RESUMO
The exact origin of neural stem cells in the adult neurogenesis niche remains unknown. Our previous studies, however, indicated an implication of both bone marrow cells as potential progenitors of hippocampal newborn neurons and polyunsaturated fatty acids as ligands of G protein-coupled receptor 40 (GPR40) signaling. Here, we aimed at studying whether bone marrow-derived stromal cells (BMSC) treated by docosahexaenoic acid (DHA) can express neuronal markers in vitro. We focused on implication of DHA/GPR40 signaling for the expression of neural markers in clonally-expanded BMSC from young macaque monkeys. Cell cycle analysis revealed that the DHA plus bFGF treatment induced a decrease of BMSC proliferation and increased the cells in the G0 resting phase. The transitions from nestin-positive progenitors via immature neuronal (beta III-tubulin-positive) to mature neuronal (NF-M and Map2-positive) phenotypes were examined using RT-PCR, Western blot and immunocytochemistry. We detected a significant increase of GPR40 mRNA and protein expression after bFGF induction, being compared with the untreated BMSC. Addition of DHA, a representative GPR40 ligand, led to a significant down-regulation of GPR40, i.e., G protein-coupled receptor-specific internalization, with a subsequent upregulation of neuronal markers such as beta III-tubulin, NF-M and Map2. These data altogether suggest that adult primate BMSC can express neuronal markers with the aid of DHA/GPR40 signaling.
Assuntos
Biomarcadores/metabolismo , Células da Medula Óssea , Ácidos Docosa-Hexaenoicos/farmacologia , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Células Estromais , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ciclo Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Macaca , Neurônios/citologia , Fenótipo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
Curative therapy for diabetes mellitus mainly involves pancreas or islet transplantation to recruit insulin-producing cells. This approach is limited, however, because of both the shortage of donor organs and allograft rejection. Intra-bone marrow bone marrow transplantation (IBM-BMT) has recently been shown to be effective in inducing donor-specific tolerance in mice and rats without the use of immunosuppressants. After induction of diabetes in 15 C3H mice with streptozotocin, the mice received both allotransplants of bone marrow cells from C57BL/6 mice by IBM-BMT and injections via the portal vein of insulin-producing cells that were induced in vitro from stem cells derived from adult C57BL/6 bone marrow. We evaluated the expression of these cells by examining the expression of not only insulin but also the crucial transcription factors insulin I and insulin II. The diabetic mice were treated with IBM-BMT and precultured insulin-producing cells. Hyperglycemia was normalized by 5 days after the treatment and remained normal for more than 45 days. This strategy might be applicable to patients with type I diabetes mellitus.
Assuntos
Células da Medula Óssea , Transplante de Medula Óssea , Diabetes Mellitus Experimental/terapia , Sobrevivência de Enxerto , Células Secretoras de Insulina/transplante , Animais , Células da Medula Óssea/metabolismo , Diabetes Mellitus Experimental/metabolismo , Regulação da Expressão Gênica , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Veia Porta , Transplante HomólogoRESUMO
We have previously demonstrated, using chimeric resistant MRL/lpr mice, that a fractionated total body irradiation (FTBI) (5 Gy x 2 with a 4 h interval on the day before allogeneic bone marrow transplantation (BMT)) is the best conditioning regimen for the treatment of autoimmune diseases in radiosensitive MRL/lpr mice. In the present study, using various standard strains of mice (not radiosensitive mice), we explore the best protocol for irradiation (doses and intervals) as the conditioning regimen for allogeneic BMT. Recipient mice were exposed to various irradiation regimens: a single total body irradiation (TBI) of 9.5 or 12 Gy and FTBI of (5+5) Gy to (7+7) Gy with a 1 to 24 h interval. The method generally utilized for humans ((2+2) Gy with a 4 h interval for 3 days (total 12 Gy)) was also used. One day after the last irradiation, donor BMCs from BALB/c, C3H, or C57BL/6 (B6) mice were transplanted into C3H or B6 mice. The irradiation protocol of (2+2) Gy for 3 days was found to be insufficient to enable the complete removal of recipient immunocompetent cells, since donor-reactive T cells were observed in the recipient spleens and many recipient-type NK and CD4(+) cells were also detected in the recipient hematolymphoid tissues. In all the combinations, the highest survival rate was achieved in the recipients irradiated with (6+6) or (6.5+6.5) Gy with a 4 h interval. In the surviving mice, the hematolymphoid tissues had been fully reconstituted with donor cells.
