RESUMO
In long-term marrow cultures, hemopoiesis can be maintained for several months, although erythropoiesis is normally suppressed at the most primitive level of development (the erythroid colony-forming cells). Infection of these cultures with a viral complex combining helper-independent murine leukemia virus (F-MuLV) and a spleen focus-forming virus (SFFVp) results in a productive infection of both the replication defective SFFVp and the F-MuLV. After infection, the cultures show a dramatic elevation in the numbers of late erythroid progenitor cells (CFU-E), many of which will grow in the absence of added erythropoietin, and a transient erythropoietin, independent erythropoiesis, including the production of mature, enucleated erythrocytes. Hemopoiesis eventually declines, with no evidence for the generation of Friend tumor cells. When erythropoiesis is induced in the long-term cultures by addition of anemic mouse serum before infection by polycythemia-inducing Friend virus, the generation of erythropoietin-independent CFU-E and erythrocyte formation is followed by the sustained production (greater than 40 wk) of primitive erythroid cells with low spontaneous levels (less than 5%) of hemoglobinization. Although these cells will produce spleen colonies in irradiated mice and can be cloned in soft-gel media, they do not produce autonomous, permanently growing cell lines in vitro, i.e., they retain a dependency upon the marrow-adherent layer for their continued growth. However, following a further passage on a "virgin" marrow environment, permanent cell lines can be established that are able to grow independently of environmental influences. Thus, this system is the first description of a complete in vitro system for the reproducible production and isolation of Friend virus-induced erythroid cell lines.
Assuntos
Vírus da Leucemia Murina de Friend , Leucemia Eritroblástica Aguda/patologia , Leucemia Experimental/patologia , Anemia/sangue , Animais , Medula Óssea/patologia , Adesão Celular , Células Cultivadas , Eritropoese , Hematopoese , CamundongosRESUMO
PURPOSE: To support multicyclic, dose-intensive chemotherapy, we assessed the effects of reinfusing hematopoietic progenitors collected at each cycle in leukapheresis product or whole blood. PATIENTS AND METHODS: Twenty-five patients with small-cell lung cancer (SCLC) were treated with six cycles of ifosfamide, carboplatin, and etoposide (ICE) with granulocyte colony-stimulating factor (G-CSF) 300 micrograms/d subcutaneously (SC) on days 4 to 15. Hematopoietic progenitors collected during each cycle were reinfused on day 3 of the next cycle. Cohort 1 (n = 6) was treated every 3 weeks, with leukapheresis after 2 weeks and cryopreservation of the leukapheresis product. Chemotherapy was given if the WBC count was > or = 3 x 10(9)/L and platelet count > or = 100 x 10(9)/L. Cohort 2 (n = 7) was treated every 2 weeks, with leukapheresis on day 1 of the next cycle and storage of the leukapheresis product at 4 degrees C. Cohort 3 (n = 12) was treated every 2 weeks, with 500 to 750 mL of blood drawn by venesection on day 1 of the next cycle and stored at 4 degrees C. In cohorts 2 and 3, chemotherapy was given if the WBC count was > or = 3 x 10(9)/L and platelet count > or = 30 x 10(9)/L. Blood and leukapheresis products were assayed for hematopoietic progenitors. RESULTS: ICE chemotherapy with G-CSF was effective in mobilizing blood progenitors (median, 120-fold). Long-term cultures showed no evidence of stem-cell depletion. The cytotoxic dose-intensity of standard every-4-weeks ICE is 100%. In the first three cycles, it was 134% (median) in cohort 1 and 200% in cohorts 2 and 3 (P < .0001). Toxicity and supportive care requirements were not increased. CONCLUSION: The dose-intensity of ICE chemotherapy can be doubled by reinfusing hematopoietic progenitors collected by leukapheresis or venesection and stored at 4 degrees C.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma de Células Pequenas/terapia , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Neoplasias Pulmonares/terapia , Adulto , Transfusão de Sangue Autóloga , Carboplatina/administração & dosagem , Carcinoma de Células Pequenas/sangue , Carcinoma de Células Pequenas/mortalidade , Causas de Morte , Terapia Combinada , Etoposídeo/administração & dosagem , Estudos de Viabilidade , Feminino , Humanos , Ifosfamida/administração & dosagem , Leucaférese , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Transfusão de PlaquetasRESUMO
