescent spleen colonies in lethally irradiated mice, while the fluorescence-negative cells yielded only dark colonies. These results indicated that GFP is a faithful marker in gene transfer into hematopoietic progenitor/stem cells, facilitating selection of the transduced cells and tracking of their progeny in vivo.
Asunto(s)
Vectores Genéticos , Células Madre Hematopoyéticas , Proteínas Luminiscentes , Retroviridae , Transducción Genética , Células 3T3 , Animales , Biomarcadores , Células Cultivadas , Citometría de Flujo , Fluorescencia , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas/virología , Leucemia Mieloide Aguda , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Bazo/citología , Células Tumorales CultivadasRESUMEN
To analyze the molecular mechanisms of the proliferation and differentiation of hematopoietic cells, we have cloned PTKs from sorted stem cells. We discuss the expression and function of receptor tyrosine kinases, STK/RON, TIE, TEK and HTK which have been cloned from these cells. STK and its ligand, MSP contributed to the motility and phagocytosis of peritoneal macrophages and bone absorption of osteoclasts. Apoptosis was induced in an erythroid cell line by the binding of MSP(MacrophageStimulating Protein). TIE, TEK and HTK were interestingly expressed in the subpopulations of stem cells and related to the myeloid differentiation. These study will indicate the heterogeneity of stem cells and their diverse differentiation.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Factor de Crecimiento de Hepatocito , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Apoptosis/efectos de los fármacos , Resorción Ósea , Diferenciación Celular , División Celular , Movimiento Celular , Clonación Molecular , Ensayo de Unidades Formadoras de Colonias , Sustancias de Crecimiento/farmacología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/fisiología , Ratones , Osteoclastos/fisiología , Fagocitosis , Receptor EphB4 , Receptor TIE-2 , Receptores de Superficie Celular/metabolismo , Receptores TIE , Proteínas Recombinantes/metabolismoRESUMEN
Tissue nonspecific alkaline phosphatase (TNAP), the product of the Akp2 locus, is expressed in mouse primordial germ cells (PGC) for an extensive period during embryogenesis. Mice with the Akp2tm1Sor mutant allele of TNAP express lacZ (beta-galactosidase; beta-gal) under control of the Akp2 locus. PGCs were purified from Akp2tm1Sor embryos using fluorescence activated cell sorting of beta-gal expressing cells (FACS-gal). Analysis of the purified cells by alkaline phosphatase staining and immunocytochemistry with anti-c-kit antibody demonstrated that highly (98%) purified PGCs can be isolated using this method. This technique will facilitate experiments that require highly purified preparations of PGCs including cell culture and gene expression analyses.
Asunto(s)
Fosfatasa Alcalina/biosíntesis , Células Germinativas/citología , Fosfatasa Alcalina/genética , Animales , Separación Celular/métodos , Cruzamientos Genéticos , Embrión de Mamíferos/citología , Femenino , Citometría de Flujo/métodos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
p59fyn is one of the Src-family kinases thought to play an important role in signaling through T cell receptor. However, Fyn deficiency has caused no overt defects in vivo on T cell development, nor has it caused any changes in the phosphorylation status of molecules such as ZAP-70 which have been proposed as p59fyn substrates. This could be explained as being due to compensation of Fyn deficiency by other Src-family kinases. Here, we have 'knocked-in' the csk gene, a negative regulator of Src-family kinases, into fyn locus to challenge the problem of redundant functions among Src-family kinases. The csk-'knock-in' mice displayed atrophy of the thymic cortex and impaired development of CD4+ CD8+ thymocytes. This was concomitant with decrease in tyrosine phosphorylation of ZAP-70 and p120cbl.
Asunto(s)
Hematopoyesis/genética , Mutagénesis Insercional , Proteínas Proto-Oncogénicas/fisiología , Subgrupos de Linfocitos T/patología , Timo/patología , Ubiquitina-Proteína Ligasas , Familia-src Quinasas/genética , Animales , Secuencia de Bases , Proteína Tirosina Quinasa CSK , Diferenciación Celular , Marcación de Gen , Genes Sintéticos , Genes src , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Proteínas Proto-Oncogénicas c-fyn , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína Tirosina Quinasa ZAP-70RESUMEN
CHK/HYL is a non-receptor tyrosine kinase that belongs to CSK (C-terminal Src kinase) family. Northern blotting and RT-PCR analyses showed that CHK/HYL was expressed in large spectrum of hematopoietic cells except for erythroid cells and brain. To explore the function of CHK/HYL in hematopoietic cells, we generated CHK/HYL deficient mice. The mutant mice were apparently normal and fertile, while CSK knockout mice died until E11.5 from a defect in the neural tube formation. Hematological observations including blood counts and FACS analysis showed no significant abnormalities in CHK/HYL mutant mice. CHK/HYL did not affect the activity of Src, Hck, and Fgr in cultured bone marrow cells, although CSK negatively regulates Src family kinases. These results suggest that CHK/HYL might not have the same function as CSK.
Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/enzimología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src) , Animales , Linfocitos B/inmunología , Secuencia de Bases , Recuento de Células Sanguíneas , Médula Ósea/enzimología , Células Cultivadas , Quimera , Cruzamientos Genéticos , Cartilla de ADN , Femenino , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Mutagénesis , Especificidad de Órganos , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , Bazo , Linfocitos T/inmunología , TimoRESUMEN
CD34 is expressed on the surface of hematopoietic stem/progenitor cells, stromal cells, and on the surface of high-endothelial venules (HEV). CD34 binds L-selectin, an adhesion molecule important for leukocyte rolling on venules and lymphocyte homing to peripheral lymph nodes (PLN). We generated CD34-deficient mutant animals through the use of homologous recombination. Wild-type and mutant animals showed no differences in lymphocyte binding to PLN HEV, in leukocyte rolling on venules or homing to PLN, in neutrophil extravasation into peritoneum in response to inflammatory stimulus, nor in delayed type hypersensitivity. Anti-L-selectin monoclonal antibody (MEL-14) also inhibited these immune responses similarly in both CD34-deficient and wild-type mice. However, eosinophil accumulation in the lung after inhalation of a model allergen, ovalbumin, is several-fold lower in mutant mice. We found no abnormalities in hematopoiesis in adult mice and interactions between mutant progenitor cells and a stromal cell line in vitro were normal. No differences existed in the recovery of progenitor cells after 5-fluorouracil treatment, nor in the mobilization of progenitor cells after granulocyte colony-stimulating factor treatment compared with wild-type animals. Surprisingly, although CD34 was not expressed in these mice, a portion of its 90-kD band crossreactive with MECA79 remained after Western blot. Thus, we have identified an additional molecule(s) that might be involved in leukocyte trafficking. These results indicate that CD34 plays an important role in eosinophil trafficking into the lung.
Asunto(s)
Alérgenos/inmunología , Antígenos CD34/inmunología , Movimiento Celular/inmunología , Eosinófilos/inmunología , Hematopoyesis/inmunología , Animales , Antígenos CD34/genética , Secuencia de Bases , Recuento de Células , Reacciones Cruzadas , Eosinófilos/citología , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Proteínas/inmunología , Proteínas/aislamiento & purificaciónRESUMEN
p59fyn is an Src family nonreceptor tyrosine kinase that has been suggested to play an important role in T-cell development and function. p125FAK is a unique nonreceptor tyrosine kinase and has been known to respond to integrin-extracellular matrix interactions. To examine their roles in thymocytes, heterozygous fak mutation was introduced into homozygous Fyn deficiency. The double mutation, but neither Fyn deficiency nor FAK heterozygosity alone, displayed impaired development of CD4+CD8+ thymocytes with atrophy of the thymic cortex, suggesting a unique cooperation between p59fyn and p125FAK in CD4+CD8+ T-cell development.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Subgrupos de Linfocitos T/citología , Animales , Atrofia , Secuencia de Bases , Antígenos CD4 , Antígenos CD8 , Diferenciación Celular , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Genotipo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas/deficiencia , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas c-fyn , Timo/patologíaRESUMEN
The interleukin-2 (IL-2) receptor gamma chain is indispensable for IL-2-, IL-4-, IL-7-, IL-9-, and IL-15-mediated signaling. Mutations of the human gamma chain cause the X-linked severe combined immunodeficiency (XSCID), showing that T and natural killer cells absolutely require the gamma chain for their development in humans. To elucidate the roles of the gamma chain in hematopoiesis, we have generated mice, by gene targeting, that express a form of the gamma chain lacking the cytoplasmic region. Male mice carrying the truncated gamma-chain mutant, which mimics mutations in patients with XSCID, showed a decrease in the number of lymphocytes and an increase in monocytes; the number of T cells was profoundly reduced and no natural killer cells were detected, which is similar to the characteristic of human XSCID. Unlike human XSCID, the levels of B cells were also reduced. In spite of the severe decrease in CD45R+/sIgM+ B cells, the level of IgM in serum of the 8-week-old mutant mice was higher than that of control littermates. Interestingly, the stem cell population with surface phenotypes of CD34, c-kit, and Sca-1 was significantly increased. Furthermore, the colony-forming assay showed that the mutant mice had 15-fold higher numbers of hematopoietic progenitor cells in the spleen as compared with that of controls. These results indicate that functional loss of the gamma chain causes significant effects on the immunological system in mice.