Assuntos
Transplante de Medula Óssea/veterinária , Camundongos Endogâmicos , Condicionamento Pré-Transplante/veterinária , Transplante Homólogo/veterinária , Irradiação Corporal Total/veterinária , Animais , Relação Dose-Resposta à Radiação , Feminino , Sobrevivência de Enxerto , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos/genética , Lesões Experimentais por Radiação/etiologia , Tolerância a Radiação/genética , Especificidade da Espécie , Organismos Livres de Patógenos Específicos , Irradiação Corporal Total/efeitos adversos , Irradiação Corporal Total/métodosRESUMO
We have recently established a new bone marrow transplantation (BMT) method for the treatment of intractable autoimmune diseases in MRL/lpr mice; the method consists of fractionated irradiation (5.5 Gy x 2), followed by BMT of whole bone marrow cells (BMCs) from allogeneic C57BL/6 mice via the portal vein (abbreviated as 5.5 Gy x 2 + PV). In the present study, we investigate the mechanisms underlying the early engraftment of donor-derived cells in MRL/lpr mice by this method. In the mice treated with this method, the number of donor-derived cells possessing the mature lineage (Lin) markers rapidly increased in the BM, spleen, and liver; almost 100% were donor-derived cells by 14 days after the treatment. The number of donor-derived hemopoietic progenitor cells (defined as c-kit(+)/Lin(-) cells) increased in the BMCs, hepatic mononuclear cells, and especially spleen cells by 14 days after the treatment. Simultaneously, hemopoietic foci adjoining donor-derived stromal cells were observed in the liver when injected via the PV, but not via the peripheral vein (i.v.). When adherent cell-depleted BMCs were injected via the PV, recipients showed a marked reduction in the survival rate. However, when mice were transplanted with adherent cell-depleted BMCs with cultured stromal cells, all the recipients survived. These findings suggest that not only donor hematopoietic stem cells (HSCs) but also donor stromal cells administered via the PV were trapped in the liver, resulting in the early engraftment of donor HSCs in cooperation with donor-derived stromal cells. This new strategy to facilitate the early recovery of hemopoiesis would therefore be of great advantage in human application.
Assuntos
Doenças Autoimunes/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Veia Porta/metabolismo , Células Estromais/citologia , Animais , Doenças Autoimunes/diagnóstico por imagem , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Leucócitos Mononucleares/metabolismo , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Cintilografia , Baço/citologia , Baço/patologia , Fatores de TempoRESUMO
Intractable autoimmune diseases in chimeric resistant MRL/lpr mice were treated by a new bone marrow transplantation (BMT) method consisting of fractionated irradiation, 5.5 Gy x 2, followed by intra-bone marrow (IBM) injection of whole bone marrow cells (BMCs) from allogeneic normal C57BL/6 (B6) mice (5.5 Gy x 2 + IBM). In MRL/lpr mice treated with this method, the number of donor-derived cells in the bone marrow, spleen, and liver rapidly increased (almost 100% donor-derived cells by 14 days after the treatment), and the number of donor-derived hemopoietic progenitor cells concomitantly increased. Furthermore, donor-derived stromal cells were clearly detected in the cultured bone pieces from MRL/lpr mice treated with 5.5 Gy x 2 + IBM. All the recipients thus treated survived more than 1 year (> 60 weeks after birth) and remained free from autoimmune diseases. Autoantibodies decreased to almost normal levels, and abnormal T cells (Thy1.2(+)/B220(+)/CD4(-)/CD8(-)) disappeared. Hematolymphoid cells were reconstituted with donor-derived cells, and newly developed T cells were tolerant to both donor (B6)-type and host (MRL/lpr)-type major histocompatibility complex determinants. Successful cooperation was achieved among T cells, B cells, and antigen-presenting cells when evaluated by in vitro antisheep red blood cell responses. These findings clearly indicate that this new strategy (IBM-BMT) creates the appropriate hemopoietic environment for the early recovery of hemopoiesis and donor cell engraftment, resulting in the complete amelioration of intractable autoimmune diseases in chimeric resistant MRL/lpr mice without recourse to immunosuppressants. This strategy would therefore be suitable for human therapy.