PURPOSE: This was the first randomized study to investigate the efficacy of peripheral-blood progenitor cell (PBPC) mobilization using stem-cell factor (SCF) in combination with filgrastim (G-CSF) following chemotherapy compared with filgrastim alone following chemotherapy. PATIENTS AND METHODS: Forty-eight patients with ovarian cancer were treated with cyclophosphamide and randomized to receive filgrastim 5 microg/kg alone or filgrastim 5 microg/kg plus SCF. The dose of SCF was cohort-dependent (5, 10, 15, and 20 microg/kg), with 12 patients in each cohort, nine of whom received SCF plus filgrastim and the remaining three patients who received filgrastim alone. On recovery from the WBC nadir, patients underwent a single apheresis. RESULTS: SCF in combination with filgrastim following chemotherapy enhanced the mobilization of progenitor cells compared with that produced by filgrastim alone following chemotherapy. This enhancement was dose-dependent for colony-forming unit-granulocyte-macrophage (CFU-GM), burst-forming unit-erythrocyte (BFU-E), and CD34+ cells in both the peripheral blood and apheresis product. In the apheresis product, threefold to fivefold increases in median CD34+ and progenitor cell yields were obtained in patients treated with SCF 20 microg/kg plus filgrastim compared with yields obtained in patients treated with filgrastim alone. Peripheral blood values of CFU-GM, BFU-E, and CD34+ cells per milliliter remained above defined threshold levels longer with higher doses of SCF. The higher doses of SCF offer a greater window of opportunity in which to perform the apheresis to achieve high yields. CONCLUSION: SCF (15 or 20 microg/kg) in combination with filgrastim following chemotherapy is an effective way of increasing progenitor cell yields compared with filgrastim alone following chemotherapy.
Assuntos
Antineoplásicos/administração & dosagem , Carcinoma/terapia , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Mobilização de Células-Tronco Hematopoéticas , Neoplasias Ovarianas/terapia , Fator de Células-Tronco/administração & dosagem , Adulto , Idoso , Antígenos CD34/análise , Antineoplásicos/efeitos adversos , Remoção de Componentes Sanguíneos , Carcinoma/sangue , Ensaio de Unidades Formadoras de Colônias , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Células Precursoras Eritroides , Feminino , Filgrastim , Fator Estimulador de Colônias de Granulócitos/efeitos adversos , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/sangue , Proteínas Recombinantes , Fator de Células-Tronco/efeitos adversosRESUMO
Recombinant purified human haemopoietic growth factors are available for clinical trials and some have been licensed for therapeutic use. Some haemopoietic lineages (erythroid, neutrophilic, monocyte-macrophagic) can be selectively stimulated in order to ameliorate the cytopenias that follow cytotoxic treatment, or that characterize some haematological syndromes, and to stimulate mature cell function. Advances in the knowledge of receptor-ligand interactions and of transduction mechanisms, plus the production of synthetic or mutant molecules that may mimic, potentiate or antagonize the effects of the natural growth factors, should make novel therapeutic approaches possible.
Assuntos
Fatores Estimuladores de Colônias/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Animais , Antineoplásicos/efeitos adversos , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Substâncias de Crescimento/uso terapêutico , Humanos , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Neutropenia/induzido quimicamente , Neutropenia/terapiaRESUMO
Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and are unable to isolate specific cell sub-populations. This report describes the development and validation of a cellular, immunofluorescence-based flow cytometry assay that allows detection of intranuclear hTERT while maintaining identifiable cell population characteristics. The assay was shown to be both sensitive to changes in telomerase expression and was semi-quantitative. In both cell line differentiation experiments and in primary cells, a good correlation existed between hTERT expression measured by flow cytometry and telomerase activity detected by the telomeric repeat amplification protocol (TRAP). The method developed offers a quick, simple and reproducible cellular-based assay for hTERT expression. This assay will provide a useful, new tool for future investigations, facilitating the analysis of hTERT expression in mixed cell populations.
Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , RNA , Telomerase/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA , Citometria de Fluxo , Células HL-60 , Humanos , Leucemia Linfocítica Crônica de Células B/patologiaRESUMO
Human haemopoietic stem and progenitor cells may be distinguished by the pattern of cell surface markers they display. The cells defined as 'stem' cells are heterogeneous and lack specific markers for their detection. However, they may be identified in in vitro assays such as the long-term culture initiating cell (LTC-IC) and in transplant assays involving immunosuppressed NOD/SCID mice. It is still not clear to what extent, if any, these cell populations overlap. The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) prolongs survival of LTC-IC in suspension cultures and we now show that in longterm bone marrow cultures (LTBMC) maintenance of haemopoiesis was significantly better from the CD34+ cells which possess MIP-1alpha receptors (P < 0.006). We examined one MIP-1alpha receptor, CCR1, which is present on CD34+ cells from haemopoietic tissues. In LTBMC the production of GM-CFC from CD34+CCR1- cells was significantly higher (P < 0.02) than that from CD34+CCR1+ cultures and the incidence of LTC-IC was 3- to 6-fold higher in the CD34+CCR1- cell fraction. In contrast, the cells responsible for high levels of engraftment in NOD/SCID mice were contained in the CD34+CCR1+ cell fraction. The CD34+CCR1+ cells engrafted to high levels in NOD/SCID and generated large numbers of progenitor cells. Therefore, we conclude that LTC-IC and SRC may be distinguished on the basis of expression of the chemokine receptor CCR1.
Assuntos
Antígenos CD34/análise , Células-Tronco Hematopoéticas/química , Receptores de Quimiocinas/análise , Animais , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , Sangue Fetal/citologia , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Proteínas Inflamatórias de Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores CCR1RESUMO
The megakaryopoietic potential in the bone marrow (BM) of patients in first remission after treatment for acute myelogenous leukaemia (AML) was investigated using long-term bone marrow cultures (LTC) stimulated with megakaryocyte growth and development factor (MGDF). The baseline number of megakaryocyte colony-forming cells (Meg-CFC) was very low. However, there was a 10 to 100-fold increase of Meg-CFC in cultures treated with 10 ng/ml MGDF with mean numbers within the normal range for the first 4 weeks of culture with a 24-fold increase in their cumulative numbers. Similarly, a 12-fold increase in the numbers of megakaryocytes (MKs) was found by CD61 immunostaining. These effects were lost at the dose of 100 ng/ml. In contrast, the cumulative mean numbers of Meg-CFC in the control cultures from normal bone marrow (NBM) were not significantly different from those in cultures treated with 10 or 100 ng/ml MGDF. These results demonstrate that MGDF stimulates megakaryocytopoiesis in patients with AML in first remission, restoring the Meg-CFC compartment to normal values, a result with potential clinical implications for their treatment with autologous transplantation.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Leucemia Mieloide Aguda/sangue , Megacariócitos/efeitos dos fármacos , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/farmacologia , Receptores de Citocinas , Adulto , Contagem de Células , Técnicas de Cultura , Feminino , Hematopoese/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Trombopoetina , Proteínas Recombinantes/farmacologia , Valores de ReferênciaRESUMO
