RESUMEN
BACKGROUND AIMS: We explored the potential therapeutic value of transplanting bone marrow (BM)-derived mesenchymal stromal cells (MSC) into utrophin/dystrophin-deficient double knock-out (dko) mice, a murine model of Duchenne muscular dystrophy. METHODS: MSC from male rats were isolated and transplanted into female dko mice via the caudal vein. Behavior and locomotor function were later evaluated, along with the expression of dystrophin and utrophin in the sarcolemma of myofiber tissues. The presence of grafted cells was confirmed via polymerase chain reaction for the sex-determining region of the Y-chromosome. RESULTS: Locomotor activity improved significantly (P < 0.05) from 5 to 15 weeks after cell transplantation, as measured by traction, rotating rod and running wheel tests. We also found that the expression of dystrophin and utrophin increased significantly (P < 0.05) and progressively in the sarcolemma from 5 to 15 weeks after transplantation. The median lifespan of mice in the normal group (74.1 weeks) was significantly (P < 0.001) higher than those in the control (22.0 weeks) and transplantation (35.0 weeks) groups, and the median lifespan of mice in the transplantation group was significantly (P < 0.001) higher than that in the control group. CONCLUSIONS: Results of this study demonstrate that BM MSC have potential value in xenogeneic transplantation therapy for muscular dystrophy.
Asunto(s)
Células de la Médula Ósea/citología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Actividad Motora/fisiología , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular Animal/terapia , Animales , Células Cultivadas , Distrofina/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Genotipo , Inyecciones Intravenosas , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Noqueados , Músculo Estriado/patología , Músculo Estriado/fisiopatología , Distrofia Muscular Animal/patología , Ratas , Ratas Sprague-Dawley , Prueba de Desempeño de Rotación con Aceleración Constante , Células del Estroma/citología , Células del Estroma/trasplante , Utrofina/metabolismoRESUMEN
BACKGROUND: The prospects of using immature CD8a(+) dendritic cells (DC2) to establish transplant immunologic tolerance and treatments for autoimmune diseases in the future are promising. However, the methods for inducing DC2 are still being explored. The present study was aimed to investigate the optimal in vitro conditions for preparing large numbers of predominant DC2 from murine bone marrow cells. METHODS: Three groups of bone marrow cells cultured under different conditions were examined, namely a cytokine-induced experimental group (cytokine group), a control group with a low concentration of granulocyte-macrophage colony stimulating factor (GM-CSF, low GM-CSF group) and a control group without endogenous cytokines. The cytokine group was cultured with 5 ng/ml GM-CSF, 25 ng/ml Flt3 ligand (Flt3L), 20 ng/ml interleukin 4 (IL-4) and 100 ng/ml stem cell factor (SCF). The low GM-CSF control group was cultured with 0.4 ng/ml GM-CSF, 25 ng/ml Flt3L and 100 ng/ml SCF, without IL-4. The control group without exogenous cytokines was cultured without additional cytokines. All cells were cultured at 37 degrees C under 5% CO2. On days 3, 7 and 16, 4-color flow cytometry was carried out to analyze the cell phenotypes, and the total cell numbers were counted to analyze the cell yields. Phase-contrast microscopy was used to observe the cell morphologies. RESULTS: The cytokine group exhibited higher proportions of typical immature CD8a(+) DC, especially on day 3, but the total cell number and DC2 proportion decreased during prolonged culture. The low GM-CSF control group showed the same tendencies as the cytokine group on days 16 and 22, but produced higher total cell numbers (P<0.05) with lower DC2 proportions and cell numbers. The control group without exogenous cytokines spontaneously generated a certain proportion of DC2, but with low total cell and DC2 numbers that decreased rapidly, especially during prolonged culture (days 7 and 16, P<0.05). CONCLUSIONS: Culture in the presence of 5 ng/ml GM-CSF, 25 ng/ml Flt3L, 20 ng/ml IL-4 and 100 ng/ml SCF can rapidly induce large quantities of predominant immature CD8a(+) DC from murine bone marrow cells. Therefore, these represent optimal culture conditions for preparing murine immature DC2 in vitro.
