RESUMO
Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166), is expressed on osteoblasts (OB) and hematopoietic stem cells (HSC) residing in the hematopoietic niche, and may have important regulatory roles in bone formation. Because HSC numbers are reduced 77% in CD166(-/-) mice, we hypothesized that changes in bone phenotype and consequently the endosteal niche may partially be responsible for this alteration. Therefore, we investigated bone phenotype and OB function in CD166(-/-) mice. Although osteoclastic measures were not affected by loss of CD166, CD166(-/-) mice exhibited a modest increase in trabecular bone fraction (42%), and increases in osteoid deposition (72%), OB number (60%), and bone formation rate (152%). Cortical bone geometry was altered in CD166(-/-) mice resulting in up to 81% and 49% increases in stiffness and ultimate force, respectively. CD166(-/-) OB displayed elevated alkaline phosphatase (ALP) activity and mineralization, and increased mRNA expression of Fra 1, ALP, and osteocalcin. Overall, CD166(-/-) mice displayed modestly elevated trabecular bone volume fraction with increased OB numbers and deposition of osteoid, and increased OB differentiation in vitro, possibly suggesting more mature OB are secreting more osteoid. This may explain the decline in HSC number in vivo because immature OB are mainly responsible for hematopoiesis enhancing activity.
Assuntos
Molécula de Adesão de Leucócito Ativado/metabolismo , Osso e Ossos/fisiologia , Hematopoese/fisiologia , Osteoblastos/metabolismo , Absorciometria de Fóton , Animais , Fenômenos Biomecânicos , Diferenciação Celular/fisiologia , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteogênese/fisiologia , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Microtomografia por Raio-XRESUMO
Homing of transplanted hematopoietic stem cells to recipient bone marrow is a critical step in engraftment and initiation of marrow reconstitution. At present, only partial understanding of the cellular and molecular mechanisms governing homing exists. Likewise, only an incomplete list of adhesion molecules implicated in directing the trafficking of stem cells to the marrow microenvironment is available. Opposing hypotheses that attribute homing to an orderly and orchestrated cascade of events or to random migration of circulating cells find ample experimental support. Also unsettled is the fate of marrow-homed cells shortly after transplantation and the rapidity at which they begin to proliferate in their new marrow microenvironment. The limited number of studies in this field and disparities in their experimental design intensifies the confusion surrounding these critical aspects of stem cell biology. However, this area of research is moving forward rapidly and results capable of clarifying many of these issues are forthcoming.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Animais , Medula Óssea/fisiologia , Ciclo Celular , Divisão Celular , Movimento Celular , Células-Tronco Hematopoéticas/citologia , Humanos , Cinética , CamundongosRESUMO
It was hypothesized that during mammalian development, the extensive need for hematopoietic cells requires equal contribution to blood cell production from both quiescent and cycling hematopoietic stem cells (HSCs) while maintaining the stem cell pool. To investigate this hypothesis, the engraftment potential of umbilical cord blood (UCB) CD34(+) cells residing in either G(0) (G(0)CD34(+) cells) or G(1) (G(1)CD34(+) cells) phases of the cell cycle was assessed in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. Whereas the level of chimerism in mice transplanted with UCB G(0)CD34(+) cells was 69.9% +/- 24.0%, mice receiving equal numbers of G(1)CD34(+) cells harbored 46.7% +/- 21.3% human cells 8 weeks posttransplantation. Both groups of cells sustained multilineage differentiation and the production of CD34(+) cells in recipient animals. The relationship between the number of transplanted G(0)CD34(+) or G(1)CD34(+) cells and the level of chimerism was analyzed by a general linear models procedure. Although the initial level of chimerism following transplantation of G(0)CD34(+) cells was higher than that sustained by G(1)CD34(+) cells, the increment in the degree of chimerism obtained with each additional 10(3) cells of either phenotype was identical, suggesting that the reconstitution potential of these 2 types of cells was similar. Of interest is that human cells recovered from primary recipients of both G(0)CD34(+) and G(1)CD34(+) cells engrafted in secondary NOD/SCID recipients, albeit at a substantially lower level, confirming the primitive nature of UCB CD34(+) cells residing in G(1).