The clonogenic cells of chronic myeloid leukaemia (CML), unlike normal haemopoietic colony forming cells (CFC), are resistant to the growth inhibitory effects of the chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha). Here, we tested the hypothesis that MIP-1alpha protects normal, but not CML, CFC from the cytotoxic effects of the cell-cycle active drug cytosine arabinoside (Ara-C). Using a 24-h Ara-C protection assay we showed that MIP-1alpha confers protection to normal CFC but also sensitizes CML CFC to Ara-C. The differential MIP-1alpha responsiveness was not due to a down-regulation of MIP-1alpha receptors on CML CD34+ cells as flow cytometric analysis showed similar binding of a biotinylated MIP-1alpha molecule to normal and CML CD34+ cells. Flow cytometric analysis of the MIP-1alpha receptor subtype CCR-5 revealed comparable CCR-5 expression levels on normal and CML CD34+ cells. Furthermore, culture of CD34+ cells for 10 h in the presence of TNF-alpha resulted in an increased MIP-1alpha receptor expression on both normal and CML CD34+ cells. Our data suggest that the unresponsiveness of CML CFC to the growth inhibitory effect of MIP-1alpha is not caused by a lack of MIP-1alpha receptor or total uncoupling of the MIP-1alpha responsiveness but may be due to an intracellular signalling defect downstream of the receptors.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Inflamatórias de Macrófagos/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Adolescente , Adulto , Idoso , Adesão Celular , Ciclo Celular/efeitos dos fármacos , Quimiocina CCL3 , Quimiocina CCL4 , Criança , Citarabina/toxicidade , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Receptores CCR5/análise , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Simple methods that separate progenitor cells of different hemopoietic lineages would facilitate studies on lineage commitment and differentiation. We used an antibody specific for the chemokine receptor CCR1 to examine mononuclear cells isolated from cord blood samples. When CD34(+) cells were separated into CD34(+)CCR1(+) and CD34(+)CCR1(-) cells and plated in colony-forming assays, the granulocyte/macrophage progenitors were found almost exclusively in the CD34(+)CCR1(+) cells. In contrast, the CD34(+)CCR1(-) cells contained the majority of the erythroid progenitors. There was a highly significant difference (P<0.002) in the total percentage distribution of both granulocyte-macrophage colony-forming cells and erythroid burst-forming units between the two populations. This is the first report of separation of erythroid progenitors from granulocyte/macrophage progenitors using a chemokine receptor antibody in cord blood samples. These results suggest that at the clonogenic progenitor cell stage the expression of CCR1 might be lineage-specific. This method should prove useful for studies on erythroid progenitor and granulocyte/macrophage differentiation.
Assuntos
Técnicas de Cultura de Células/métodos , Células Precursoras Eritroides/citologia , Células Progenitoras Mieloides/citologia , Receptores de Quimiocinas/biossíntese , Anticorpos/imunologia , Antígenos CD34/análise , Biomarcadores/análise , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Quimiocina CCL4 , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura Livres de Soro , Células Precursoras Eritroides/química , Sangue Fetal/citologia , Citometria de Fluxo , Granulócitos/citologia , Granulócitos/imunologia , Humanos , Proteínas Inflamatórias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Células Progenitoras Mieloides/química , Células Progenitoras Mieloides/imunologia , RNA Mensageiro/biossíntese , Receptores CCR1 , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologiaRESUMO
Repeated whole-body irradiation of adult mice induced hemopoiesis in liver, as shown by the presence of stem cells (CFUS), progenitor cells of granulocytes and macrophages (CFUC) and foci of granulocytic cells. The largest numbers of CFUS (up to 700) were found 24 to 47 days after four doses of 450 rad x-rays given at 24 day intervals and 15-17 days after 2310 rad gamma radiation given at a low dose-rate (70 rad per day). CFUS were still present (although in smaller numbers) up to 210 days after four doses of 375 rad x-rays or 225 rad neutrons. CFUC were also present in liver after four doses of 450 rad x-rays, but their numbers could not be calculated accurately because of the marked inhibitory effect of liver cells on in vitro colony growth. Irradiation with one dose of 450 rad x-rays did not result in the appearance of CFUS in liver, suggesting that hepatic hemopoiesis can be induced by radiation only after repeated or prolonged bone marrow injury.
