RESUMO
Successful stem cell transplantation depends on cell dose, and this is particularly true for placental/cord blood transplantation in which it has been clearly shown that both the success of engraftment as well as the speed of white cell and platelet recovery are dependent on the nucleated cell dose in the graft. Thus, if stem cell numbers could be increased, the speed as well as the likelihood of engraftment might be improved. We studied the effect of two different cytokine combinations--kit ligand (KL), interleukin-3 (IL-3), and Flt-3 ligand supplemented with thrombopoietin and IL-11 (combination 1) or granulocyte/macrophage colony-stimulating factor (GM-CSF) and G-CSF (combination 2)--for their ability to affect speed and extent of engraftment using limited numbers (5 x 10(4)) of murine bone marrow (BM) light-density (LD) cells or their progeny expanded ex vivo in the presence one or the other cytokine combination for 6 days. With combination 1, we found that speed of platelet recovery was enhanced, but at the expense of white blood cell (WBC) recovery and percent donor engraftment. Furthermore, the cytokine combination that best maintained donor engraftment, combination 2, did so at the expense of platelet recovery. In no case was percent donor engraftment improved over 5 x 10(4) unmanipulated LD BM cells. These results are consistent with the interpretation that immediate recovery of blood cells of different lineages and longterm donor engraftment are separate functions that can be influenced by the choice of cytokines used during the ex vivo expansion process.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Transplante de Células-Tronco Hematopoéticas , Interleucina-3/metabolismo , Proteínas de Membrana/metabolismo , Fator de Células-Tronco/metabolismo , Trombopoetina/metabolismo , Animais , Plaquetas/metabolismo , Linhagem da Célula , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The desire to improve engraftment following transplantation of limited numbers of hematopoietic stem cells (HSC) has spurred the investigation of ex vivo stem cell expansion techniques. While surrogate outcomes, such as an increase in SCID-repopulating cells, suggest successful stem cell expansion in some studies, it is not clear that such assays predict outcomes using a more clinically relevant approach (e.g., myeloablation). We have addressed this by testing three cytokine combinations for their ability to increase the radioprotective and long-term marrow reconstitution capacity of hematopoietic cells cultured ex vivo. Low numbers of light-density (LD) mouse bone marrow (BM) cells or their expanded product were injected into lethally irradiated (9 Gy) congenic recipients. Survival rates and percent donor engraftment were compared at 2, 5, and 7 months post-transplant. The three cytokine combinations used were: (i) kit-ligand (L), thrombopoietin (Tpo), Flt-3 L; (ii) cytokines in (i) plus interleukin-11 (IL-11); (iii) cytokines in (ii) plus IL-3. At 7 months post-transplant, LD cell doses of 10(4), 2-2.5 x 10(4), and 0.5-1.0 x 10(5) gave predictable survivals of 20-30%, 40-70%, and 100%, respectively. Mean percent donor engraftments were 54.9% (SEM 36%), 55.7% (SEM 36%), and 76.3% (SEM 21%), respectively. When cells expanded for 3 or 5-7 days with the various cytokine combinations were transplanted into different groups of mice, survival rates and percent donor engraftment were almost uniformly poorer than results obtained with unmanipulated cells, and cells expanded for 5-7 days led to poorer outcomes than cells expanded for 3 days. Overall, ex vivo expansion of LD BM cells with the cytokine combinations chosen failed to improve transplant outcomes in this model.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Citocinas/biossíntese , Transplante de Células-Tronco/métodos , Animais , Divisão Celular , Sobrevivência Celular , Transplante de Células , Citocinas/metabolismo , Citocinas/uso terapêutico , Feminino , Técnicas In Vitro , Interleucina-11/uso terapêutico , Proteínas de Membrana/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Fator de Células-Tronco/metabolismo , Trombopoetina/uso terapêutico , Fatores de TempoRESUMO