Assuntos
Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34 , Contagem de Células Sanguíneas , Diferenciação Celular , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MitoseRESUMO
Engraftment potential of hematopoietic stem cells (HSCs) is likely to be dependent on several factors including expression of certain adhesion molecules (AMs) and degree of mitotic quiescence. The authors investigated the functional properties and engraftment potential of Sca-1(+)lin(-) cells subfractionated on the basis of expression, or lack thereof, of CD11a, CD43, CD49d, CD49e, or CD62L and correlated that expression with cell cycle status and proliferative potential of engrafting fractions. Donor-derived chimerism in mice receiving CD49e(+) or CD43(+) Sca-1(+)lin(-) cells was greater than that in mice receiving cells lacking these 2 markers, while Sca-1(+)lin(-) cells positive for CD11a and CD62L and bright for CD49d expression mediated minimal engraftment. AM phenotypes enriched for engraftment potential contained the majority of high proliferative potential-colony forming cells, low proliferative potential-colony forming cells, and cells providing rapid in vitro expansion. Cell cycle analysis of AM subpopulations revealed that, regardless of their bone marrow repopulating potential, Sca-1(+)lin(-) AM(-) cells contained a higher percentage of cells in G(0)/G(1) than their AM(+) counterparts. Interestingly, engrafting phenotypes, regardless of the status of their AM expression, were quicker to exit G(0)/G(1) following in vitro cytokine stimulation than their opposing phenotypes. When engrafting phenotypes of Sca-1(+)lin(-) AM(+) or AM(-) cells were further fractionated by Hoechst 33342 into G(0)/G(1) or S/G(2)+M, cells providing long-term engraftment were predominantly contained within the quiescent fraction. These results define a theoretical phenotype of a Sca-1(+)lin(-) engrafting cell as one that is mitotically quiescent, CD43(+), CD49e(+), CD11a(-), CD49d(dim), and CD62L(-). Furthermore, these data suggest that kinetics of in vitro proliferation may be a good predictor of engraftment potential of candidate populations of HSCs. (Blood. 2000;96:1380-1387)
Assuntos
Sobrevivência Celular , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Antígenos CD/fisiologia , Moléculas de Adesão Celular , Ciclo Celular , Diferenciação Celular/fisiologia , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.
Assuntos
Fibronectinas/genética , Técnicas de Transferência de Genes , Genes MDR , Vetores Genéticos , Germinoma/terapia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Retroviridae , Adolescente , Adulto , Antígenos CD34 , Seguimentos , Terapia Genética/métodos , Humanos , Pessoa de Meia-Idade , Projetos Piloto , Fatores de Tempo , Resultado do TratamentoRESUMO
Ex vivo expansion of hematopoietic stem and progenitor cells is a very ambitious idea that would have major implications in the areas of stem cell transplantation and somatic gene therapy. However, successful ex vivo expansion has evaded and frustrated scientists for a number of years. The goal of ex vivo expansion is to induce cell division and proliferation of stem cells while maintaining their primary functional characteristic, namely, their ability to engraft and sustain long-term hematopoiesis. Only when a balance between these two requirements is reached can ex vivo expansion of stem cells be considered successful. Establishing such a balance has not been easy. However, many lessons have been learned along the way, and today we have a more profound understanding of the potential obstacles facing ex vivo expansion than we did only a few years ago. In this review, we discuss these obstacles and evaluate the current status of ex vivo expansion of stem and progenitor cells both from the perspective of basic stem cell biology and from the viewpoint of clinical utility of these cells in transplantation.