Asunto(s)
Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/citología , Técnicas de Cultivo de Célula/métodos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía de Contraste de FaseRESUMEN
The aim of this study was to examine in vitro the response of human mesenchymal stem cells (hMSCs) on the novel biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine (BG-COL-HYA-PS) composite scaffold for potential use in bone tissue engineering. The initial attachment, the proliferation, migration and differentiation behavior of the cells on the BG-COL-HYA-PS composites were assessed in comparison with those on pure 58sBG, BG-COL, and BG-COL-HYA composites in either growth medium (L-DMEM supplemented with 10% fetal bovine serum) or osteogenic medium (growth medium supplemented with 0.1 microM dexamethasone, 10 mM beta-glycerophosphate, and 50 microM ascorbic acid). HMSCs attached, and subsequently proliferated and migrated on the BG-COL-HYA-PS composites to a significantly higher degree. The alkaline phosphatase (ALP) staining, ALP activity and the expression of the bone associated gene ALP, osteocalcin (OC), and osteopontin (OPN) was also significantly higher in the hMSCs on the BG-COL-HYA-PS scaffolds than those on the BG-COL, BG-COL-HYA composites and the pure 58sBG. These findings suggest that the BG-COL-HYA-PS composite porous scaffolds have high potential for use as scaffolds in bone tissue engineering and repair.
Asunto(s)
Materiales Biomiméticos/síntesis química , Huesos/citología , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos/métodos , Médula Ósea , Cerámica , Humanos , Ácido Hialurónico , FosfatidilserinasRESUMEN
The thymus provides a unique cellular and hormonic microenvironment for the development of immunocompetent T cells. Thymic polypeptides have been widely used clinically for the treatment of tumors, infectious diseases and immune deficiency diseases. They have already shown the ability to stimulate the maturation of hematopoietic stem cells towards the CD3+CD4+ T cell lineage. However, their effects on the thymopoiesis of embryonic stem cells are still unexplored. In this paper, we compared the effects of three thymic polypeptides, thymopentin (TP5), thymosin alpha-1 (Talpha-1) and thymopeptides on the in vitro thymopoiesis of mouse embryonic stem (ES) cells. Using the embryoid body induction system, we found that both Talpha-1 and thymopeptides effectively induced ES cells to differentiate sequentially into the CD3+ and CD4+/CD8+ T cells. These T cells had T cell receptor (TCR) Vbeta gene rearrangement and most were TCRalphabeta T cells. We also found that the expression of the Notch receptor and its ligands Delta-like-1 and Delta-like-4 gradually increased during the induction. However, TP5 failed to induce the T cell differentiation of the ES cells. In summary, this is the first report to demonstrate that Talpha-1 can stimulate the T cell early stage differentiation from ES cells using the embryoid body protocol. These findings provide a powerful model for studying T cell development and may open new venues for the clinical application of Talpha-1.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/fisiología , Timopentina/farmacología , Timosina/análogos & derivados , Timo , Animales , Complejo CD3/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Madre Embrionarias/citología , Ratones , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Timalfasina , Timopentina/metabolismo , Timosina/metabolismo , Timosina/farmacología , Timo/metabolismo , Timo/fisiologíaRESUMEN
The full potential of embryonic stem (ES) cells to generate precise cell lineages and complex tissues can be best realized when they are differentiated in vivo-i.e. in developing blastocysts. Owing to various practical and ethical constraints, however, it is impossible to introduce ES cells of certain species into blastocysts of the same species. One solution is to introduce ES cells into blastocysts of a different species. However, it is not known whether ES cells can contribute extensively to chimerism when placed into blastocysts of a distantly related species. Here, we address this question using two divergent species, Apodemus sylvaticus and Mus musculus, whose genome sequence differs by approximately 18% from each other. Despite this considerable evolutionary distance, injection of Apodemus ES cells into Mus blastocysts led to viable chimeras bearing extensive Apodemus contributions to all major organs, including the germline, with Apodemus contribution reaching approximately 40% in some tissues. Immunostaining showed that Apodemus ES cells have differentiated into a wide range of cell types in the chimeras. Our results thus provide a proof of principle for the feasibility of differentiating ES cells into a wide range of cell types and perhaps even complex tissues by allowing them to develop in vivo in an evolutionarily divergent host-a strategy that may have important applications in research and therapy. Furthermore, our study demonstrates that mammalian evolution can proceed at two starkly contrasting levels: significant divergence in genome and proteome sequence, yet striking conservation in developmental programs.