There is evidence that the total cellular content of placental cord blood (PCB) grafts is related to the speed of engraftment, though the total nucleated cell (TNC) dose is not a precise predictor of the time of neutrophil or platelet engraftment. It is important to understand the reasons for the quantitative association and to improve the criteria for selecting PCB grafts by using indices more precisely predictive of engraftment. The posttransplant course of 204 patients who received grafts evaluated for hematopoietic colony-forming cell (CFC) content among 562 patients reported previously were analyzed using univariate and multivariate life-table techniques to determine whether CFC doses predicted hematopoietic engraftment speed and risk for transplant-related events more accurately than the TNC dose. Actuarial times to neutrophil and platelet engraftment were shown to correlate with the cell dose, whether estimated as TNC or CFC per kilogram of recipient's weight. CFC association with the day of recovery of 500 neutrophils/microL, measured as the coefficient of correlation, was stronger than that of the TNC (R = -0.46 and -0.413, respectively). In multivariate tests of speed of platelet and neutrophil engraftment and of probability of posttransplantation events, the inclusion of CFC in the model displaced the significance of the high relative risks associated with TNC. The CFC content of PCB units is associated more rigorously with the major covariates of posttransplantation survival than is the TNC and is, therefore, a better index of the hematopoietic content of PCB grafts. (Blood. 2000;96:2717-2722)
Assuntos
Contagem de Células Sanguíneas , Sangue Fetal/citologia , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Placenta/irrigação sanguínea , Adolescente , Adulto , Anemia Aplástica/terapia , Peso Corporal , Criança , Pré-Escolar , Ensaio de Unidades Formadoras de Colônias , Feminino , Seguimentos , Doenças Genéticas Inatas/terapia , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Células-Tronco Hematopoéticas , Humanos , Lactente , Recém-Nascido , Tábuas de Vida , Masculino , Análise Multivariada , Síndromes Mielodisplásicas/terapia , Neoplasias/terapia , Gravidez , Sistema de Registros , Reticulócitos , Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
Thrombopoietin (Tpo) is the major physiologic regulator of platelet production. Its gene is expressed in many organs and appears constitutive in liver and kidney. However, inducible gene expression in the bone marrow and spleen have been reported as well as the presence of a number of isoforms, presumably arising from alternative splicing. We have identified five new murine Tpo isoforms, designated Tpo 5 to Tpo 9. Alternative splicing of these isoforms, in addition to the already-reported four isoforms, occurs around exon 7, the last exon, with insertion of some intron sequences or deletion of exon sequences. Studies of tissue distribution indicate that Tpo 4 is the major isoform in lymph nodes and bone marrow. The roles of these isoforms in hematopoietic regulation are unknown, but the presence of inducible Tpo mRNA in the marrow microenvironment may contribute to platelet or stem cell homeostasis.
Assuntos
Processamento Alternativo , Trombopoetina/genética , Animais , Camundongos , Isoformas de Proteínas/genética , RNA Mensageiro/genéticaRESUMO
CFC numbers have shown to correlate with success of engraftment and speed of neutrophil recovery following cord blood (CB) transplantation. To investigate whether the number of Meg-CFC in a CB stem cell preparation might correlate with time to platelet engraftment, we evaluated the frequency of Meg-CFC among all CFC types in 134 CB, 21 adult bone marrow (BM) and 52 cytokine-mobilized peripheral blood (PB) stem cell preparations. The correlation of Meg-CFC with the total number of CFC and mixed cell-CFC was also assessed. The frequency of Meg-CFC was highest in CB and correlated significantly with total CFC numbers (mean 20.8%, correlation coefficient (r) 0.84, P = 0.0001) compared with Meg-CFC from mobilized PB (mean 13.1%, r = 0.29, P = 0.07) and BM (mean 4%, r = 0. 39, P = 0.13). In addition, mixed-cell CFC numbers in CB were highly correlated with the total number of Meg-CFC (r = 0.7, P = 0.0001). No such correlations were found with mobilized PB or BM. We conclude that, based on the high degree of correlation between Meg-CFC and non-Meg-CFC numbers in CB, no additional information concerning time to platelet engraftment would be gained by routinely performing Meg-CFC assays in addition to non-Meg-CFC assays. The fact that CB Meg-CFC and mixed-cell CFC are strongly correlated suggests that CB Meg-CFC are more primitive than their counterparts in BM and PB and may reflect the number of stem cells in CB. Bone Marrow Transplantation (2000).