Assuntos
Doenças Autoimunes/cirurgia , Transplante de Medula Óssea/métodos , Medula Óssea , Animais , Autoanticorpos/sangue , Doenças Autoimunes/mortalidade , Células da Medula Óssea , Contagem de Células , Feminino , Células-Tronco Hematopoéticas , Injeções , Fígado/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Baço/citologia , Células Estromais , Taxa de Sobrevida , Linfócitos T/patologia , Doadores de TecidosRESUMO
We examined the importance of the coadministration of bone marrow (BM) stromal cells with BM cells via the portal vein. A significant increase in the number of day-14 colony-forming unit-spleen (CFU-S) was observed in the recipient mice injected with hemopoietic stem cells (HSCs) along with donor BM stromal cells obtained after three to four weeks of culture. Histological examination revealed that hematopoietic colonies composed of both donor hemopoietic cells and stromal cells coexist in the liver of these mice. However, when donor HSCs plus BM stromal cells were administered i.v., neither the stimulatory effects on CFU-S formation nor the hemopoietic colonies in the recipient liver were observed. These findings suggest that the interaction of HSCs with stromal cells in the liver is the first crucial step for successful engraftment of allogeneic HSCs. It is likely that donor stromal cells and HSCs trapped in the liver migrate into the recipient BM and spleen, where they form CFU-BM and CFU-S, respectively.
Assuntos
Células da Medula Óssea/fisiologia , Transplante de Medula Óssea/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/fisiologia , Células Estromais/fisiologia , Animais , Células da Medula Óssea/citologia , Células-Tronco Hematopoéticas/citologia , Injeções Intravenosas , Fígado , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Veia Porta , Células-Tronco , Células Estromais/citologia , Fatores de Tempo , Distribuição Tecidual , Transplante HomólogoRESUMO
We have previously found that a significant number of hematopoietic progenitors accumulate in engrafted bones with the same major histocompatibility complex (MHC) as the transplanted bone marrow cells. In the present study, to further clarify the MHC restriction between hematopoietic stem cells (HSC) and microenvironment, we carried out cobblestone colony formation assays by culturing HSCs with MHC-matched or -mismatched stromal cell monolayers. The formation of cobblestone colonies under MHC-mismatched stromal cells significantly decreased in comparison with MHC-matched stromal cells. However, the decrease in cobblestone colony formation under MHC-mismatched stromal cells was not significant when using MHC class I-deficient HSC or stromal cells. Taken together with the results using B10 congenic strains, it is suggested that the MHC preference is restricted by MHC class Ia molecules. Treatment with monoclonal antibodies (mAbs) against MHC class Ia molecules of stromal cell phenotypes significantly enhanced the cobblestone colony formation, whereas treatment with mAbs against HSC phenotypes significantly inhibited it. The expression of cytokines to promote hematopoiesis was enhanced by the mAbs against stromal cell phenotypes. The enhancement of cytokine expression was also observed when stromal cells and HSCs were MHC-matched. These results suggest that signaling via the MHC molecules augments stromal cell activity and elicits the MHC restriction.
Assuntos
Células-Tronco Hematopoéticas/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Células Estromais/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Transplante de Medula Óssea/imunologia , Citocinas/análise , Citometria de Fluxo , Doença Enxerto-Hospedeiro/imunologia , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/citologia , Técnicas In Vitro , Isoantígenos/análise , Isoantígenos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Transdução de Sinais/imunologia , Especificidade da Espécie , Células Estromais/química , Células Estromais/citologiaRESUMO
To minimize contamination of bone marrow cells (BMCs) with T cells from the peripheral blood, a new "perfusion method" for collecting BMCs is proposed using cynomolgus monkeys. Two BM puncture needles are inserted into a long bone such as the humerus, femur, or tibia. One needle is connected to an extension tube and the end of the tube is inserted into a culture flask to collect the BM fluid. The other needle is connected to a syringe containing 30 ml of phosphate-buffered saline. The solution is pushed gently from the syringe into the medullary cavity, and the medium containing the BM fluid is collected into the culture flask. There is significantly less contamination with peripheral blood, determined from the frequencies of CD4(+) and CD8(+) T cells, when using this method (<6%) than when using the conventional method (>20%) consisting of multiple BM aspirations from the iliac crest. Furthermore, the number and progenitor activities of the cells harvested using this "perfusion method" are greater than those harvested using the conventional aspiration method. This perfusion method was carried out 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. These findings suggest that the "perfusion method" is safe and simple and would be of great advantage in obtaining pure BMCs, resulting in a less frequent occurrence of acute graft-versus-host-disease in allogeneic BM transplantation.