Asunto(s)
Células Madre Embrionarias/citología , Quimera por Trasplante/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Evolución Biológica , Blastocisto/citología , Diferenciación Celular , Cartilla de ADN/genética , Desarrollo Embrionario/genética , Células Madre Embrionarias/trasplante , Femenino , Células Germinativas , Proteínas Fluorescentes Verdes/genética , Masculino , Ratones , Murinae/embriología , Murinae/genética , Especificidad de Órganos , Filogenia , Reacción en Cadena de la Polimerasa , Embarazo , Proteínas Recombinantes/genética , Especificidad de la Especie , Teratoma/genética , Teratoma/patología , Quimera por Trasplante/embriología , Trasplante HeterólogoRESUMEN
Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into adipocytes in the presence of a hormone cocktail. These cells thus provide a promising model for studying the early events of adipogenesis. Here, we examine the involvement of the PI3K/Akt and mTOR/p70S6K signaling pathways in human MSC adipogenesis. We found that the two pathways were strongly activated with a similar temporal profile under the adipogenesis-inducing hormone cocktail and this activation could be blocked by LY294002, a specific inhibitor of PI3K. Furthermore, rapamycin, a specific inhibitor of mTOR, blocked the activation of mTOR/p70S6K but not PI3K/Akt. Both LY294002 and rapamycin severely suppressed lipid accumulation, as well as the expression of adipogenic markers, including PPAR gamma 2 and C/EBP alpha, two master adipogenic transcription factors. Together, these data indicate that the mTOR/p70S6K pathway acts downstream of the PI3K/Akt pathway in mediating the adipogenic conversion of MSCs. In conclusion, our data suggest that the PI3K/Akt and mTOR/p70S6K signaling pathways are essential for adipogenesis of human MSCs.
Asunto(s)
Adipogénesis , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Adipogénesis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Serina-Treonina Quinasas TOR , Transcripción Genética/efectos de los fármacosRESUMEN
Human mesenchymal stem cells (hMSC) are a population of multipotent cells that can differentiate into osteoblasts, chondrocytes, adipocytes, and other cells. The exact mechanism governing the differentiation of hMSC into osteoblasts remains largely unknown. Here, we analyzed protein expression profiles of undifferentiated as well as osteogenic induced hMSC using 2-D gel electrophoresis (2-DE), mass spectrometry (MS), and peptide mass fingerprinting (PMF) to investigate the early gene expression in osteoblast differentiation. We have generated proteome maps of undifferentiated hMSC and osteogenic induced hMSC on day 3 and day 7. 2-DE revealed 102 spots with at least 2.0-fold changes in expression and 52 differently expressed proteins were successfully identified by MALDI-TOF-MS. These proteins were classified into 7 functional categories: metabolism, signal transduction, transcription, calcium-binding protein, protein degradation, protein folding and others. The expression of some identified proteins was confirmed by further RT-PCR analyses. This study clarifies the global proteome during osteoblast differentiation. Our results will play an important role in better elucidating the underlying molecular mechanism in hMSC differentiation into osteoblasts.
Asunto(s)
Diferenciación Celular , Perfilación de la Expresión Génica/métodos , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteoblastos/metabolismo , Proteómica , Células Cultivadas , Análisis por Conglomerados , Electroforesis en Gel Bidimensional , Humanos , Células Madre Mesenquimatosas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Human embryonic stem cells can propagate indefinitely in vitro and are able to differentiate into derivatives of all three embryonic germ layers. The excitement surrounding human embryonic stem cells lies largely in their potential to produce specialized cells that can be used for transplant therapies. However, further investigation requires additional cell lines with varying genetic background. Therefore, efforts to derive and establish more human embryonic stem cell lines are highly warranted. METHODS: Surplus embryos (blastocysts) from donors were used to isolate the inner cell mass by immunosurgery. All cells were cultured continuously on irradiated murine embryonic fibroblasts feed layer and likely human embryonic stem cell colonies were subsequently characterized by cell surface marker staining, karyotyping and teratoma formation. RESULTS: Two human embryonic stem cell lines (SYSU-1 and SYSU-2) were established from surplus embryos. The two lines express several pluripotency markers including alkaline phosphatase, SSEA-4, Tra-1-60, Oct-4, Nanog and Rex-1. They remain in undifferentiated state with normal karyotype after prolonged passages and can form embryoid bodies in vitro and teratoma in vivo. CONCLUSION: Two new human embryonic stem cell lines have been established from surplus embryos. They can be used to understand selfrenewal and differentiating mechanisms and provide more choices for regenerative medicine.
