RESUMEN
Hematopoietic stem cells play the vital role of maintaining appropriate levels of cells in blood. Therefore, regulation of their fate is essential for their effective therapeutic use. Here we report the role of polyunsaturated fatty acids (PUFAs) in regulating hematopoiesis which has not been explored well so far. Mice were fed daily for 10 days with n-6/n-3 PUFAs, viz. linoleic acid (LA), arachidonic acid (AA), alpha-linolenic acid and docosahexanoic acid (DHA) in four separate test groups with phosphate-buffered saline fed mice as control set. The bone marrow cells of PUFA-fed mice showed a significantly higher hematopoiesis as assessed using side population, Lin-Sca-1+ckit+, colony-forming unit (CFU), long-term culture, CFU-spleen assay and engraftment potential as compared to the control set. Thrombopoiesis was also stimulated in PUFA-fed mice. A combination of DHA and AA was found to be more effective than when either was fed individually. Higher incorporation of PUFAs as well as products of their metabolism was observed in the bone marrow cells of PUFA-fed mice. A stimulation of the Wnt, CXCR4 and Notch1 pathways was observed in PUFA-fed mice. The clinical relevance of this study was evident when bone marrow-transplanted recipient mice, which were fed with PUFAs, showed higher engraftment of donor cells, suggesting that the bone marrow microenvironment may also be stimulated by feeding with PUFAs. These data indicate that oral administration of PUFAs in mice stimulates hematopoiesis and thrombopoiesis and could serve as a valuable supplemental therapy in situations of hematopoietic failure.
Asunto(s)
Trasplante de Médula Ósea/efectos adversos , Suplementos Dietéticos , Ácidos Grasos Omega-3/uso terapéutico , Ácidos Grasos Omega-6/uso terapéutico , Hematopoyesis , Trombopoyesis , Regulación hacia Arriba , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Suplementos Dietéticos/efectos adversos , Ácidos Grasos Omega-3/efectos adversos , Ácidos Grasos Omega-6/efectos adversos , Femenino , Regulación de la Expresión Génica , Supervivencia de Injerto , Hematínicos/uso terapéutico , Ratones Congénicos , Ratones Endogámicos C57BL , Receptor Notch1/agonistas , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores CXCR4/agonistas , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Acondicionamiento Pretrasplante/efectos adversos , Proteínas Wnt/agonistas , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
We have recently demonstrated that the mannose-binding lectins, namely banana lectin (BL) and garlic lectin (GL), interacted with the insulin receptors on M210B4 cells--an established mesenchymal cell line of murine marrow origin--and initiate mitogen-activated protein kinase kinase (MEK)-dependent extracellular signal-regulated kinase (ERK) signaling in them. In this study, we show that this lectin-mediated active ERK signaling culminates into an adipogenic differentiation of these cells. Gene expression studies indicate that the effect takes place at the transcriptional level. Experiments carried out with pharmacological inhibitors show that MEK-dependent ERK and phosphatidylinositol 3-kinase-dependent AKT pathways are positive regulators of the lectin- and insulin-mediated adipogenic differentiation, while stress-activated kinase/c-jun N-terminal kinase pathway acts as a negative one. Since both lectins could efficiently substitute for insulin in the standard adipogenic induction medium, they may perhaps serve as molecular tools to study the mechanistic aspects of the adipogenic process that are independent of cell proliferation. Our study clearly demonstrates the ability of BL and GL to activate insulin-like signaling in the mesenchymal cells in vitro leading to their adipocytic differentiation. The dietary origin of these lectins underscores an urgent need to examine their in vivo effects on tissue homeostasis.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Ajo , Células Madre Mesenquimatosas/efectos de los fármacos , Musa , Lectinas de Plantas/farmacología , Células 3T3-L1 , Adipogénesis/efectos de los fármacos , Animales , Antígenos de Diferenciación/metabolismo , Células de la Médula Ósea/citología , Proteínas Potenciadoras de Unión a CCAAT/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Glicerolfosfato Deshidrogenasa/genética , Humanos , Insulina/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , Células Madre Mesenquimatosas/citología , Ratones , PPAR alfa/genética , PPAR gamma/genética , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND AIMS: Ex vivo generation of megakaryocytes (MK) from hematopoietic stem cells (HSC) is important for both basic research, to understand the mechanism of platelet biogenesis, and clinical infusions, for rapid platelet recovery in thrombocytopenic patients. We investigated the role of two nutraceuticals, docosahexanoic acid (DHA) and arachidonic acid (AA), in the in vitro generation of MK. METHODS: Umbilical cord blood (UCB)-derived CD34+cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO) in the presence (test) or absence (control) of the two additives. On day 10, MK and platelets generated were quantitated by morphologic, phenotypic and functional assays. RESULTS: The cell yield of MK and platelet numbers were significantly higher in test compared with control cells. Phenotypic analyzes and gene expression profiles confirmed these findings. Functional properties, such as colony-forming unit (CFU)-MK formation, chemotaxis and platelet activation, were found to be enhanced in cells cultured with nutraceuticals. The engraftment potential of ex vivo-expanded cells was studied in NOD/SCID mice. Mice that received MK cultured in the presence of DHA/AA engrafted better. There was a reduction in apoptosis and total reactive oxygen species (ROS) levels in the CD41(+) compartment of the test compared with control sets. The data suggest that these compounds probably exert their beneficial effect by modulating apoptotic and redox pathways. CONCLUSIONS: Use of nutraceuticals like DHA and AA may prove to be a useful strategy for efficient generation of MK and platelets from cord blood cells, for future use in clinics and basic research.