Assuntos
Terapia Genética , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Divisão Celular , Mobilização de Células-Tronco Hematopoéticas/métodos , Mobilização de Células-Tronco Hematopoéticas/tendências , Células-Tronco Hematopoéticas/patologia , HumanosRESUMO
Merocyanine 540 (MC540) is a membrane probe that inserts preferentially into loosely packed domains in the phospholipid bilayer of intact cells. Previous experiments have demonstrated that MC540 will bind to human bone marrow (BM) hematopoietic progenitor cells (HPC). Fractions of mononuclear BM cells expressing high MC540 fluorescence have been shown to be enriched for myeloid progenitors and cells residing in the S/G2 + M phases of the cell cycle. We rationalized that MC540 uptake could be used to distinguish between quiescent and metabolically active cells and, therefore, to fractionate normal and leukemic BM cells and normal mobilized peripheral blood (MPB) cells into functionally distinct groups of progenitors. BM and MPB cells were separated into fractions ranging in fluorescence from MC540Bright to MC540Dim. Cell cycle analysis of these fractions revealed that the MC540Dim fraction of normal and CML BM CD34+ cells constituted the most quiescent fraction, and the MC540Bright fractions from these cell types contained the most actively cycling cells. However, no differences in the percentage of cells in G/G1 were observed between MC540Bright and MC540Dim fractions of MPB CD34+ cells. To investigate if these cell cycle status differences translated into distinct functional properties, the hematopoietic potential of BM CD34+MC540Bright and CD34+MC540Dim cell fractions was analyzed in vitro in long-term BM cultures and limiting dilution analysis (LDA) assays. CD34+MC540Dim cells produced more total and committed progenitor cells in long-term cultures than did the CD34+MC540Bright fraction. The CD34+MC540Dim fraction also contained a 2-fold higher number of long-term hematopoietic culture-initiating cells (LTHCIC) than the CD34+MC540Bright fraction, as defined by LDA assays. These data demonstrate that MC540 can be a useful probe for the isolation of primitive HPC from some hematopoietic tissues and may assist in monitoring structural changes in the phospholipid bilayer during proliferation and differentiation of HPC.
Assuntos
Medula Óssea/patologia , Separação Celular/métodos , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/patologia , Pirimidinonas , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas , HumanosRESUMO
We have investigated the expression and functional competence of folate receptor (FR) isoforms on human hematopoietic cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) methodology, we find that a substantial fraction of low-density mononuclear and CD34(+) cells express both the beta and gamma isoforms of FR. The alpha isoform of FR (the form most commonly found on cancer cells) was surprisingly absent from all hematopoietic cells examined. Compared with KB cells (a human cell line known for its elevated expression of FR-alpha), the abundance of FR-beta on CD34(+) cell surfaces was relatively low (approximately 8% of KB cell levels). Because many antifolates and folic acid-linked chemotherapeutic agents enter malignant cells at least partially via FR endocytosis, it was important to evaluate the ability of FR on CD34(+) cells to bind folic acid (FA). Based on three FR binding assays, freshly isolated CD34(+) cells were found to display no affinity for FA. Thus, regardless of whether steps were taken to remove endogenous folates before receptor binding assays, FR on primitive hematopoietic cells failed to bind 3H-FA, fluorescein isothiocyanate (FITC)-linked FA, or FA-derivatized liposomes. In contrast, analogous studies on KB cells showed high levels of receptor binding for all three FR probes. These studies show that although multipotent hematopoietic progenitor cells express FR, the receptor does not transport significant amounts of FA. Consequently, antifolates and FA-linked chemotherapeutic agents that can be engineered to enter malignant cells exclusively through the FR should not harm progenitor/stem cell function.
Assuntos
Proteínas de Transporte/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Antígenos CD34 , Linhagem Celular , Citometria de Fluxo , Receptores de Folato com Âncoras de GPI , Humanos , Isoformas de Proteínas/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
In the use of autologous PBPC transplantation in patients with multiple myeloma, contamination of PBPC with myeloma cells is commonly observed. Enrichment for CD34+ cells has been employed as a method of reducing this contamination. In this study the reduction of myeloma cells in PBPC was accomplished by the positive selection of CD34+ cells using immunomagnetic bead separation (Isolex 300 system). PBPC were mobilized from 18 patients using cyclophosphamide (4.5 g/m2) and G-CSF (10 microg/kg/day). A median of two leukaphereses and one selection was performed per patient. The median number of mononuclear cells processed was 3.50 x 10(10) with a recovery of 1.11 x 10(8) cells after selection. The median recovery of CD34+ cells was 48% (range 17-78) and purity was 90% (29-99). The median log depletion of CD19+ cells was 3.0. IgH rearrangement, assessed by PCR, was undetectable in 13 of 24 evaluable CD34+ enriched products. Patients received 200 mg/m2 of melphalan followed by the infusion of a median of 2.91 x 10(6)/kg CD34+ cells (1.00-16.30). The median time to absolute neutrophil count >0.5 x 10(9)/l was 11 days, and sustained platelet recovery of >20 x 10(9)/l was 14 days. We conclude that immunomagnetic-based enrichment of CD34+ cells results in a marked reduction in myeloma cells without affecting engraftment kinetics.