Assuntos
Hematopoese/efeitos da radiação , Fígado/efeitos da radiação , Animais , Divisão Celular/efeitos da radiação , Células Cultivadas , Células Clonais , Granulócitos , Células-Tronco Hematopoéticas , Fígado/fisiologia , Macrófagos , Camundongos , Camundongos Endogâmicos , Doses de RadiaçãoRESUMO
To investigate the persistence of spleen colonies from day 8 to day 11 of their development, we injected low numbers of marrow cells in order to obtain single colonies on the spleens of irradiated mice. Colonies were isolated on either half of the spleen on the eighth day. The position of day-11 colonies, determined relative to the ligature, indicated where novel colonies appear between those times. The results showed no evidence of the persistence of colonies from day 8 to day 11. The self-reproduction capacity of CFU-S that survive various cytotoxic drugs depends on the specific subpopulations that are affected by the drug. Using cyclophosphamide, busulphan, or BCNU, the self-renewal capacity of surviving CFU-S was manipulated. The results show that after cytotoxic treatments, a high day-11-day-8 ratio is not necessarily a reflection of a high self-renewal capacity of the CFU-S population that forms the day-11 colonies.
Assuntos
Células-Tronco Hematopoéticas/citologia , Baço/citologia , Animais , Células da Medula Óssea , Transplante de Medula Óssea , Ensaio de Unidades Formadoras de Colônias , Ciclofosfamida/farmacologia , Camundongos , Quimera por Radiação , Fatores de TempoRESUMO
The cycling state of granulocyte-macrophage progenitor cells (GM-CFC) was measured using the [3H]thymidine suicide technique in individual male B6D2F1 mice. The proportion of the GM-CFC population in S phase was found to decrease from 25%-35% at 1-6 months of age to about 10% in mice greater than 2 years old.
Assuntos
Envelhecimento/fisiologia , Ciclo Celular , Células-Tronco Hematopoéticas/citologia , Animais , Células da Medula Óssea , Células Cultivadas , Granulócitos , Humanos , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Although much progress has been made in defining primitive cell phenotypes using flow cytometry and clonogenic assays, the direct study of marrow repopulating cells remains elusive. Long-term culture initiating cells (LTC-ICs) are arguably the most primitive human hematopoietic cells detectable by in vitro functional assays. Two endpoints have been reported for scoring LTC-ICs in limiting dilution assays. The first endpoint described was the generation of colony forming cells (CFCs) after 5 to 8 weeks of culture. An alternative method for scoring the LTC-IC assay is to identify cobblestone area forming cells. In the present study, estimations of LTC-IC frequency were made by measuring both endpoints and by comparing LTC-IC frequencies measured using limiting dilution assays of normal human bone marrow stroma with the measurements for murine bone marrow stromal cell line M2-10B4. For assays established on normal human bone marrow stroma, there was an equivalence between the two endpoint measures. Likewise, there was an equivalence between the two types of stroma when scoring CFC generation after 5 weeks. However, a consistently higher frequency of LTC-ICs was estimated when scoring cobblestone areas compared with that found when scoring CFC generation on the M2-10B4 stroma (p < 0.0001). Although the murine bone marrow stromal cell line M2-10B4 remains a very useful and consistently reliable alternative to normal human bone marrow stroma, these data indicate that the LTC-IC populations defined by scoring cobblestone areas or by measuring the generation of CFCs on this cell line are, in contrast to those measured using bone marrow stroma, not identical.
Assuntos
Células-Tronco Hematopoéticas/citologia , Animais , Medula Óssea/patologia , Técnicas de Cultura de Células/métodos , Ensaio de Unidades Formadoras de Colônias , Feminino , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Mobilização de Células-Tronco Hematopoéticas , Humanos , Técnicas de Diluição do Indicador , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Células Estromais/citologiaRESUMO
A single dose of Myleran induced a prolonged depletion of multi-potential haemopoietic cells and of early erythroid precursors to levels between 2 and 6 of control. In these mice, a priming dose of highly purified erythropoietin (Epo) or of any of 3 Epo preparations from different sources, and having different specific activities, increased the magnitude of the response to a test dose of Epo by erythropoietin responsive cells (ERC) in the hypertransfused mouse assay. As significant feeding into the ERC compartment from the most immature cells is unlikely because of the depletion induced by the Myleran, it may be concluded that highly purified Epo, not contaminated with other biologically active molecules, has a dual effect on the ERC: it causes increased amplification besides inducing differentiation into cells with the capacity to synthesize haemoglobin.