Assuntos
Células da Medula Óssea/citologia , Separação Celular/métodos , Animais , Antígenos CD/análise , Antígenos de Superfície/análise , Células da Medula Óssea/imunologia , Contagem de Células , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Macaca fascicularisRESUMO
Intrathymic T-cell differentiation is characterized by two selection events: positive and negative selection. It has been shown that thymic epithelial cells in the cortex are involved in the positive selection, while macrophages and dendritic cells, derived from hemopoietic stem cells, are involved in the negative selection. Here we investigate whether donor-derived bone marrow stromal cells can migrate into the thymus and participate there in positive selection after bone marrow transplantation plus bone grafts (to recruit bone marrow stromal cells). Allogeneic bone marrow transplantation with or without bone grafts was carried out in the [C57BL/6-->C3H] combination. Fluorescence-activated cell sorter analyses of recipient thymic adherent cells showed that donor-type bone marrow stromal cells exist in the thymus of mice that received bone marrow plus bone grafts but not in the mice that received bone marrow cells alone. Histological examination using confocal microscopy also confirmed the existence of donor-type stromal cells in the thymus of mice that received bone marrow cells plus bones. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice show donor-type major histocompatibility complex-restriction. These findings strongly suggest that stromal cells can migrate from the bone marrow to the thymus, where they participate in the positive selection of thymocytes.
Assuntos
Células da Medula Óssea/citologia , Transplante de Medula Óssea , Transplante Ósseo , Células Estromais/citologia , Células Estromais/fisiologia , Timo/citologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Adesão Celular , Movimento Celular , Antígenos H-2/imunologia , Humanos , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Microscopia Confocal , Linfócitos T/imunologia , Doadores de TecidosRESUMO
The role of the thymic microenvironment in the development of murine thymic B cells has yet to be fully clarified. We therefore investigate the microenvironment that supports the development of mature thymic B cells (sIg+/B220+/CD43-B cells) from thymic B cell progenitors with immunophenotypes of sIg-/B220med/CD43+ cells. As we have previously reported, thymic B cells generated from these progenitors in the thymus are CD5+ B cells. We next study the in vitro condition that supports the differentiation of thymic B cell progenitors. Stromal cells (from the bone marrow or thymus), thymus-derived cell lines with the character of thymic nurse cells (TNCs) or thymic epithelial cells (TECs), or the bone marrow-derived cell line (MS-5) are tested for their ability to support B-lymphopoiesis from thymic B cell progenitors. Interestingly, thymic stromal cells (but neither stromal cells from the bone marrow nor stromal cell lines) support the differentiation of thymic B cell progenitors into thymic B cells in the presence of IL-7. Cortical epithelia (but not medullary epithelia, thymic macrophages or dendritic cells) are found to contribute to thymic B cell differentiation. Surface phenotype and Ig rearrangement analyses reveal that mature B cells generated in this condition are primarily CD5+ B cells, indicating that the thymic microenvironment (particularly cortical epithelia) determines the differentiation of thymic B cells.