Asunto(s)
Células Madre Embrionarias/citología , Diferenciación Celular , Línea Celular , Humanos , CariotipificaciónRESUMEN
Graft-vs.-host disease (GVHD) remains the major limitation of allogeneic bone marrow transplantation (BMT) and stem cell transplantation, and leukemia recurrence is another important complication in leukemia treatment. Immature CD8alpha+ dendritic cells (DC) have good potential in GVHD treatment and immunological tolerance studies. To find a new way to prevent GVHD, not increasing the risk of leukemia recurrence, in this study, predominant CD8alpha+ immature DC were induced from murine bone marrow (BM) cells by 5 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF) plus 20-ng/mL interleukin (IL)-4, 100-ng/mL stem cell factor (SCF) and 25-ng/mL Flt3L, and 97.09 of prepared DC were CD8alpha+ on day 3. These DC were identified as morphologically and phenotypically immature CD8alpha+ DC. The suppressive function was observed in vitro, and then in vivo on allo-BMT leukemia model. The prepared predominant immature CD8alpha+ DC were weak syngeneic lymphocyte stimulators and could suppress mixed leukocyte reaction in vitro. In vivo, they prevented the pathological changes of GVHD and prolonged the surviving time of allo-BMT leukemia mice. The effect showed a dose-effect relationship. 86.7% of allo-BMT plus 1 million predominant CD8alpha+ DC leukemia mice reached long-term survival. Although predominant immature CD8alpha+ DC had the function of GVHD suppression, they did not increase leukemia recurrence. The method and findings may have important potency for GVHD treatment in clinical application.
Asunto(s)
Antígenos CD8/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Leucemia/inmunología , Leucemia/patología , Animales , Trasplante de Médula Ósea , Antígenos CD8/inmunología , Diferenciación Celular , Células Dendríticas/inmunología , Enfermedad Injerto contra Huésped/patología , Leucemia/metabolismo , Leucemia/cirugía , Ratones , Microscopía Electrónica de Transmisión , Factores de Riesgo , Prevención Secundaria , Tasa de Supervivencia , Trasplante HomólogoRESUMEN
Embryonic stem cells can proliferate indefinitely and are capable of differentiating into derivatives of all three embryonic germ layers in vitro, including the neural lineage. The main objective of this study is to test the effects of neural stem cell conditioned medium on the neural differentiation of mouse embryonic stem cells. When cultured in neural stem cell conditioned medium, mouse embryonic stem cells can form floating cell spheres composed of many nestin-positive cells. After trypsinization and growth on gelatin, these embryonic stem cell-derived neural progenitor cells can be expanded for more than 3 months without loss of neural progenitor characteristics. Both neuronal and glial cells can be readily generated from these cells under differentiation conditions. Thus, neural stem cell conditioned medium is a highly potent reagent for inducing the development of mouse embryonic stem cells into the neural lineage, especially neural progenitor cells.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Neuronas/efectos de los fármacos , Células Madre/efectos de los fármacos , Animales , Recuento de Células/métodos , Proliferación Celular/efectos de los fármacos , Separación Celular/métodos , Células Cultivadas , Embrión de Mamíferos , Inmunohistoquímica/métodos , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuroglía/efectos de los fármacos , Neuronas/citología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Células Madre/fisiología , Factores de TiempoRESUMEN
Neural stem cells are multipotent and self-renewing cells with important potential application in cell replacement therapy in brain damage. Many studies have shown that nestin-positive cells represent neural stem and progenitor cells in the central neural system. Here, we derived neural stem cells from the subventricular zone of a newborn nestin-promoter-driven green fluorescent protein mouse, and found that the percentage of nestin-positive cells decreased continuously at each passage in neurosphere culture. Using the relative proliferation ratio and relative division ratio analysis, we concluded that the slower cycling of nestin-positive cells was responsible for this decrease.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Ciclo Celular/fisiología , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Trasplante de Células Madre/métodos , Células Madre/metabolismo , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Recuento de Células , División Celular/fisiología , Proliferación Celular , Separación Celular/métodos , Células Cultivadas , Proteínas Fluorescentes Verdes , Hipocampo/citología , Hipocampo/embriología , Hipocampo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nestina , Neuronas/citología , Esferoides Celulares , Células Madre/citología , Factores de TiempoRESUMEN
OBJECTIVE: To investigate the association between Arg16Gly polymorphism of beta(2)-adrenergic receptor (ADRB2) gene and blood pressure levels. METHODS: A total of 487 hypertensive individuals were recruited from YueXi county of Anhui province. 672 patients' parents and siblings were also invited to take part in the study and used as genomic control. Blood pressure was measured and a standardized questionnaire regarding social-economic characteristics, general health status, occupational history and life-style and dietary factors was administered for each participant. The ADRB2 Arg16Gly polymorphism was genotyped by using a PCR-Restriction Fragment Length Polymorphism (RFLP) method. The association between the ADRB2 polymorphism and hypertension in hypertensive adults was determined by utilizing a family-based association test analysis. RESULTS: In this study population, carriers of the ADRB2 Arg16 allele had lower systolic (P < 0.01) and diastolic (P < 0.01) blood pressure, suggesting that the genetic effect on blood pressure was more likely to fit an additive model. CONCLUSION: Our results suggest a probable association between Arg16Gly polymorphism of ADRB2 gene and hypertension.