Asunto(s)
Modelos Animales de Enfermedad , Hematopoyesis/fisiología , Receptores de Interleucina-2/genética , Inmunodeficiencia Combinada Grave/genética , Transducción de Señal/fisiología , Animales , Subgrupos de Linfocitos B/inmunología , Médula Ósea/patología , Concanavalina A/farmacología , Expresión Génica , Marcación de Gen , Células Madre Hematopoyéticas/patología , Humanos , Inmunoglobulina M/sangre , Inmunofenotipificación , Activación de Linfocitos , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interleucina-2/química , Receptores de Interleucina-2/fisiología , Eliminación de Secuencia , Inmunodeficiencia Combinada Grave/fisiopatología , Bazo/patología , Subgrupos de Linfocitos T/inmunología , Timo/patología , Cromosoma XRESUMEN
A receptor tyrosine kinase (RTK), TIE (tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor [EGF] homology domains), is expressed in vascular endothelial and hematopoietic cells. We generated monoclonal antibodies (MoAbs) against the extracellular domain of TIE and a polyclonal antibody against the TIE carboxyterminus and used them to analyze expression of TIE in hematopoietic cells. Western blotting detected two forms of TIE protein with a molecular mass of 135 and 130 kD in hematopoietic and endothelial cells. Northern blotting analysis revealed that TIE was expressed preferentially in undifferentiated cell lines, especially when megakaryocytic, but not erythroid differentiation was induced. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that TIE was predominantly expressed in the human hematopoietic progenitor fraction, CD34+ cells. Fluorescence-activated cell sorting (FACS) showed that 42% of CD34+ and 17% of KIT-positive (KIT+) cells were TIE-positive (TIE+). The majority (81%) of the primitive hematopoietic stem cells, CD34+CD38- cells, were TIE+. Assays of progenitor cells and long-term culture-initiating cells (LTC-IC) showed that the TIE+ fraction contained more primitive cells than the TIE- fraction. Some TIE+ cells were in the CD34- fraction, which were CD19+ and CD20+ (B cells). These findings indicate that TIE has a unique spectrum of expression in primitive hematopoietic stem cells and B cells. Although its ligand has not been identified, TIE and its ligand may establish a novel regulatory pathway not only in early hematopoiesis, but also in the differentiation and/or proliferation of B cells.
Asunto(s)
Linfocitos B/enzimología , Células Madre Hematopoyéticas/enzimología , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores de Superficie Celular/biosíntesis , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Diferenciación Celular , Línea Celular , ADN Complementario/genética , Inducción Enzimática , Fibroblastos/enzimología , Citometría de Flujo , Humanos , Leucemia Eritroblástica Aguda/enzimología , Leucemia Eritroblástica Aguda/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores TIE , Células Tumorales CultivadasRESUMEN
Interleukin-13 (IL-13) is a pleiotropic cytokine that inhibits the production of inflammatory cytokines of monocytes. We investigated the effects of IL-13 on the clonal growth of human hematopoietic progenitors. IL-13 alone did not support any colony formation. IL-13 markedly suppressed macrophage colonies that were formed in the presence of IL-3 and erythropoietin, granulocyte-macrophage colony-stimulating factor, or macrophage colony-stimulating factor. Macrophage colony cells showed dendritic cell-line morphology and cellular aggregates. IL-13 did not affect granulocyte colony and erythroid burst formation. Delayed addition of IL-13 and replating onto the culture dishes with IL-13 showed that macrophage colony formation was suppressed during days 8 and 14 of culture. These results indicate that IL-13 affects the growth of the late stage of committed macrophage progenitors. Single-cell culture of isolated CD34+CD33+ cells with IL-13 confirmed that macrophage colony formation was significantly suppressed. These results show that IL-13 directly suppresses the proliferation of differentiating macrophages. In addition, these suppressive effects of IL-13 were synergistic with IL-4. Furthermore, in the liquid culture of bone marrow cells in the presence of IL-13, the number of CD14 (monocyte-macrophage antigen)-positive cells decreased and CD18 (LFA-1 beta)-positive cells increased. It is concluded that IL-13 affects the growth of the late stage of macrophage precursors as well as mature monocytes. Induction of differentiation of human monocytes may be correlated with the suppression of their progenitors.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-13/farmacología , Células de la Médula Ósea , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Sinergismo Farmacológico , Células Madre Hematopoyéticas/citología , Humanos , Interleucina-4/farmacología , Macrófagos/citologíaRESUMEN
To identify receptor tyrosine kinases (RTKs) critical to early hematopoiesis, we performed polymerase chain reaction-based cloning from yolk sac and highly enriched bone marrow hematopoietic stem cells (HSCs). Characterization of two novel genes of their full-length cDNA sequences revealed that they were mouse homologues of the endothelial cell RTK genes, TIE and TEK. They shared a unique structural property of coexistent immunoglobulin-like domain, epidermal growth factor-like repeats, and fibronectin type III repeats in their extracellular domains. Both genes were expressed in a similar fashion in adult tissues and primitive hematopoietic cells, predominantly in the bone marrow HSCs.