Assuntos
Antígenos CD , Linfócitos B/citologia , Células-Tronco/citologia , Timo/citologia , Animais , Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Antígenos CD5/análise , Comunicação Celular , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/imunologia , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Interleucina-7/farmacologia , Antígenos Comuns de Leucócito/análise , Leucossialina , Masculino , Camundongos , Camundongos Endogâmicos C3H , Receptores de Antígenos de Linfócitos B/análise , Sialoglicoproteínas/análise , Células Estromais/citologia , Células Estromais/metabolismo , Timo/imunologia , Fatores de TempoRESUMO
The (NZW x BXSB)F1 (W/BF1) mouse is known as an autoimmune-prone strain which develops lupus nephritis, thrombocytopenia due to platelet-specific autoantibodies, leukocytosis, and myocardial infarction. In this experiment, we investigated the age-dependent abnormalities of the hematopoietic stem cells (HSCs) and hematopoiesis in this mouse. White blood cell counts (especially Mac-1- or Gr-1-positive cells) in the peripheral blood of 12-week-old W/BF1 mice increased in comparison with those of four-week-old W/BF1 or normal mice. To investigate whether the abnormal hematopoiesis can be attributed to the HSCs of W/BF1 mice, colony-forming unit in spleen (CFU-S) and colony-forming unit in culture (CFU-C) assays were performed. Day 12 CFU-S counts of 12-week-old W/BF1 mice significantly increased in comparison with those of four-week-old W/BF1 mice or normal mice. In the CFU-C assay, CFU-GEMM and CFU-GM counts in 12-week-old W/BF1 mice increased in comparison with those of four-week-old W/BF1 or control mice. The bone marrow cells (BMCs) from 12-week-old W/BF1 mice showed a high level of G-CSF and a low level of GM-CSF in mRNA expression. To examine the effect of HSCs from 12-week-old W/BF1 mice on the onset of autoimmune diseases and the abnormal hematopoiesis, T- and B-cell-depleted BMCs of four-week-old or 12-week-old W/BF1 mice were transplanted to C3H mice. Recipient C3H mice that had received the BMCs from 12-week-old W/BF1 mice showed an earlier onset of autoimmune diseases and a shorter survival rate than those that had received the BMCs from four-week-old W/BF1 mice. These data suggest that the HSCs from 12-week-old W/BF1 mice showing the symptoms of autoimmune diseases have the capacity to induce autoimmune diseases earlier than the HSCs from four-week-old W/BF1 mice.
Assuntos
Envelhecimento/patologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Nefrite Lúpica/patologia , Trombocitopenia/patologia , Idade de Início , Animais , Modelos Animais de Doenças , Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Hematopoese/imunologia , Células-Tronco Hematopoéticas/química , Contagem de Leucócitos , Leucocitose/mortalidade , Leucocitose/patologia , Leucocitose/terapia , Nefrite Lúpica/mortalidade , Nefrite Lúpica/terapia , Fator Estimulador de Colônias de Macrófagos/genética , Antígeno de Macrófago 1/análise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos NZB , Contagem de Plaquetas , RNA Mensageiro/análise , Análise de Sobrevida , Trombocitopenia/mortalidade , Trombocitopenia/terapiaRESUMO
Using Ly5 congenic mice, we characterized the early differentiation step of pluripotent hemopoietic stem cells. Lineage- (Lin-)/CD71- cells in the bone marrow cells were separated into major histocompatibility complex (MHC) class I(high)/c-kit(low) and MHC class I(high)/c-kitAssuntos
Células-Tronco Hematopoéticas/fisiologia
, Baço/citologia
, Fator de Células-Tronco/farmacologia
, Animais
, Antígenos CD4/imunologia
, Antígenos CD8/imunologia
, Ensaio de Unidades Formadoras de Colônias
, Células Precursoras Eritroides/imunologia
, Feminino
, Citometria de Fluxo
, Fluoruracila/farmacologia
, Granulócitos/imunologia
, Antígenos Comuns de Leucócito/imunologia
, Macrófagos/imunologia
, Masculino
, Camundongos
, Camundongos Congênicos
, Camundongos Endogâmicos C57BL
, Baço/química
, Fator de Células-Tronco/sangue
, Fatores de Tempo
RESUMO
Hepatocyte growth factor (HGF) is a multifunctional cytokine with early hematopoiesis-stimulatory activity. Here, we focus on its erythropoiesis-stimulatory effect on highly purified human hematopoietic progenitor cells (CD34+/CD45+ cells) derived from the cord blood. In immunoblot analyses, c-met protein (a receptor of HGF) was detected in the CD34+/CD45+ cells, although the expression levels were different among samples. The c-met expression was facilitated by incubation of the cells with stem cell factor (SCF) or interleukin 3 (IL-3), even if the expression level had been low. IL-6, G-CSF, or erythropoietin (EPO) did not show such a stimulatory effect on the c-met expression of the cells. When HGF was added to the CD34+/CD45+ cells in the presence of SCF, the numbers of CD36+/CD11b- cells (very early erythroid lineage cells) and BFU-E increased. EPO-dependent tyrosine phosphorylation of Stat 5 also increased, but the EPO receptor (EPO-R) expression remained unchanged in the CD34+/CD45+ cells treated with SCF + HGF. Our present study suggests that stimulation of the HGF/c-met signal is concomitant with induction of c-met protein by SCF. The subsequent enhancement of signal transduction via the activation of Stat 5 from the EPO-R plays a crucial role in the commitment of hematopoietic stem cells into erythroid lineage cells.