Assuntos
Antígenos CD34 , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Mieloma Múltiplo/terapia , Adulto , Idoso , Antineoplásicos Alquilantes/administração & dosagem , Antineoplásicos Alquilantes/uso terapêutico , Terapia Combinada , Ciclofosfamida/administração & dosagem , Ciclofosfamida/uso terapêutico , Esquema de Medicação , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Separação Imunomagnética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Condicionamento Pré-Transplante/métodos , Transplante AutólogoRESUMO
Murine fetal liver (FL) and adult bone marrow (BM) hematopoietic stem cells (HSCs) are characterized by cell surface expression of CD38 and c-kit. Because murine yolk sac (YS) HSC activity precedes the initiation of FL hematopoiesis, we investigated whether YS-derived HSCs also expressed c-kit and CD38. c-Kit+ CD38+ lineage- cells derived from day 9 YS as well as adult BM were found to be enriched in high proliferative potential colony-forming cells. c-Kit+ CD38+ lineage- YS or adult BM cells were capable of long-term reconstitution (>6 months) of busulfan-conditioned newborn or lethally irradiated adult mice, respectively. In contrast, c-kit+ CD38- lineage- populations from both tissues were enriched in lineage-committed progenitors and had no long-term HSC activity. We concluded that c-kit and CD38 are cell surface markers of HSCs expressed throughout murine ontogeny.
Assuntos
Antígenos CD/biossíntese , Antígenos de Diferenciação/biossíntese , Proteínas Sanguíneas/biossíntese , Células-Tronco Hematopoéticas/imunologia , NAD+ Nucleosidase/biossíntese , Proteínas Proto-Oncogênicas c-kit/biossíntese , Saco Vitelino/imunologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Animais Recém-Nascidos , Linhagem da Célula , Células Cultivadas , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas , Glicoproteínas de Membrana , Camundongos , Saco Vitelino/citologiaRESUMO
Most primitive hematopoietic progenitor cells reside in vivo within the G0/G1 phase of the cell cycle. By simultaneous DNA/RNA staining it is possible to distinguish G0 and G1 states and to isolate cells in defined phases of the cell cycle. We report here the use of cell cycle fractionation to separate human mobilized peripheral blood (MPB) CD34(+) cells capable of repopulating the bone marrow (BM) of non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice. In freshly isolated MPB, repopulating cells were predominant within the G0 phase, because transplantation of CD34(+) cells residing in G0 (G0CD34(+)) resulted on average in a 16.6- +/- 3.2-fold higher BM chimerism than infusion of equal numbers of CD34(+) cells isolated in G1. We then investigated the effect of ex vivo cell cycle progression, in the absence of cell division, on engraftment capacity. Freshly isolated G0CD34(+) cells were activated by interleukin-3 (IL-3), stem cell factor (SCF), and flt3-ligand (FL) for a 36-hour incubation period during which a fraction of cells progressed from G0 into G1 but did not complete a cell cycle. The repopulating capacity of stimulated cells was markedly diminished compared with that of unmanipulated G0CD34(+) cells. Cells that remained in G0 during the 36-hour incubation period and those that traversed into G1 were sorted and assayed separately in NOD/SCID recipients. The repopulating ability of cells remaining in G0 was insignificantly reduced compared with that of unstimulated G0CD34(+) cells. On the contrary, CD34(+) cells traversing from G0 into G1 were largely depleted of repopulating capacity. Similar results were obtained when G0CD34(+) cells were activated by the combination of thrombopoietin-SCF-FL. These studies provide direct evidence of the quiescent nature of cells capable of repopulating the BM of NOD/SCID mice. Furthermore, these data also demonstrate that G0-G1 progression in vitro is associated with a decrease in engraftment capacity.