Asunto(s)
Hipertensión/genética , Polimorfismo de Nucleótido Simple , Receptores Adrenérgicos beta 2/genética , Adulto , Pueblo Asiatico/genética , Presión Sanguínea , China/epidemiología , Femenino , Genotipo , Humanos , Hipertensión/epidemiología , Masculino , Persona de Mediana Edad , Linaje , Adulto JovenRESUMEN
To investigate effects of rat bone marrow mesenchymal stem cells (rBMMSC) on hematopoiesis after allo-hematopoietic stem cell transplantation (HSCT), allogeneic BMT model from Fischer 344 rats (RT-1Al) to Wistar rats (RT-1Au) was established; effects of MSCs on hematopoietic reconstitution were studied by survival rate, peripheral blood counts, histological analysis and FACS at day 30 after transplantation. The results showed that (1) MSCs from donor Fisher344 could survive in recipient irradiated by lethal dose and could be found in the thymus, spleen and bone marrow of the recipient at 30 days after cotransplantation with BM by measuring EGFP gene. (2) Cotransplanation of MSCs and BM improved hematopoietic reconstitution. Lymphocyte and platelet counts of peripheral blood in cotransplantation group were higher than those in the control group. Active hematopoiesis and increase of bone marrow nucleated cells were observed in cotransplantation group. MSCs significantly enhanced hematopoiesis of B lymphocyte and megakaryocytopoietic lineages by FACS analysis. It is concluded that (1) MSCs of Fisher344 can be found in the thymus, spleen, bone marrow of the recipients at 30 days after cotransplantion by measuring EGFP gene. (2) hematopoietic reconstitution is significantly enhanced by MSCs cotransplanted with BM.
Asunto(s)
Trasplante de Médula Ósea/métodos , Hematopoyesis/fisiología , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/fisiología , Animales , Diferenciación Celular/fisiología , Citometría de Flujo , Recuento de Linfocitos , Masculino , Células Madre Mesenquimatosas/citología , Modelos Animales , Recuento de Plaquetas , Ratas , Ratas Endogámicas F344 , Ratas WistarRESUMEN
AIM: Recent reports have shown the capacity of mesenchymal stem cells (MSCs) to differentiate into hepatocytes in vitro and in vivo. MSCs administration could repair injured liver, lung, or heart through reducing inflammation, collagen deposition, and remodeling. These results provide a clue to treatment of liver fibrosis. The aim of this study was to investigate the effect of infusion of bone marrow (BM)-derived MSCs on the experimental liver fibrosis in rats. METHODS: MSCs isolated from BM in male Fischer 344 rats were infused to female Wistar rats induced with carbon tetrachloride (CCl4) or dimethylnitrosamine (DMN). There were two random groups on the 42nd d of CCl4:CCl4/MSCs, to infuse a dose of MSCs alone; CCl4/saline, to infuse the same volume of saline as control. There were another three random groups after exposure to DMN: DMN10/MSCs, to infuse the same dose of MSCs on d 10; DMN10/saline, to infuse the same volume of saline on d 10; DMN20/MSCs, to infuse the same dose of MSCs on d 20. The morphological and behavioral changes of rats were monitored everyday. After 4-6 wk of MSCs administration, all rats were killed and fibrosis index were assessed by histopathology and radioimmunoassay. Smooth muscle alpha-actin (alpha-SMA) of liver were tested by immunohistochemistry and quantified by IBAS 2.5 software. Male rats sex determination region on the Y chromosome (sry) gene were explored by PCR. RESULTS: Compared to controls, infusion of MSCs reduced the mortality rates of incidence in CCl4-induced model (10% vs 20%) and in DMN-induced model (20-40% vs 90%). The amount of collagen deposition and alpha-SMA staining was about 40-50% lower in liver of rats with MSCs than that of rats without MSCs. The similar results were observed in fibrosis index. And the effect of the inhibition of fibrogenesis was greater in DMN10/MSCs than in DMN20/MSCs. The sry gene was positive in the liver of rats with MSCs treatment by PCR. CONCLUSION: MSCs treatment can protect against experimental liver fibrosis in CCl4-induced or DMN-induced rats and the mechanisms of the anti-fibrosis by MSCs will be studied further.