Assuntos
Antígenos CD34/metabolismo , Sangue Fetal/metabolismo , Fator de Crescimento de Hepatócito/fisiologia , Proteínas do Leite , Receptores de Complemento 3b/metabolismo , Receptores da Eritropoetina/fisiologia , Transdução de Sinais , Western Blotting , Antígenos CD36/metabolismo , Proteínas de Ligação a DNA/imunologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas , Humanos , Interleucina-3/metabolismo , Testes de Precipitina , Proteínas Proto-Oncogênicas c-met/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5 , Fator de Células-Tronco/metabolismo , Fatores de Tempo , Transativadores/imunologiaRESUMO
Dendritic cells (DCs), which are the most effective professional antigen-presenting cells (APCs), initiate and regulate immune responses. In this report, we examine the role of DCs in the induction of autoimmune thyroiditis. Experimental autoimmune thyroiditis (EAT) induced by immunization with thyroglobulin (Tg) plus adjuvant is considered to be an animal model of autoimmune thyroiditis, and is categorized as a T cell-mediated autoimmune disease. To examine the contribution of DCs to EAT, naive DCs were purified from high responder B10BR mice and pulsed with various concentrations of porcine Tg (pTg). These pTg-pulsed DCs were transferred without adjuvant to syngenic BIOBR mice to induce EAT. Mice that had received pTg-pulsed DCs showed thyroiditis, and the degree of thyroiditis induced was positively correlated to the amounts of pTg used for the incubation (pulsing) of DCs. The severity of thyroiditis was also correlated to the amounts of anti-pTg IgG2a antibodies and IFN-gamma in the recipient sera, but not to IL-4 or IL-10, indicating that Th1 cells are mainly activated by pTg-pulsed DCs and attributable to the pathogenesis of EAT.
Assuntos
Células Dendríticas/imunologia , Tireoglobulina/imunologia , Tireoidite Autoimune/induzido quimicamente , Animais , Apresentação de Antígeno , Linfócitos T CD4-Positivos/imunologia , Transplante de Células , Técnicas de Cocultura , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Ativação Linfocitária , Camundongos , Baço/citologia , Baço/imunologiaRESUMO
In this study, we report that W/W mutant mice, which have severe macrocytic anemia caused by a deficit of extracellular domain in c-kit molecules and therefore die perinatally, have hemopoietic stem cells (HSCs) and mature hematolymphoid cells in the bone marrow (BM), thymus, and spleen, although there are significant decreases in cell counts. Moreover, the mitogen-induced proliferative response, mixed lymphocyte reaction, and anti-SRBC plaque formation of spleen cells in W/W mice are similar to those in age-matched +/? littermates and normal mice, suggesting that the SCF/c-kit system is necessary for cell proliferation but not essential for HSCs to differentiate. We next examine the stimulatory effects of hepatocyte growth factor (HGF) on hemopoiesis in W/W mice. HGF has a stimulatory effect on the colony formation (CFU-C) of W/W BM cells when cultured using either a methylcellulose assay (containing cytokines) or a long-term culture (LTC) assay. A similar stimulatory effect of HGF is observed in the other W or SI locus-mutant mice (W/Wv and SI/SId mice), which show less severe anemia than W/W. The numbers of nonadherent cells and cobblestone colonies significantly increase in the LTCs using their BM cells. In addition, in vivo administration of HGF shows a transient increase in the CFU-C counts in BM cells and peripheral blood cells. RBC, WBC, and platelet counts also increased. These results suggest that the SCF/c-kit system is not essential to hemopoiesis but that a compensatory system such as the HGF/c-met system functions in the SCF/c-kit system-deficient mice.