Assuntos
Ciclo Celular , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Glicoproteínas de Membrana , Animais , Antígenos CD/análise , Medula Óssea/patologia , Antígeno CD24 , Divisão Celular , Separação Celular , Células Cultivadas/transplante , Ensaio de Unidades Formadoras de Colônias , Sobrevivência de Enxerto , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interleucina-3/farmacologia , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Quimera por Radiação , Fase de Repouso do Ciclo Celular , Fator de Células-Tronco/farmacologia , Transplante HeterólogoRESUMO
Seventy-one patients with poor-prognosis breast cancer were enrolled after informed consent in a multicentre randomized study to evaluate the use of selected peripheral blood CD34+ cells to support haematopoietic recovery following high-dose chemotherapy. Patients who responded to conventional chemotherapy were mobilized with chemotherapy (mainly high-dose cyclophosphamide) and/or recombinant human granulocyte colony-stimulating factor (rhG-CSF). Patients who reached the threshold of 20 CD34+ cells per microl of peripheral blood underwent apheresis and were randomized at that time to receive either unmanipulated mobilized blood cells or selected CD34+ cells. For patients in the study arm, CD34+ cells were selected from aphereses using the Isolex300 device. Fifteen patients failed to mobilize peripheral blood progenitors and nine other patients were excluded for various reasons. Forty-seven eligible patients were randomized into two comparable groups. CD34+ cells were selected from aphereses in the study group. Haematopoietic recovery occurred at similar times in both groups. No side-effect related to the infusion of selected cells was observed. The frequency of epithelial tumour cells in aphereses was low (8 out of 42 evaluated patients), as determined by immunocytochemistry. We conclude that selected CD34+ cells safely support haematopoietic recovery following high-dose chemotherapy in patients with poor-prognosis breast cancer.
Assuntos
Antígenos CD34 , Antineoplásicos/administração & dosagem , Neoplasias da Mama/terapia , Transplante de Células-Tronco Hematopoéticas , Adulto , Remoção de Componentes Sanguíneos , Neoplasias da Mama/tratamento farmacológico , Separação Celular/métodos , Terapia Combinada , Feminino , Mobilização de Células-Tronco Hematopoéticas , Humanos , Pessoa de Meia-Idade , Prognóstico , Estudos ProspectivosRESUMO
To investigate the mechanisms behind the leukaemic expansion of chronic myelogenous leukaemia (CML), we examined the cell cycle status and activation kinetics of purified subpopulations of CD34+ cells from normal and CML bone marrow (BM). Propidium iodide staining was used to assess cell cycle status of fresh cells or those stimulated with cytokines. Although the cell cycle status of fresh low-density cells from CML and normal BM was similar, a larger percentage of CML CD34+ cells were cycling than those from normal BM. The HLA-DR compartment of CML CD34+ cells, a fraction enriched for normal, non-leukaemic progenitors, contained a higher percentage of quiescent cells than the CD34+ HLA-DR+ fraction. When the activation of CD34+ cells was examined in response to SCF or IL-3 alone, or SCF+IL-3+IL-6, CML CD34+ cells exited GO/G1 more rapidly than normal CD34+ cells. Interestingly, although normal BM CD34+ cells failed to cycle in response to IL-6 alone, or in the absence of exogenous cytokines, 30% of CML cells cycled under these conditions. No differences in the degree of apoptosis were documented among CML and normal CD34+ cells in these cultures. These data suggest that enhanced cell cycle activation of CML CD34+ cells, by either autocrine stimuli or via enhanced sensitivity to exogenous stimuli, may be partially responsible for the pronounced cellular expansion characteristic of CML.
Assuntos
Antígenos CD34/metabolismo , Células da Medula Óssea/patologia , Fase G1/fisiologia , Leucemia Mieloide de Fase Crônica/patologia , Fase de Repouso do Ciclo Celular/fisiologia , Adulto , Apoptose/fisiologia , Divisão Celular/fisiologia , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Pessoa de Meia-Idade , Fator de Células-Tronco/farmacologia , Células Tumorais CultivadasRESUMO
NK cells are lymphocytes which exhibit spontaneous cytotoxicity against a variety of target cells, including cancer cells. Mature NK and T cells may derive from a common precursor which differentiates into T or NK cells depending on the microenvironment. We evaluated the effect of human fetal thymic stroma on human CD34+Lin- progenitors. The culture medium was supplemented with human AB serum with or without interleukin-2 (IL2; 1000 U/ml) and interleukin-7 (IL7; 1000 U/ml). After 3 weeks of culture, CD45/56 cells were detected by flow cytometry and their activity was tested against K562 targets. In cultures with IL2 the percentage of CD56-positive cells was much higher in the Transwell cultures (60.8 +/- 12.5% from CD34+Lin-DR+ and 51% from CD34+Lin-progenitors) than in adherent cultures (25 +/- 21.9% from CD34+Lin-DR+ and 25.3 +/- 9.5% from CD34+Lin-progenitors) or suspension cultures (23 +/- 21.4% from CD34+Lin-DR+ progenitors and 43.1 +/- 14.2% from CD34+Lin-progenitors). Cytolytic activity as measured by K562 lysis was also higher in Transwell cultures with IL2. NK cells were also obtained in cultures without factors or supplemented with IL7, but in smaller numbers. These data indicate that NK cells can be obtained in vitro by coculture of immature hematopoietic progenitors with thymic stromal cells and that IL2 appears to strongly favor their development in the absence of stromal contact. This would indicate a direct inhibitory effect of the thymic stroma on NK progenitors.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Células Matadoras Naturais/fisiologia , Células Estromais/fisiologia , Timo/fisiologia , Divisão Celular , Células Cultivadas , Técnicas de Cocultura , Testes Imunológicos de Citotoxicidade , Células-Tronco Hematopoéticas/imunologia , Humanos , Imunofenotipagem , Células Matadoras Naturais/imunologia , Timo/citologiaRESUMO