Asunto(s)
Células de la Médula Ósea/citología , Trasplante de Células Madre Hematopoyéticas , Hepatocitos/citología , Cirrosis Hepática/terapia , Animales , Tetracloruro de Carbono , Diferenciación Celular , Dimetilnitrosamina , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/prevención & control , Masculino , Mesodermo/citología , Ratas , Ratas Endogámicas F344RESUMEN
OBJECTIVE: Embryonic stem cells (ESCs) are derived from totipotent cells of early embryo and they are potential to differentiate to any kind of cells of tissues in the body. Some reports showed that ESCs had broad capabilities of differentiating to variety of hematopotietic cells, such as erythroid, granulocyte/macrophage, megakaryocyte, mast and lymphocyte precursors. However, it is very difficult to control the phase of differentiation for ESCs in vitro. There is few report about hematopotietic stem cells (HSCs) from ESCs. Therefore, this research was designed to establish a culture system for generation of CD(34)(+)/Sca-1(+) HSC from ESC in vitro. METHODS: Single mouse E 14.1 cells were suspended in methylcellulose medium, containing 40 ng/ml stem cell factor (SCF) and 20 ng/ml vascular endothelial growth factor (VEGF) and incubated at 37 degrees C with 5% CO2. In order to ensure the viability of the primary differentiation cultures over an extended period of time, the cultures were fed on day 7 with a dilute methylcellulose medium containing VEGF, SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (EPO), which promoted their primary differentiation into embryoid bodies (EBs) with more CD(34)(+)/Sca-1(+) cells. Then, EBs with peak level of CD(34)(+)/Sca-1(+) cells were dispersed into single cells and replanted either in methylcellulose medium or in bone marrow stromal cells differentiation system containing 15% fetal bovine serum (FBS), 160 ng/ml SCF, 20 ng/ml VEGF, 30 ng/ml IL-3, 30 ng/ml IL-6, 3 U/ml EPO and 20% BIT for HSC into second-step differentiation. The HSCs were characterized by flow cytometric analysis, colonogenic cell assay and Wright-Giemsa stains. RESULTS: VEGF had the strongest stimulatory effect on the enhancement of the CD(34)(+)/Sca-1(+) cells population when combined with SCF, IL-3, IL-6 and EPO. It could markedly accelerate mouse E14.1 cells to differentiate into EB with more CD(34)(+)/Sca-1(+) cells. Cell cytometric analysis showed CD(34)(+)/Sca-1(+) cells were up to (1.91 +/- 0.40)% by day 5 and (8.11 +/- 1.17)% by day 8, and the peak level of CD(34)(+)/Sca-1(+) cells was (13.72 +/- 1.92)% by day 12. However, CD(34)(+)/Sca-1(+) cells could not increase in number with the prolongation of differentiation. So renewal single cells suspension from EB by day 12 was dispersed into the second step differentiation. The results showed that HSC was slowly generated with a few hematopoietic colony formations in methylcellulose medium differentiation system. CD(34)(+)/Sca-1(+) cells got (2.74 +/- 0.80)% by day 5 and (11.37 +/- 1.84)% by day 10, and apex percentage of CD(34)(+)/Sca-1(+) cells was about (20.52 +/- 2.78)% by day 14. However, EBs generated quickly for HSC with increased hematopoietic cell population by co-culture on bone marrow stromal cells feeder. Flow cytometric analysis showed that the percentages of CD(34)(+)/Sca-1(+) cells was (7.33 +/- 1.61)% by day 5, (13.28 +/- 2.59)% by day 8, and (20.81 +/- 3.19)% by day 10. EB cells were induced after 12 days to reach the peak level of (34.60 +/- 3.71)%. Hematopoietic colony formation unit (CFU) analysis showed that CFU was sufficient from cells on bone marrow stromal cells differentiation system in the second step compared to that in methylcellulose medium differentiation system, and Wright-Giemsa stain could confirm its characteristics of hematopoietic progenitors. CONCLUSION: Using two-step differentiation, the investigators got a good way to control the phase of differentiation from ESC to HSC. The bone marrow stromal cell differentiation system combining with VEGF, SCF, IL-3, IL-6 and EPO was an optimal system for the generation of HSC with CD(34)(+)/Sca-1(+) surface marker from ESC differentiated in vitro. This study demonstrated that these cells could form more hemopoietic colonies.