Assuntos
Hematopoese/fisiologia , Fator de Crescimento de Hepatócito/farmacologia , Anemia Macrocítica , Animais , Linfócitos B/imunologia , Contagem de Células Sanguíneas , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Ativação Linfocitária , Mastócitos/citologia , Mastócitos/ultraestrutura , Camundongos , Camundongos Mutantes , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/fisiologia , Proteínas Proto-Oncogênicas c-met/análise , Baço/imunologia , Fator de Células-Tronco/genética , Fator de Células-Tronco/fisiologia , Células-Tronco , Linfócitos T/imunologiaRESUMO
We have previously found that TJ-48 has the capacity to accelerate recovery from hematopoietic injury induced by radiation and the anti-cancer drug mitomycin C (MMC). The effects are found to be due to its stimulation of spleen colony-forming unit (CFU-S) counts on day 14. In the present study, we attempt to isolate and purify the active components in TJ-48 extracts using a new in vitro hematopoietic stem cell (HSC) assay method. n-Hexane extract from TJ-48 shows a significant stimulatory activity. The extract is further fractionated by silica gel chromatography and HPLC in order to identify its active components. 1H-NMR and GC-EI-MS indicate that the active fraction is composed of free fatty acids (oleic acid and linolenic acid). When 27 kinds of free fatty acids (commercially available) are tested using the HSC proliferating assay, oleic acid, elaidic acid, and linolenic acid are found to have potent activity. The administration of oleic acid to MMC-treated mice enhances CFU-S counts on days 8 and 14 to twice the control group. These findings strongly suggest that fatty acids contained in TJ-48 actively promote the proliferation of HSCs. Although many mechanisms seem to be involved in the stimulation of HSC proliferation, we speculate that at least one of the signals is mediated by stromal cells, rather than any direct interaction with the HSCs.
Assuntos
Medicamentos de Ervas Chinesas/química , Ácidos Graxos/isolamento & purificação , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Mitógenos/isolamento & purificação , Animais , Medula Óssea/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Fracionamento Químico/métodos , Clorofórmio , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Ensaio de Unidades Formadoras de Colônias , Tecido Conjuntivo/efeitos dos fármacos , Ácidos Graxos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Hexanos , Espectroscopia de Ressonância Magnética , Metanol , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mitomicina/toxicidade , Ácido Oleico/isolamento & purificação , Ácido Oleico/farmacologia , Ácidos Oleicos , Solventes , Ácido alfa-Linolênico/isolamento & purificação , Ácido alfa-Linolênico/farmacologiaRESUMO
Pluripotent hemopoietic stem cells (P-HSCs) were thought to be c-kit+, but recent reports indicate that they are c-kit(low). In the present report, we provide evidence using Ly5 congenic mice that P-HSCs are c-kit(Assuntos
Transplante de Células-Tronco Hematopoéticas
, Células-Tronco Hematopoéticas/imunologia
, Proteínas Proto-Oncogênicas c-kit/biossíntese
, Animais
, Células da Medula Óssea
, Camundongos
, Camundongos Endogâmicos C57BL
, Reação em Cadeia da Polimerase
, Proteínas Proto-Oncogênicas c-kit/análise
, Proteínas Proto-Oncogênicas c-kit/genética
, Baço/citologia
, Timo/citologia
RESUMO
To investigate whether hemopoietic stem cells (HSCs) can differentiate into all lineage cells even in the thymus, we injected two types of HSCs (c-kit+ and c-kit < low cells) obtained from C57BL/6 Ly5.1 mice directly into the thymus of 7.5 Gy-irradiated C57BL/6 Ly5.2 mice. When c-kit < low cells (low density/lineage-/CD71-/major histocompatibility complex class I high/Sca-1+/Thy-1low/ c-kit < low) were injected, donor-derived (Ly5.1) cells were detected on day 8 after intrathymic (i.t.) injection, and the number reached a maximum on day 24 after injection. Granulocytes and macrophages were also detected on day 8 after injection. However, B220+ B cells were observed on day 13. Eighteen days after i.t. injection, the injected lobes showed red color due to the synchronous development of erythroid cells. Histological studies revealed the development not only of erythroid lineage cells but also of megakaryocytes in the thymus. In contrast, when c-kit+ cells were injected, a significant number of donor-derived cells were detected on day 5 after i.t. injection (three days earlier than in the case of c-kit < low cell injection). The differentiation into erythroid lineage cells was also observed six days earlier than when c-kit < low HSCs were injected. These findings suggest that c-kit < low HSCs are more primitive than c-kit+ HSCs, although both can differentiate into all lineage cells after i.t. injection.