Exit of primitive hematopoietic progenitor cells (HPCs) from the G0 phase of the cell cycle in response to in vitro cytokine stimulation is a limiting step in successful ex vivo expansion. Simultaneous DNA/RNA staining with Hoechst 33342 and pyronin Y was used to separate human bone marrow CD34+ cells residing in G0 (G0CD34+) from those cycling in G1 and S/G2+M. Compared with CD34+ cells isolated in G1, G0CD34+ cells were characterized by a delayed response to cytokine stimulation and were enriched for long-term hematopoietic culture-initiating cells. We next compared the activation kinetics of individually sorted G0CD34+ cells stimulated with stem cell factor (SCF), flt3-ligand (FL), or interleukin-3 (IL-3) as single factors. In a novel clonal proliferation assay, the functional status of cells that had remained quiescent after an initial 7-day period and of those that had completed successive division cycles under each of these three factors was evaluated by assessment of subsequent proliferative capacity and maintenance of colony-forming cell precursor (pre-CFC) activity. All three cytokines were equally able to support the survival of primitive HPCs in the absence of cell division. Cells that did not respond to any cytokine stimulation for 7 days retained higher proliferative and pre-CFC activities than dividing cells. The hematopoietic function of cells that divided in response to SCF, FL, or IL-3 decreased after each division cycle. However, G0CD34+ cells displayed a heterogeneous response pattern to cytokine stimulation whereby SCF appeared to have a superior ability to promote the cycling of cells with high proliferative and pre-CFC activities. These results indicate that HPCs reside in opposing hierarchies of hematopoietic potential and responsiveness to cytokine stimulation. The data also begin to indicate relationships between cellular division in response to different stimuli and maintenance of hematopoietic function.
Assuntos
Antígenos CD34/análise , Células da Medula Óssea/imunologia , Fase de Repouso do Ciclo Celular , Adulto , Divisão Celular/imunologia , Separação Celular , Células Clonais , Ensaio de Unidades Formadoras de Colônias , Estudos de Avaliação como Assunto , Humanos , Mitose/imunologia , Valores de ReferênciaRESUMO
Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21(ras) (Ras) proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile chronic myelogenous leukemia (JCML) and other malignant myeloid disorders, and heterozygous Nf1 knockout mice spontaneously develop a myeloid disorder that resembles JCML. Both human and murine leukemias show loss of the normal allele. JCML cells and Nf1-/- hematopoietic cells isolated from fetal livers selectively form abnormally high numbers of colonies derived from granulocyte-macrophage progenitors in cultures supplemented with low concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF). Taken together, these data suggest that neurofibromin is required to downregulate Ras activation in myeloid cells exposed to GM-CSF. We have investigated the growth and proliferation of purified populations of hematopoietic progenitor cells isolated from Nf1 knockout mice in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF), as well as to GM-CSF. We found abnormal proliferation of both immature and lineage-restricted progenitor populations, and we observed increased synergy between SCF and either IL-3 or GM-CSF in Nf1-/- progenitors. Nf1-/- fetal livers also showed an absolute increase in the numbers of immature progenitors. We further demonstrate constitutive activation of the Ras-Raf-MAP (mitogen-activated protein) kinase signaling pathway in primary c-kit+ Nf1-/- progenitors and hyperactivation of MAP kinase after growth factor stimulation. The results of these experiments in primary hematopoietic cells implicate Nf1 as playing a central role in regulating the proliferation and survival of primitive and lineage-restricted myeloid progenitors in response to multiple cytokines by modulating Ras output.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Interleucina-3/farmacologia , Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Fator de Células-Tronco/farmacologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurofibromina 1RESUMO
Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34(+) cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol using neoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34(+) cells. BM CD34(+) cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers of neoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transduced neoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.
