RESUMEN
We have previously shown that T cells can acquire donor-type major histocompatibility complex (MHC) restriction and can interact with both donor-type antigen-presenting cells (APCs) and B cells, when adult donor bones are co-grafted with intravenous (IV) injection of bone marrow cells (BMCs) in order to supply donor bone marrow (BM) stromal cells. We have also found that the direct injection of donor BMCs into recipient BM (intra-bone marrow-bone marrow transplantation: IBM-BMT) produces more rapid reconstitution (including T-cell functions) and higher survival rates than IV injection (IV-BMT) even in chimerism-resistant combinations. In the present study, we show that the co-administration of bones from suckling (2-3 days old) donor mice is also effective in the IBM-BMT system. Even when a relatively low number of BMCs were injected into adult (more than 15 weeks old) mice, complete reconstitution was achieved in the mice that had received IBM-BMT+bone grafts, but not in the mice that had received IBM-BMT alone. Most BM and splenic adherent cells obtained from the recipients that had received IBM-BMT+bone grafts were reconstituted by donor-type cells. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice showed donor-type MHC restriction. Moreover, the analyses of thymic sections using confocal microscopy revealed that donor BM stromal cells had migrated into the thymus. Thus, the co-administration of donor bones has great advantages for allogeneic BMT in adult mice.
Asunto(s)
Células de la Médula Ósea/inmunología , Trasplante de Médula Ósea/inmunología , Trasplante Óseo/inmunología , Sistema Hematopoyético/fisiología , Linfocitos T/inmunología , Animales , Proliferación Celular , Antígenos H-2/inmunología , Hematopoyesis , Sistema Hematopoyético/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Bazo/inmunología , Bazo/metabolismo , Células del Estroma/inmunología , Células del Estroma/metabolismo , Linfocitos T/metabolismo , Timo/inmunología , Timo/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: Using various animal models for autoimmune diseases, we have previously shown that such diseases are stem cell disorders.1 In order to understand how autoimmune diseases develop, we investigated the distinct qualitative differences between hematopoietic stem cells (HSC) from normal and autoimmune-prone mice. DESIGN AND METHODS: We studied the major histocompatibility complex (MHC) restriction between HSC and stromal cells in vitro and in vivo. We also examined the ability of HSC to adhere to a stromal cell line and, using flow cytometry, analyzed the expression of various adhesion molecules in HSC before and after the onset of autoimmune disease. In addition, the effect of antibodies to anti-adhesion molecules on the proliferation of HSC was investigated. RESULTS: The abnormal HSC of MRL/lpr mice showed no MHC restriction (or preference) with stromal cells either in vitro or in vivo, although there was MHC restriction between normal HSC and stromal cells, as we previously reported.2,3 The abnormal HSC of MRL/lpr mice exhibited enhanced adhesion to stromal cells in vitro and expressed a higher amount of adhesion molecules such as neural cell adhesion molecule (NCAM). Interestingly, the proliferation of HSC in MRL/lpr mice was significantly suppressed by anti-NCAM monoclonaal antibodies. INTERPRETATION AND CONCLUSIONS: Abnormal HSC of MRL/lpr mice are more resilient than normal HSC. Furthermore, among various adhesion molecules, only NCAM shows increased expression on HSC of MRL/lpr mice after the onset of autoimmune diseases, and these molecules contribute to the enhanced proliferation capacity of abnormal HSC in MRL/lpr mice. The present findings suggest that there are intrinsic qualitative differences between HSC from normal and autoimmune-prone MRL/lpr mice.