Asunto(s)
Antígenos CD34 , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , Supervivencia Celular , Células Cultivadas , Células Madre Embrionarias/inmunología , Células Madre Hematopoyéticas/fisiología , RatonesRESUMEN
OBJECTIVE: To prevent Graft-versus-host disease (GVHD) in rat model, we evaluated the feasibility of mesenchymal stem cells (MSCs) as a gene transfer target and studied the efficiency of recombinant adenovirus mediated gene therapy. METHODS: We constructed the recombinant adenovirus containing CTLA4Ig gene. Rat MSCs of passages 3-5 were infected by the adenovirus, and the transfection efficiency was monitored by GFP markers. We performed flow cytometric analysis, immunohistochemical and Western blotting analysis to identify the CTLA4Ig expression. The gene transferred MSCs were tested for their ability to inhibit the allogeneic lymphocyte response in vitro and to prevent GVHD in a rat model. RESULTS: Recombinant adenovirus pAd-CTLA4Ig was correctly constructed and confirmed. After MSCs were infected by the adenovirus, the CTLA4Ig protein was detected not only in transgenic MSCs, but also in the culture medium. In a mixed lymphocytes response (MLR) test, the transgenic MSCs could significantly inhibit the allogeneic lymphocyte response compared with the control groups (P < 0.05). A model of GVHD was developed by transplanting bone marrow cells and spleen lymphocytes of F344 rats to lethally irradiated SD rats. The onset of GVHD could be ameliorated or prevented by co-administration of transgenic MSCs. All the rats in the control groups suffered severe acute GVHD. CTLA4Ig expression was observed in the liver, intestine, kidney and spleen 30 days post-transplantation. CONCLUSIONS: Our results indicate that adenoviral vectors could efficiently transfer CTLA4Ig gene into MSCs and sustain long-term stable expression in vitro and in vivo.
Asunto(s)
Técnicas de Transferencia de Gen , Enfermedad Injerto contra Huésped/prevención & control , Inmunoconjugados/genética , Células Madre Mesenquimatosas , Abatacept , Adenoviridae , Animales , Estudios de Factibilidad , Vectores Genéticos , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Recombinación GenéticaRESUMEN
The study was aimed at the exploration of relationship between T cells expressing killer cell inhibitor receptors (KIR, CD158 and CD94) and graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation. The expression rates of CD158a, CD158b and CD94 on T cells and NK cell were detected by flow cytometry and donor/recipient HLA-Cw was analyzed using PCR after peripheral blood stem cell transplantation (PBSCT) and umbilical cord blood transplantation (UCBT). After both PBSCT and UCBT, the rates of CD3(+)CD158a(+) and CD3(+)CD158b(+) T cells increased, especially the rate of CD8(+)CD158b(+) T cells. In both acute and chronic GVHD groups, the rate of CD3(+)CD158b(+) T cells increased, especially in acute GVHD. The CD94 mainly expressed on CD3(+)CD8(+) T cells. The percentage of the expression of CD94 on CD4(+) and CD8(+) cells after UCBT and PBSCT increased significantly. The expression of KIR in GVHD (early stage of transplantation) increased but the expression of KIR in chronic GVHD (advanced stage of transplantation) decreased. Five patients who HLA-Cw matched had no severe GVHD. In four patients who underwent allo-PBSCT and UCBT from related HLA-matched donors, only 2 patients had no aGVHD. Four patients underwent transplantation from unrelated HLA-matched donors had GVHD. These observations suggested that there is some relationship between GVHD and KIR expression on T cells. CD158b might be an inhibitory molecule of T cell activated at early stage after transplantation. Understanding the mechanism of GVHD with the expression of KIR on T cells, especially those binding the HLA-Cw might shed light on the establishment of the specific immunotolerance for the prevention of GVHD. To pay attention to HLA-Cw typing is very important to reduce GVHD and increase GVL effect in related or unrelated HLA-matched transplantation.