Assuntos
Células Sanguíneas/citologia , Plaquetas/citologia , Células da Medula Óssea/citologia , Ensaio de Unidades Formadoras de Colônias , Sangue Fetal/citologia , Sobrevivência de Enxerto , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Megacariócitos/citologia , Adulto , Contagem de Células Sanguíneas , Doadores de Sangue , Contagem de Células , Diferenciação Celular , Estudos de Avaliação como Assunto , Humanos , Recém-Nascido , Neutrófilos/citologia , Especificidade de Órgãos , Fatores de Tempo , Doadores de Tecidos , Obtenção de Tecidos e ÓrgãosRESUMO
Vascular endothelial cells (VEC) transduce mitogenic and chemoattractant signals in response to erythropoietin (Epo). An analysis of changes in gene expression in VEC would be helpful to understanding the molecular nature of mitogenic signals. An effective method for analysis of gene expression is through differential display. Using this approach, we obtained from Epo-treated human microvascular endothelial cells (HMVEC) a cDNA fragment with characteristics of the 3'end of mRNA. Using the cDNA fragment, we then isolated a full-length clone from a HMVEC cDNA library. The cDNA of interest encodes a protein consisting of 404 amino acids with a carboxy-terminal end sequence identical to glialblastoma cell differentiation factor-related protein (GBDR1). Northern blot analysis showed that GBDR1 mRNA was ubiquitously expressed in human tissues. In Southern blot analysis, GBDR1 cDNA identified a single gene on chromosome 9. Since analysis of the amino acid sequence revealed several putative phosphorylation sites for different protein kinases, the GBDR1 protein was expressed and purified from bacterial extracts and, as predicted, casein kinase II phosphorylated GBDR1 in vitro. Immunofluorescence and biochemical data revealed that the GBDR1 protein is not entirely localized in the cytosolic fraction, suggesting that it may interact with another protein(s). These findings demonstrate that GBDR1 is an intracellular signaling molecule that may play a role in the regulation of endothelial cell growth.
Assuntos
Diferenciação Celular/genética , Endotélio Vascular/citologia , Substâncias de Crescimento/genética , Peptídeos , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Mapeamento Cromossômico , Clonagem Molecular , Cricetinae , DNA Complementar , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Eritropoetina/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Substâncias de Crescimento/química , Substâncias de Crescimento/metabolismo , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Microcirculação , Dados de Sequência Molecular , Fosforilação , RNA Mensageiro/genética , Distribuição Tecidual , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: Anemic patients with chronic renal failure receiving recombinant human erythropoietin (rHuEPO) therapy frequently develop hypertension through an unknown mechanism. We hypothesize that EPO receptors (EPORs) on endothelial cells (ECs) in various sites of vasculature may mediate the activities of nitric oxide synthase (NOS) and/or the release of endothelin-1 (ET-1), contributing to blood pressure changes. We tested this hypothesis using primary cultures of ECs obtained from human coronary artery (HCAEC), pulmonary artery (HPAEC), dermis (HDEC), and umbilical vein (HUVEC). METHODS: EPORs were measured by 125I-EPO binding. The effect of EPO on EPOR, ET-1, and NOS mRNA levels was assessed by quantitative reverse transcription-polymerase chain reaction. Cellular NOS activity and ET-1 release into the medium was measured by the NOSdetect assay and by radioimmunoassay kits. RESULTS: Short-term (4 h) treatment with EPO (4 U/mL) did not change the number or affinity of EPOR per cell. Neither were there any changes in the amount of EPOR, ET-1, and NOS transcripts (cDNA/microg of mRNA) nor in ET-1 release and NOS activity. In HUVEC only, 24-hour exposure to EPO caused a threefold increase in NOS transcript. In other cells, EPO treatment for six days increased NOS activity by twofold to fourfold. CONCLUSIONS: We show that upon extended exposure, EPO induces NOS activity but does not affect ET-1 release. These findings indicate that the hypertensive effect of EPO is not likely to be caused by a direct effect on ECs.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Eritropoetina/farmacologia , Óxido Nítrico Sintase/biossíntese , Células Cultivadas , Endotelina-1/genética , Endotelina-1/metabolismo , Endotélio Vascular/enzimologia , Indução Enzimática/efeitos dos fármacos , Eritropoetina/metabolismo , Humanos , Óxido Nítrico Sintase/genética , RNA Mensageiro/análise , Receptores da Eritropoetina/genética , Proteínas RecombinantesRESUMO
Placental/umbilical cord blood (PCB) stem cells for transplantation provide a potentially useful alternative for patients who do not have an HLA-matched family or unrelated bone marrow donor. Concerns regarding this source of stem cells include the limited number of stem cells in a PCB unit and the delayed time to platelet engraftment. Because of the limited number of stem cells, there is a very clear cell dose effect for both success of engraftment and time to engraftment. As a result, many transplant centers will only consider PCB stem cells as a second choice for transplanting adults, despite the very favorable profile of post-transplant graft-versus-host disease (GvHD). This has resulted in considerable interest in the development of ex vivo stem cell expansion strategies. This review outlines the current status of PCB transplant outcomes as well as the status of our understanding of stem cell expansion with the currently available technologies. A stem cell dose-limiting effect on outcome will result in a narrower window of clinical indications for the use of this stem cell source, despite the acknowledged reduction in GvHD. The trade-offs between poor engraftment and reduction in fatal or severe chronic GvHD remain to be quantitated.