Asunto(s)
Células Madre Hematopoyéticas/patología , Lupus Eritematoso Sistémico/patología , Ratones Endogámicos MRL lpr/anatomía & histología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Factores de Edad , Animales , Anticuerpos Monoclonales/farmacología , Médula Ósea/embriología , Adhesión Celular , División Celular , Células Cultivadas/citología , Células Cultivadas/metabolismo , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Femenino , Antígenos H-2/inmunología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/inmunología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Nefritis Lúpica/genética , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/patología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr/genética , Ratones Endogámicos MRL lpr/inmunología , Ratones Endogámicos NOD , Ratones Endogámicos NZB , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Moléculas de Adhesión de Célula Nerviosa/inmunología , Quimera por Radiación , Tolerancia a Radiación/genética , Células del Estroma/inmunología , Células del Estroma/patologíaRESUMEN
AIM: To prepare and characterize antibody against Memo protein and to detect the tissue distribution of Memo in mice. METHODS: Fusion protein GST-Memo was expressed and purified, and polyclonal antibody against Memo was prepared by immunizing mice. A FLAG-tagged eukaryotic expression vector pcDNA3-FLAG-Memo was constructed. The specificity of the antibody was detected by Western blot. RESULTS: An eukaryotic expression vector pcDNA3-FLAG-Memo was obtained. The polyclonal antibody was found to be specific to Memo. Memo protein was widely expressed in mouse tissues using the obtained antibody in Western blot. CONCLUSION: Antibody specific to Memo has been successfully obtained, which provides useful tool for investigation into Memo-associated mechanisms of tumor metastasis and invasiveness.
Asunto(s)
Anticuerpos/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas de Hierro no Heme/inmunología , Proteínas de Hierro no Heme/metabolismo , Animales , Western Blotting , Línea Celular , Escherichia coli/genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Ratones , Proteínas de Hierro no Heme/biosíntesis , Proteínas de Hierro no Heme/aislamiento & purificación , Especificidad de Órganos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Mesenchymal stem cells (MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells. MSCs have some features (surface molecules and cytokine production, etc.) common to so-called traditional bone marrow (BM) stromal cells, which have the capacity to support hemopoiesis. In the present study, we isolated murine MSCs (mMSCs) from the fetal BM using an anti-PA6 monoclonal antibody (mAb) that is specific for bone marrow stromal cells. The mMSCs, called FMS/PA6-P cells, are adherent, fibroblastic, and extensively expanded and have the ability to differentiate not only into osteoblasts and adipocytes but also into vascular endothelial cells. The FMS/PA6-P cells produce a broad spectrum of cytokines and growth factors closely related to hemopoiesis and show good hemopoiesis-supporting capacity both in vivo and in vitro, suggesting that they are a component of the hemopoietic stem cell niche in vivo. Interestingly, although the FMS/PA6-P cells express a high level of the PA6 molecule, which is reactive with anti-PA6 mAb, they gradually lose their ability to express this molecule during the course of differentiation into osteoblasts and adipocytes, indicating that the PA6 molecule might serve as a novel marker of mMSCs.
Asunto(s)
Células de la Médula Ósea/fisiología , Diferenciación Celular/fisiología , Feto/fisiología , Hematopoyesis/fisiología , Células Madre Mesenquimatosas/fisiología , Adipocitos/citología , Adipocitos/fisiología , Animales , Antígenos de Diferenciación/biosíntesis , Células de la Médula Ósea/citología , Células Cultivadas , Femenino , Feto/citología , Sustancias de Crecimiento/biosíntesis , Masculino , Células Madre Mesenquimatosas/citología , Ratones , Ratones Desnudos , Osteoblastos/citología , Osteoblastos/fisiología , Células del Estroma/citología , Células del Estroma/fisiologíaRESUMEN
To clarify mechanisms underlying cell-to-cell interactions between hemopoietic stem cells (HSCs) and stromal cells, we established a stromal cell line (FMS/PA6-P) from day-16 fetal bone marrow (BM) adherent cells using an anti-PA6 monoclonal antibody (mAb) specific for BM stromal cells. Importantly, this FMS/PA6-P cell line, showing homogenous fibroblastic morphology, is absent from hematolymphoid and endothelial lineage markers and maintains a high level of expression of PA6 molecule, recognized by the anti-PA6 mAb, for approximately 20 passages. Further, the cell line expressing a high level of PA6 molecule has a better hemopoiesis-supporting capacity in vitro than other stromal cell lines such as PA6 and MS-5. In fact, the PA6 molecule is closely related to the hemopoiesis-supporting capacity of the stromal cells because the proliferation of HSCs was suppressed to a great extent by the anti-PA6 mAb. Affinity chromatography and mass peptide fingerprinting revealed that the protein reacting with the anti-PA6 mAb is neural cell adhesion molecule (NCAM). The frequencies of long-term cobblestone area-forming cells and long-term culture-initiating cells were significantly suppressed by repression of NCAM in the FMS/PA6-P cells using NCAM small interfering RNA. Our findings clearly indicate that NCAM functions on the maintenance of HSCs.