Assuntos
Eritropoese , Eritropoetina/fisiologia , Células-Tronco Hematopoéticas/citologia , Animais , Bussulfano/farmacologia , Ensaio de Unidades Formadoras de Colônias , Eritropoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Policitemia/sangue , Policitemia/etiologiaRESUMO
Persistent reductions in the femoral content of hemopoietic colony-forming cells (CFU-S and GM-CFC) were observed after four doses of irradiation, delivered with three weeks between doses. In general, the reductions were dose dependent, and similar reductions were produced by single doses and repeated doses using the same total dose. After the lowest doses investigated, 4 X 0.75 Gy 300-kVp x-rays or 4 X 0.38 Gy 14.7-MeV neutrons, the recovered levels remained at 60%-80% of control for at least one year after irradiation. The relative biological effectiveness (RBE) of neutrons is about 2 for these long-term hemopoietic deficiencies.
Assuntos
Células-Tronco Hematopoéticas/efeitos da radiação , Animais , Ensaio de Unidades Formadoras de Colônias , Relação Dose-Resposta à Radiação , Feminino , Granulócitos/efeitos da radiação , Macrófagos/efeitos da radiação , Camundongos , Nêutrons , Doses de Radiação , Eficiência Biológica Relativa , Fatores de Tempo , Irradiação Corporal Total , Raios XRESUMO
The ectopic implantation of mouse marrow to the kidney capsule offers considerable scope as an assay of the hemopoietic microenvironment. Our previous work has shown that whole-body irradiation of the graft recipient prior to implantation results in superior ossicle formation in the kidney of the host. Here we report that a range of irradiation doses over a 4-Gy threshold are equivalent with respect to conditioning the graft recipient. We also show that two distinct and separable influences affect graft growth in the irradiated recipient, namely, a local effect brought about in the irradiated kidney (and restricted to it) and secondly, a systemic effect resulting from irradiation of sites other than the kidney, which nevertheless affects ossicle growth in the shielded renal capsule.
Assuntos
Transplante de Medula Óssea , Rim/efeitos da radiação , Animais , Ensaio de Unidades Formadoras de Colônias/métodos , Relação Dose-Resposta à Radiação , Feminino , Células-Tronco Hematopoéticas/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos , Fatores Sexuais , Transplante/métodos , Irradiação Corporal TotalRESUMO
Injury inflicted upon the bone marrow stroma following whole-body irradiation and its repair over a 1-year period has been assessed in murine long-term bone marrow cultures established at increasing time intervals after irradiation. Different doses at different dose rates (10 Gy at 0.05 cGy/min, 4.5 Gy and 10 Gy at 1.6 cGy/min, and 4 x 4.5 Gy [3 weeks between doses] at 60 cGy/min) were chosen so as to maximize differences in effect in the stroma. The cellularity of the adherent layer in long-term cultures established 1 month after irradiation was reduced by 40%-90% depending on the dose and dose rate. Simultaneous with the poor ability of the marrow to form adherent layers, the cumulative spleen colony-forming unit (CFU-S) and granulocyte-macrophage colony-forming cell (GM-CFC) production over a 7-week period was reduced to 0% and 30% of control cultures, respectively. The slow recovery of the adherent layer was paralleled by an increase in the numbers of CFU-S and GM-CFC in the supernatant. Cultures established from repeatedly irradiated mice performed poorly over the entire 1-year period. Whereas the regeneration of the stroma was near complete 1 year after irradiation, the CFU-S and GM-CFC levels reached only between 50% and 80% of control cultures, respectively. Also, the concentration of CFU-S and GM-CFC in the supernatant remained persistently lower in cultures established from irradiated mice as compared to control cultures. The levels of sulfated glycosaminoglycans, which have been implicated in the establishment of the functional integrity of the microenvironment, were not reduced in the adherent layers at any time after irradiation. These results indicate that the regeneration of the stroma is accompanied by an incomplete recovery of active hemopoiesis in vitro. However, no evidence was found for persistent functional defects in the stroma after irradiation, using the present endpoints.