Assuntos
Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Adulto , Animais , Divisão Celular , Feminino , Células-Tronco Hematopoéticas/citologia , Teste de Histocompatibilidade , Humanos , Técnicas In Vitro , Recém-Nascido , Placenta/irrigação sanguínea , GravidezRESUMO
The transplantation of placental/umbilical cord blood (P/CB) has been used successfully to reconstitute bone marrow function in both related and unrelated recipients. We report here the experience of the New York Blood Center P/CB Program. Since its inception in 1992, over 400 unrelated transplants were supported between July 1993 and September 1997. Overall, event-free survival for all diagnoses and ages approached 0.45. Success and rapidity of engraftment correlated most strongly with the degree of human leukocyte antigen (HLA) disparity and cell dose/kg body weight recipient. Acute graft-versus-host disease (GVHD) was common in all patients but, surprisingly, did not differ between those patients who received grafts having one or more antigen mismatches. Chronic GVHD was uncommon and only rarely contributed to death. These results demonstrate the feasibility of large-scale P/CB banking for the provision of cryopreserved stem cell preparations for unrelated transplants. The degree of the program's success argues strongly for additional P/CB banks in order to increase the likelihood of finding a suitable stem cell preparation for patients for whom related matched donors do not exist.
Assuntos
Bancos de Sangue/organização & administração , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Anemia/terapia , Bancos de Sangue/estatística & dados numéricos , Preservação de Sangue , Criopreservação , Doença Enxerto-Hospedeiro/epidemiologia , Teste de Histocompatibilidade , Humanos , Recém-Nascido , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Cidade de Nova Iorque , PlacentaRESUMO
Iron overload was a common complication in patients with chronic renal failure treated with dialysis prior to the availability of recombinant human erythropoietin (rHuEPO) therapy. Iron overload was the result of hypoproliferative erythroid marrow function coupled with the need for frequent red blood cell transfusions to manage symptomatic anemia. The repetitive use of intravenous iron with or without the use of red blood cell transfusions also contributed to iron loading and was associated with iron deposition in liver parenchymal and reticuloendothelial cells; however, there were no abnormal liver function tests or evidence of cirrhosis unless viral hepatitis resulted from the transfusions. With rHuEPO therapy, the excess iron stores were shifted back into circulating red blood cells as the anemia was partially corrected, and red blood cells were lost from circulation by the hemodialysis procedure. After several years of rHuEPO therapy, most hemodialysis patients required iron supplements to replace the continuing blood losses related to hemodialysis. The potential complications of iron overload (parenchymal iron deposition, permanent organ damage, increased risk of bacterial infections, and increased free radical generation) are reviewed in the context of this setting.