Asunto(s)
Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas , Receptores Inmunológicos/análisis , Linfocitos T/inmunología , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Genotipo , Antígenos HLA-C/genética , Humanos , Lectinas Tipo C , Receptores de Interleucina-2/análisis , Receptores KIR , Receptores KIR2DL1 , Receptores KIR2DL3RESUMEN
AIM: To explore in-vivo regulation of T cells in pulmonary tissues of ovalbumin(OVA)-sensitized mice by BCG and its effect on airway inflammatory reaction. METHODS: Thirty BALB/c mice (male and female, 6 to 8 weeks of age) were divided into 3 groups. For the mice of first group, aerosol OVA was inhaled daily for 20 minutes once for 10 days running, so as to establish a OVA-sensitized mouse model. In the mice of second group, aerosol normal solution was inhaled according to the time and frequency of the first group. In the third group, the mice were intradermally injected with BCG of 0.04 to 0.06 mg on 10 and 14 days before OVA sensitization, respectively, and then aerosol purified protein derivative (PPD) was inhaled to the mice on 6 days after OVA sensitization. The changes in the number of CD4(+), CD8(+) and IFN-gamma(+) T cells in the inflammatory tissues and changes in the inflammatory cells in the inflammatory tissues and broncho-alveolar lavage fluid (BALF) were detected by SABC immuno-histochemical staining. RESULTS: The number of CD4(+) T cells significantly increased in the pulmonary tissues of OVA-sensitized mice, which mainly was the increased number of CD4(+)/IFN-gamma(-) T cells, While the number of CD8(+) T cells had no notable variation, thus the ratio of CD4(+)/IFN-gamma(+) T cells descended. After BCG treatment, the number of CD8(+) T cells markedly increased, so the ratio of CD4(+)/ IFN-gamma(+) T cells variously rose, whereas the number of inflammatory cells decreased in BALF. CONCLUSION: BCG can upregulate the number of Th1 cells and lighten the airway inflammatory reaction of experimental asthma mice.
Asunto(s)
Asma , Mycobacterium bovis , Animales , Asma/inmunología , Líquido del Lavado Bronquioalveolar , Linfocitos T CD8-positivos , Interferón gamma/inmunología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunologíaRESUMEN
This study was undertaken to establish a murine model for unrelated allogeneic umbilical cord blood transplantation (UCBT). The characteristics and percentage of hematopoietic stem/progenitor cells between near-term fetal and neonatal murine peripheral blood (FNPB) and bone marrow (BM) were evaluated by flow cytometry and semisolid methylcellulose culture. BABL/c (H-2(d)) recipient mice conditioned with high dose CTX were transplanted with FNPB form C57BL/6 (H-2(b)) mice and the survival rate, hematopoietic and immunological reconstruction, graft versus host disease (GVHD) and engraftment level were observed. The results showed that the numbers of day 14 CFU-GM and CFU-GEMM in FNPB (176.40 +/- 78.39)% and (141.40 +/- 56.57)%, respectively were much higher than those in BM (75.20 +/- 26.41)% and (68.80 +/- 23.95)%, respectively. Moreover the percentage of Sca-1(+) CD34(+) cell subsets in FNPB (3.63 +/- 1.13)% was also higher than that in BM (1.41 +/- 0.8 7)%. FNPB transplantation improved survival rate and reconstituted hematopoietic and immune function in recipients. There was no evidence of GVHD. Chimeric analysis showed that the proportion of donor cells in BM of recipients was 27.94% at 21 days after transplantation. It was concluded that FNPB contains more hematopoietic stem and progenitor cells with high expansion ability and weak allogeneic immunity, which was similar to human UCB. The murine model for allogeneic UCBT (C57BL/6-->BALB/c) was established successfully.
Asunto(s)
Sangre Fetal/citología , Trasplante de Células Madre Hematopoyéticas , Animales , Animales Recién Nacidos , Citometría de Flujo , Enfermedad Injerto contra Huésped/etiología , Inmunidad , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Animales , Trasplante HomólogoRESUMEN
AIM:To determine whether antisense insulin-like growth factor-I(IGF-I) gene can modulate CEA and AFP expression in human hepatoma cells (HepG2).METHODS: Transfection of HepG2 cells was accomplished using Lipofectin reagent. Northern blot analysis confirmed the antisense IGF-I RNA of the transfected cells. CEA and AFP levels were measured using radioimmunoassay.RESULTS: Human hepatoma cell lines (HepG2) were transfected with antisense IGF-I gene. Northern blot analysis confirmed that antisense IGF-I RNA was expressed in the transfected cells. The effect of antisense IGF-I gene on CEA and AFP expression was demonstrated by the fact that the CEA and AFP levels in the supernatant of transfected cell culture were significantly lower as compared with the parent cells, (CEA 7.0&mgr;g/L plus minus 0.76&mgr;g/L and 3.29&mgr;g/L plus minus 1.80&mgr;g/L (P < 0.05) and AFP 53.63&mgr;g/L plus minus 6.02&mgr;g/L and 9.0&mgr;g/L plus minus 5.26&mgr;g/L (P<0.01), respectively).CONCLUSION: The malignant potentiality of the transfected cells was partially suppressed.Antisense IGF-I gene can modulate the expression of CEA and AFP in human hepatoma cell lines (HepG2)