Assuntos
Células da Medula Óssea , Irradiação Corporal Total , Animais , Medula Óssea/metabolismo , Medula Óssea/fisiologia , Medula Óssea/efeitos da radiação , Divisão Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta à Radiação , Feminino , Glicosaminoglicanos/metabolismo , Granulócitos/citologia , Granulócitos/fisiologia , Granulócitos/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Hematopoéticas/efeitos da radiação , Macrófagos/citologia , Macrófagos/fisiologia , Macrófagos/efeitos da radiação , Camundongos , Fatores de TempoRESUMO
The addition of burst forming activity (BFA) from mouse spleen conditioned medium (MSCM) to serum-free long-term cultures induced the formation of day 4 BFUE demonstrating that MSCM has an erythropoietin-independent activity on erythroid development. Conditioned medium (CM) from long-term mouse marrow cell cultures was inhibitory to burst formation at concentrations as low as 0.01% when bone marrow cells were used as the external source of BFA, whereas significant levels of inhibition were apparent only at 100-fold greater concentrations of CM in cultures containing MSCM. CM did not inhibit erythropoietin-stimulated erythropoiesis in liquid subcultures of long-term culture cells nor did it stimulate erythroid maturation in the absence of erythropoietin. Adherent cells from the long-term cultures, used as feeder cells, were more active than normal bone marrow cells in enhancing erythroid burst formation.
Assuntos
Células da Medula Óssea , Ensaio de Unidades Formadoras de Colônias , Eritropoese , Animais , Adesão Celular , Células Cultivadas , Meios de Cultura , Eritropoetina/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Baço/metabolismo , Fatores de TempoRESUMO
Combinations of murine recombinant interleukin 3 (IL-3), purified murine macrophage colony-stimulating factor (M-CSF), and human recombinant interleukin 1 alpha (IL-1 alpha) were used to determine the effects of growth factors on the measured radiosensitivity of different populations of murine colony-forming cells (CFC). The data showed that combinations of growth factors resulted in different values of CFC radiosensitivity, being less than values observed when colony growth was stimulated using a single factor. For various combinations of growth factors, Do values ranged from 106 +/- 8 to 175 +/- 24 cGy for progenitor cells in normal bone marrow; 74 +/- 3 to 171 +/- 18 cGy for primitive multipotent CFC enriched using fluorescence-activated cell sorting; and from 46 +/- 4 to 131 +/- 10 cGy for more mature granulocyte-macrophage CFC, enriched by counterflow centrifugal elutriation. Only combinations of three factors produced the high values of Do reported in experiments using unpurified conditioned medium as a stimulus for colony formation.
Assuntos
Interleucina-3/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Tolerância a Radiação , Células-Tronco/fisiologia , Animais , Medula Óssea/fisiologia , Combinação de Medicamentos , Feminino , Citometria de Fluxo , Interleucina-3/administração & dosagem , Fator Estimulador de Colônias de Macrófagos/administração & dosagem , Masculino , Camundongos , Células-Tronco/efeitos dos fármacosRESUMO
We evaluated the role of high-dose granulocyte colony stimulating factor (G-CSF) in vitro, in inducing the generation of high-proliferative potential colony forming cells (HPP-CFC), from either mononuclear cells or purified CD34+ cells. Both normal controls and patients undergoing peripheral blood stem cell (PBSC) mobilization and transplantation were studied. In serum-driven agar cultures, G-CSF stimulated the proliferation of HPP-CFC in a dose dependent manner (r = 0.92). The number of HPP-CFC was four-fold greater in mobilized patients than in normal controls. Purified CD34+ cells yielded 11-fold more colonies than mononuclear cells. HPP-CFC from mobilized patients showed replating capacity, giving rise to secondary colonies of more mature appearance. In serum-free cultures, the effect of G-CSF appeared to be mediated by synergistic interaction with stem cell factor. Our results suggest that G-CSF stimulates primitive hematopoietic cells that are detectable in increased amounts in patients receiving mobilization therapy. Therefore, determination of G-CSF induced HPP-CFC could be a useful tool in the evaluation of mobilization strategies.