Assuntos
Eritropoetina/uso terapêutico , Sobrecarga de Ferro/etiologia , Falência Renal Crônica/complicações , Falência Renal Crônica/tratamento farmacológico , Anemia/tratamento farmacológico , Anemia/etiologia , Infecções Bacterianas/etiologia , Radicais Livres/metabolismo , Humanos , Ferro/fisiologia , Sobrecarga de Ferro/complicações , Sobrecarga de Ferro/fisiopatologia , Proteínas RecombinantesRESUMO
Recombinant human erythropoietin (Epoetin alfa) is effective in increasing hemoglobin concentration and hematocrit, and in significantly reducing transfusion requirements in the majority of patients with either the anemia of chronic renal failure or chemotherapy-induced anemia of cancer. Identification of factors that could enable the clinician to predict individual patient hematological responses to Epoetin alfa therapy would be of great value. Changes in levels of serum transferrin receptor protein, hemoglobin, ferritin and reticulocyte count, following a short course of Epoetin alfa therapy, were useful markers for predicting later hematopoietic responses to Epoetin alfa. In addition, recent data suggest that low baseline erythropoietin levels, in association with increases of either >0.5 g/dl in hemoglobin or >/=25% in circulating levels of transferrin receptor protein after 2 weeks of Epoetin alfa therapy, are highly predictive of a response (>/=2 g/dl increase in hemoglobin) to Epoetin alfa. Progress has clearly been made in the development of predictive models that can identify those patients most likely to respond to Epoetin alfa by monitoring several specific hematological parameters at baseline and early in therapy. Future studies will focus on nonhematological measures of response, such as transfusion requirement and quality of life benefit.
Assuntos
Anemia/sangue , Anemia/tratamento farmacológico , Eritropoetina/uso terapêutico , Hematínicos/uso terapêutico , Hematopoese/efeitos dos fármacos , Neoplasias/sangue , Anemia/etiologia , Ensaios Clínicos como Assunto , Epoetina alfa , Humanos , Neoplasias/complicações , Valor Preditivo dos Testes , Proteínas Recombinantes , Resultado do TratamentoRESUMO
First used successfully to correct the anemia associated with chronic renal failure, epoetin alfa has been shown to be highly effective in many patients with either hematologic or nonhematologic malignancies. Multiple studies have demonstrated effective response rates, with increases in hemoglobin concentration and reduction or elimination of transfusion requirements in up to 75% or 80% in such patients. Nevertheless, as clinical experience has grown, several issues have arisen. First, not all cancer patients respond to epoetin alfa and, consequently, it is important to identify those patients most likely to respond to make early clinical decisions regarding dose adjustment or drug withdrawal. Second, experience in patients with renal failure has revealed a state of "functional iron deficiency" and, thus, highlighted the importance of iron supplementation to optimize the response to epoetin alfa. Does "functional iron deficiency" complicate epoetin alfa therapy of patients with the anemia of cancer, and could such patients benefit from iron supplementation? Finally, some hematologic malignancies, especially myelodysplastic syndromes, can be resistant to epoetin alfa monotherapy. Can the effective response rates in such patients be improved by combining epoetin alfa therapy with the administration of other hematopoietic growth factors? Epoetin alfa has made substantial contributions to the care of patients with cancer and, with time, additional uses for this very valuable drug will become apparent.
Assuntos
Anemia/tratamento farmacológico , Eritropoetina/uso terapêutico , Hematínicos/uso terapêutico , Anemia/etiologia , Transfusão de Sangue , Epoetina alfa , Eritropoetina/economia , Hematínicos/economia , Humanos , Ferro/uso terapêutico , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Qualidade de Vida , Proteínas RecombinantesRESUMO
Cord blood banking for the purpose of stem cell transplantation is a rapidly growing area of medical interest. Based on the fact that cord blood contains large numbers of stem and progenitor cells, transplantation of cord blood for marrow reconstitution was first attempted in 1988. The success of this initial transplant between related donor and patient rapidly led to the establishment of efforts to collect, store and eventually transplant unrelated cord blood samples. A collection and storage program established by the New York Blood Center has led to more than 400 such transplants. The results demonstrate acceptable rates of engraftment and little graft-versus-host disease compared to the results employing adult marrow. As a consequence of these observations, considerable effort is being made to establish cord blood banks around the world.