Assuntos
Marcação de Genes/métodos , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/virologia , Canamicina Quinase/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Retroviridae/genética , Transfecção , Adulto , Animais , Antígenos CD34/análise , Divisão Celular , Separação Celular , Ensaio de Unidades Formadoras de Colônias , Resistência Microbiana a Medicamentos , Fibronectinas , Citometria de Fluxo , Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Separação Imunomagnética , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Canamicina Quinase/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Fator de Células-Tronco/farmacologiaRESUMO
Bone marrow cells expressing the surface antigen CD34 comprise approximately 1% of harvested marrow and are highly enriched for marrow progenitor cells, including the cells believed to be responsible for long-term engraftment following bone marrow transplantation (BMT). Selection of CD34-expressing cells was applied in allogeneic BMT (alloBMT) to decrease the number of T lymphocytes in the infused marrow in an attempt to prevent severe graft-versus-host disease (GVHD). We report 14 patients who underwent HLA-identical sibling-matched alloBMT with marrow-enriched for CD34 cells using the Isolex 300 SA device. Patients received total body irradiation, thiotepa, cyclophosphamide, antithymocyte globulin and methylprednisolone prior to marrow infusion. No post-transplantation immunosuppressive therapy was given except for a 5-week course of steroids. The purity of the infused marrow was 64.9+/-6.0% (mean +/- s.e.m.) CD34-positive cells and patients received a mean of 1.24+/-0.21 x 10(6) CD34 cells/kg. A mean of 9.4+/-1.7 x 10(4) CD3 T cells/kg were present in the CD34-enriched product, representing a 2.7+/-0.1 log depletion. There were no graft rejections and patients achieved a sustained absolute granulocyte count of >500 in a median of 10.5 days and a sustained platelet engraftment of >20000 untransfused in a median of 27 days. Patients were discharged a median of 21.5 days after marrow infusion. There were no instances of grade III or IV graft-versus-host disease (GVHD) and no unexpected adverse events during the transplant hospitalization. With a median follow-up of 12 months, the estimated 100 day survival is 86+/-9%. CD34 selection in alloBMT permits rapid engraftment without unanticipated toxicities.
Assuntos
Antígenos CD34/análise , Transplante de Medula Óssea , Adolescente , Adulto , Feminino , Doença Enxerto-Hospedeiro/etiologia , Humanos , Depleção Linfocítica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Transplante HomólogoRESUMO
We examined the decline in hematopoietic potential observed when human CD34+ cells are cultured in vitro by evaluating the association between proliferation history and the fate of long-term hematopoietic culture-initiating cells (LTHC-ICs) as well as the onset of programmed cell death. The membrane dye PKH2 was used to track ex vivo expanded human CD34+ cells from bone marrow, cord blood, and mobilized peripheral blood, and to identify and isolate CD34+ cells that had divided once, twice, three, or four times or more, as well as cells that had remained cytokine nonresponsive and therefore failed to proliferate. These isolated groups of cells were assayed for their hematopoietic potential, cell cycle status, and percentage of apoptotic cells. A gradual decline in the content of LTHC-ICs, as well as in their ability to initiate and sustain in vitro hematopoiesis, was found to correlate with the number of in vitro cellular divisions, such that the hematopoietic potential of CD34+ cells dividing four or more times was nearly depleted. DNA analysis revealed that cells dividing more than three times resided predominantly in G0/G1 phases of the cell cycle. In addition, the percentage of CD34+ cells undergoing apoptosis was found to increase concomitantly with the number of in vitro cellular divisions; less than 10% of cells dividing once were apoptotic, whereas more than 25% of CD34+ cells dividing four or more times underwent programmed cell death. Together, these data suggest that a proliferation-associated, and possibly activation-induced, loss of hematopoietic potential among dividing CD34+ cells may result from an increase in programmed cell death among dividing primitive hematopoietic progenitor cells.