Asunto(s)
Comunicación Celular/fisiología , Hematopoyesis/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Células del Estroma/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Procesos de Crecimiento Celular/fisiología , Línea Celular , Citocinas/biosíntesis , Femenino , Hematopoyesis/genética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Moléculas de Adhesión de Célula Nerviosa/antagonistas & inhibidores , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Moléculas de Adhesión de Célula Nerviosa/genética , Embarazo , ARN Interferente Pequeño/genética , Células del Estroma/citología , Células del Estroma/metabolismo , TransfecciónRESUMEN
OBJECTIVE: To explore the way to induce mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons in vitro. METHODS: MSCs were obtained from rat bone marrow, cultured and passaged. MSCs used in this experiment had multipotency, which was indirectly proved by being induced to differentiate into chondrocytes and adipocytes. MSCs were cultured in medium containing 0.5 mmol/L IBMX for 2 days. Then the medium was replaced with induction medium, which contained GDNF, IL-1beta, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments. The surface markers of the differentiated neurons, such as NSE, nestin, MAP-2a, b and TH were detected by immunocytochemistry and Western blot after MSCs were cultured in induction medium for 7 days and 15 days. RESULTS: MSCs differentiated into neural progenitors and expressed nestin after MSCs were incubated with medium containing IBMX for 2 d. After the medium was replaced with induction medium containing many inducing agents, MSCs differentiated into neuron-like cells and dopaminergic neuron-like cells and expressed NSE, MAP-2a, b and TH. The percentage of NSE-positive cells, MAP-2a, b-positive cells and TH-positive cells was 30.032 +/- 2.489%, 41.580 +/- 5.101% and 34.958 +/- 5.534%, respectively after MSCs were induced in medium containing GDNF, IL-1beta, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments for 15 days. CONCLUSION: MSCs can differentiate into dopaminergic neuron-like cells and are a new cell source for the treatment of neurodegeneration diseases and have a great potential for wide application.
Asunto(s)
Células de la Médula Ósea , Diferenciación Celular , Células Madre Mesenquimatosas/citología , Neuronas/citología , Adipocitos/citología , Animales , Western Blotting , Carboxilesterasa/análisis , Células Cultivadas , Condrocitos/citología , Medios de Cultivo Condicionados , Dopamina/análisis , Proteínas de Filamentos Intermediarios/análisis , Mesencéfalo/citología , Proteínas del Tejido Nervioso/análisis , Nestina , Neuronas/metabolismo , Fosfoproteínas Fosfatasas/análisis , Ratas , Ratas WistarRESUMEN
Human cord blood (CB) contains hematopoietic stem cells and progenitors. Because the major limitation to a widespread use of CB for transplantation lies in its limited volume, it is necessary to combine the CB from several donors. In this study, we show that lethally irradiated mice can be reconstituted with the injection of a mixture of T cell-depleted bone marrow cells (BMCs; total, 3 x 10(6)) obtained from three fully allogeneic mouse strains in two different mouse combinations. A higher survival rate was obtained in the triple injection group than in mice injected with BMCs (1 x10(6)) obtained from a single mouse strain. In the mixed chimeric mice, three kinds of donor-type and recipient-type cells were detected in all the hematopoietic organs 1 month after bone marrow transplantation (BMT). Mixed-lymphocyte reaction showed that the tolerance to both recipient-type and donor-type major histocompatibility complex determinants was induced in the chimeric mice. In the peripheral blood (PB) of these mice, only one type of cells from the three different donor strains became dominant in most chimeric mice and reached a stable level about 4 months after BMT. Polymerase chain reaction analyses, however, revealed that the skins from all the donors were accepted even when no cells with their phenotypes could be detected in the PB. These results suggest that both hemato-lymphoid reconstitution and stable tolerance to not only the recipient strain but also all the donor strains can be achieved in chimeric mice, indicating the possibility of mixed CB transplantation in humans.