Assuntos
Bancos de Sangue , Sangue Fetal , Transplante de Células-Tronco Hematopoéticas , Adolescente , Criança , Pré-Escolar , Humanos , Lactente , New York , Sistema de RegistrosRESUMO
Erythropoietin, which is produced by peritubular capillary lining cells of the kidney, is critical to the production of red blood cells. Endogenously produced erythropoietin circulates in the plasma to act on specific target cells in the marrow through cell surface receptors. The primary target of erythropoietin action is the erythroid colony-forming cell. In addition to proliferative effects, high circulating erythropoietin levels result in the premature release of marrow reticulocytes and the mobilization of marrow progenitor cells. Production of endogenous erythropoietin increases in response to the stress of anemia or hypoxemia. Iron, an important element in hemoglobin synthesis, is bound to the iron transport protein transferrin, and the complex is internalized, along with the transferrin receptor, by the developing erythroid cell. Within the cell, the iron molecule is subsequently split off and either used for hemoglobin synthesis or stored within the cytoplasm as ferritin. Administration of recombinant human erythropoietin induces changes in iron metabolism, which are reflected by decreases in both the serum iron level and transferrin saturation. These reductions can be marked, even in healthy individuals, and can occur in the presence of normal or even increased iron stores.
Assuntos
Eritrócitos/fisiologia , Epoetina alfa , Eritropoetina/fisiologia , Humanos , Ferro/metabolismo , Proteínas RecombinantesRESUMO
In this study, a primitive progenitor cell, the blast-cell colony-forming cell (BC-CFC), which is thought by some to be equivalent to the hematopoietic stem cell (HSC), those cells capable of long-term marrow repopulation, has been isolated from normal murine marrow. The cell separation method we employed has, as its final step, the purification of marrow cells based on their ability to take up (bright) or exclude (dull) the mitochondrial dye, Rhodamine (Rho)-123. Rho-bright and Rho-dull cells are enriched for multipotential progenitor cells or for HSC, respectively. It was found that Rho-bright cells contain more BC-CFC than Rho-dull cells (310 +/- 50 v 120 +/- 40 per 10(5) purified cells, respectively). However, the BC-CFC that copurified with the Rho-bright and the Rho-dull cells were different in terms of replating efficiency and response to interleukin-3 (IL-3) and stem cell factor (SCF). In fact, on replating, the blast-cell colonies cultured from the Rho-dull population give rise to many more secondary colonies and had a greater replating efficiency than those obtained from Rho-bright cells (replating efficiency: 29.0 +/- 6.3 v 19.5 +/- 3.7, respectively, P < .01). Furthermore, while the same numbers of blast-cell colonies were detected in culture of Rho-bright cells stimulated with IL-3 alone or in combination with SCF, blast-cell colonies were generated in cultures of Rho-dull cells only in the presence of both IL-3 and SCF. After 5 days in suspension culture stimulated with IL-3 and SCF, Rho-dull cells generated BC-CFC whose replating potential was similar to the replating potential of BC-CFC contained in the Rho-bright population. These results indicate that BC-CFC contained in the Rho-bright and -dull populations are qualitatively different. Because the Rho-dull population contains HSC, the results indicate that few, if any, BC-CFC are HSC.
Assuntos
Células da Medula Óssea , Células-Tronco Hematopoéticas/citologia , Animais , Separação Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Sinergismo Farmacológico , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/efeitos dos fármacos , Interleucina-3/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Rodamina 123 , Rodaminas , Fator de Células-Tronco/farmacologiaRESUMO
Clinical evidence of hematopoietic restoration with placental/umbilical cord blood (PCB) grafts indicates that PCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. In the unrelated setting, human leukocyte antigen (HLA)-matched donors must be obtained for candidate patients and, hence, large panels of frozen HLA-typed PCB units must be established. The large volume of unprocessed units, consisting mostly of red blood cells, plasma, and cryopreservation medium, poses a serious difficulty in this effort because storage space in liquid nitrogen is limited and costly. We report here that almost all the hematopoietic colony-forming cells present in PCB units can be recovered in a uniform volume of 20 ml by using rouleaux formation induced by hydroxyethyl starch and centrifugation to reduce the bulk of erythrocytes and plasma and, thus, concentrate leukocytes. This method multiples the number of units that can be stored in the same freezer space as much as 10-fold depending on the format of the storage system. We have also investigated the proportion of functional stem/progenitor cells initially present that are actually available to the recipient when thawed cryopreserved PCB units are infused. Progenitor cell viability is measurably decreased when thawed cells, still suspended in hypertonic cryopreservative solutions, are rapidly mixed with large volumes of isotonic solutions or plasma. The osmotic damage inflicted by the severe solute concentration gradient, however, can be averted by a simple 2-fold dilution after thawing, providing almost total recovery of viable hematopoietic progenitor cells.