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Timo/citologia , Animais , Separação Celular , Feminino , Transplante de Células-Tronco Hematopoéticas , Camundongos , Camundongos Endogâmicos C57BL , RatosRESUMO
c-kit, a receptor for stem cell factor, has been widely accepted as a distinctive marker for hematopoietic stem cells. However, the level of c-kit expression on pluripotent hematopoietic stem cells is still controversial in mice and humans. We purified CD34+/c-kit < low cells (phenotypically c-kit-negative but only detectable at the message level) from human cord blood and examined their maturational steps in relation to the expression of c-kit molecules. When the CD34+/c-kit < low cells were cultured with cytokines (flt 3 ligand, interleukin 6 and interleukin 7) plus immobilized anti-CD34 monoclonal antibody (to crosslink CD34 molecules), c-kit molecules were clearly induced within 24 h. The c-kit expression gradually increased until day 8. When CD34+/c-kit(low) or CD34+/c-kit+ cells that had been induced from CD34+/c-kit < low cells were resorted and recultured using a methylcellulose culture system, they showed the same colony-forming ability as the freshly isolated CD34+/c-kit(low) or CD34+/c-kit+ cells, respectively. Furthermore, CD34+/c-kit < low cells have a similar hematopoietic potential to CD34+/c-kit(low) cells in assays for long-term culture initiating cell and colony-forming unit culture generated from long-term cultures. These findings suggest that CD34+/c-kit < low cells mature into CD34+/c-kit(low) and CD34+/c-kit+ cells, and acquire the reactivity to various humoral hematopoietic stimuli. Moreover, CD34+/c-kit < low cells showed a low level of rhodamine 123 retention, suggesting that CD34+/c-kit < low cells have multidrug resistance. Therefore, the CD34+/c-kit < low cells without colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte activity are also a pluripotent hematopoietic stem cell population, and the expression of c-kit on c-kit < low cells is the first maturational step of hematopoiesis.
Assuntos
Antígenos CD34/imunologia , Células-Tronco Hematopoéticas/citologia , Proteínas Proto-Oncogênicas c-kit/biossíntese , Separação Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Corantes Fluorescentes/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro , Rodamina 123 , Rodaminas/metabolismoRESUMO
To clarify major histocompatibility complex (MHC) restriction between hematopoietic stem cells (HSCs) and microenvironments, T cell-depleted bone marrow cells (BMCs) were transplanted into MHC-compatible and MHC-incompatible recipients. A significantly larger number of spleen colony-forming units (CFU-S) on day 12 were noted in MHC-compatible recipients, while only a small number were observed in MHC-incompatible recipients. There was, however, no significant difference in CFU-S counts on day 8 between the two groups. A large number of CFU-S counts on day 12 were also observed in F1 hybrid recipients, as seen in syngeneic recipients. The decrease in CFU-S counts on day 12 in MHC-incompatible recipients was also observed even after in vivo abrogation of T and NK cells. The difference in CFU-S counts on day 12 became more prominent when HSC-enriched cells were transferred. These results suggest that an MHC restriction exists between pluripotent HSCs (P-HSCs) and spleen microenvironments. Furthermore, experiments using B10. A recombinant strains revealed that H-2D and S loci play a crucial role in the MHC restriction. The experiments of serial transplantation suggest that the differentiation and proliferation of P-HSCs are inhibited in MHC-incompatible microenvironments. It is therefore likely that the MHC-compatible microenvironment is essential to the differentiation and proliferation of P-HSCs.