Asunto(s)
Antígenos CD34/metabolismo , Ácido Araquidónico/farmacología , Medios de Cultivo/química , Citocinas/farmacología , Ácidos Docosahexaenoicos/farmacología , Sangre Fetal/citología , Megacariocitos/citología , Animales , Apoptosis/efectos de los fármacos , Plaquetas/citología , Plaquetas/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Suplementos Dietéticos , Humanos , Megacariocitos/trasplante , Ratones , Ratones SCID , Fenotipo , Activación Plaquetaria/efectos de los fármacos , Ploidias , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Lectins form an important constituent of our daily diet, and thus, it is essential that their effect(s) on various tissues be examined systematically in order to assess whether they are beneficial or detrimental to human health. We examined the effect of oral administration of two dietary lectins that were isolated from banana (BL) and garlic (GL)-two quite commonly consumed food items-on the hematopoiesis of mice. Balb/c mice were fed weekly with lectins and their marrow mononuclear cells (MNCs) were subjected to various hematopoietic stem/progenitor (HSPC)-specific phenotypic and functional assays. It was observed that the lectin-fed mice harbored a considerably increased HSPC pool in their marrow. Marrow-derived MNCs isolated from these lectin-fed mice gave rise to large-sized colony-forming unit-fibroblast (CFU-F) colonies indicating that the lectins had a salutary effect on the stromal compartment. The molecular mechanisms involved in the process were examined by using a stromal cell line model, M210B4. The lectins pulled down pro-insulin and insulin receptors in an immunoprecipitation experiment and activated extracellular signal-regulated kinase (ERK) signaling in the treated cells, in a manner comparable to insulin, both in terms of kinetics as well as extent. M210B4 cells incubated with BL, GL, or insulin showed reduced levels of reactive oxygen species, suggesting that perhaps the lectins protected the stem cell pool of mice by activating ERK signaling and reducing the oxidative stress in the niche. Our data suggest that these lectins may serve as micronutrients for therapeutic purposes in hematological deficiencies.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Lectinas de Plantas/administración & dosificación , Receptor de Insulina/metabolismo , Células Madre/efectos de los fármacos , Administración Oral , Animales , Western Blotting , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea/métodos , Línea Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ajo/química , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/trasplante , Ratones , Ratones Endogámicos BALB C , Musa/química , Lectinas de Plantas/metabolismo , Unión Proteica , Transducción de Señal/efectos de los fármacos , Bazo/citología , Bazo/metabolismo , Células Madre/citología , Células Madre/metabolismo , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismoRESUMEN
Our previous studies had shown that a combination of the bio-antioxidant catalase and the membrane stabilizer trehalose in the conventional freezing mixture affords better cryoprotection to hematopoietic cells as judged by clonogenic assays. In the present investigation, we extended these studies using several parameters like responsiveness to growth factors, expression of growth factor receptors, adhesion assays, adhesion molecule expression, and long-term culture-forming ability. Cells were frozen with (test cells) or without additives (control cells) in the conventional medium containing 10% dimethylsulfoxide (DMSO). Experiments were done on mononuclear cells (MNC) from cord blood/fetal liver hematopoietic cells (CB/FL) and CD34(+) cells isolated from frozen MNC. Our results showed that the responsiveness of test cells to the two early-acting cytokines, viz. interleukin-3 (IL-3) and stem cell factor (SCF) in CFU assays was better than control cells as seen by higher colony formation at limiting concentrations of these cytokines. We, therefore, analyzed the expression of these two growth factor receptors by flow cytometry. We found that in cryopreserved test MNC, as well as CD34(+) cells isolated from them, the expression of both cytokine receptors was two- to three-fold higher than control MNC and CD34(+) cells isolated from them. Adhesion assays carried out with CB/FL-derived CD34(+) cells and KG1a cells showed significantly higher adherence of test cells to M210B4 than respective control cells. Cryopreserved test MNC as well as CD34(+) cells isolated from them showed increased expression of adhesion molecules like CD43, CD44, CD49d, and CD49e. On isolated CD34(+) cells and KG1a cells, there was a two- to three-fold increase in a double-positive population expressing CD34/L-selectin in test cells as compared to control cells. Long-term cultures (LTC) were set up with frozen MNC as well as with CD34(+) cells. Clonogenic cells from LTC were enumerated at the end of the fifth week. There was a significantly increased formation of CFU from test cells than from control cells, indicating better preservation of early progenitors in test cells. Our results suggest that use of a combination of catalase and trehalose as a supplement in the conventional freezing medium results in better protection of growth factor receptors, adhesion molecules, and functionality of hematopoietic cells, yielding a better graft quality.