Assuntos
Preservação de Sangue/métodos , Medula Óssea , Criopreservação/métodos , Sangue Fetal , Células-Tronco Hematopoéticas/citologia , Contagem de Células Sanguíneas , Sedimentação Sanguínea , Separação Celular/métodos , Sobrevivência Celular , Feminino , Antígenos HLA/sangue , Teste de Histocompatibilidade , Humanos , Leucócitos/citologia , Linfócitos/citologia , Neutrófilos/citologia , Placenta , GravidezRESUMO
The generation of murine mast cells is supported by several cytokines, and mast cell lines are frequently established in long-term cultures of normal murine marrow cells. In contrast, growth of human mast cells was initially dependent on coculture with murine fibroblasts. The growth factor produced by murine fibroblasts and required to observe differentiation of human mast cells is attributable in part to stem cell factor (SCF). However, other factors are likely involved. We have previously shown that the combination of SCF and interleukin-3 (IL-3) efficiently sustains proliferation and differentiation of colony-forming cells (CFCs) from pre-CFC enriched from human umbilical cord blood by CD34+ selection. With periodic medium changes and the addition of fresh growth factors, five consecutive cultures of different cord blood samples gave rise to differentiated cells and CFCs for more than 2 months. Although differentiated cells continued to be generated for more than 5 months, CFCs were no longer detectable by day 50 of culture. The cells have the morphology of immature mast cells, are Toluidine blue positive, are karyotypically normal, are CD33+, CD34-, CD45+, c-kit-, and c-fms-, and die in the absence of either SCF or IL-3. These cells do not form colonies in semisolid culture and are propagated in liquid culture stimulated with SCF and IL-3 at a seeding concentration of no less than 10(4) cells/mL. At refeedings, the cultures contain a high number (> 50%) of dead cells and have a doubling time ranging from 5 to 12 days. This suggests that subsets of the cell population die because of a requirement for a growth factor other than SCF or IL-3. These results indicate that the combination of cord blood progenitor and stem cells, plus a cocktail of growth factors including SCF and IL-3, is capable with high efficiency of giving rise in serum-deprived culture to human mast cells that behave like factor-dependent cell lines. These cells may represent a useful tool for studies of human mast cell differentiation and leukemia.
Assuntos
Sangue Fetal/citologia , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Interleucina-3/farmacologia , Mastócitos , Antígenos CD , Antígenos CD34 , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro , Técnicas de Cultura/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Recém-Nascido , Proteínas Recombinantes/farmacologia , Fator de Células-TroncoRESUMO
32D Epo and 32D GM cells are subclones of the murine 32D cell line which are selectively dependent for proliferation and survival on erythropoietin (Epo) or granulocyte/macrophage colony-stimulating factor (GM-CSF), respectively. 32D GM cells were previously shown to express significant levels of the Epo receptor mRNA and protein which was retained intracellularly and did not appear on the cell surface. We have now analyzed the EpoR mRNA from the 32D GM line, using PCR followed by direct sequencing. Several alternatively spliced products were detected. In some molecules, intron 5 (I5) or part of I6 or both were retained. Retention of I5 results in a mRNA potentially encoding an almost complete extracellular domain, while retention of I6 gives rise to a mRNA encoding the complete extracellular and transmembrane domains. A different type of splicing results in the loss of exon 5 (E5), giving rise to a sequence encoding a truncated extracellular domain. These alternatively spliced sequences are differentially represented in 32D Epo versus 32D GM cells. All are additionally present in normal bone marrow cells. Apart from these alternatively spliced EpoR RNAs, no other abnormalities were detected in EpoR RNA from 32D GM cells that could account for the intracellular retention of EpoR in the non-erythroid subclones of 32D.