RESUMO
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
Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity (micro-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function. To this end, BM CD34+ cells were cultured for 4-6 d in rotating wall vessels for simulation of micro-g, and assessed for expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34+ cells cultured in normal gravity (1-g) proliferated up to threefold by day 4-6, cells cultured in micro-g did not increase in number. As a possible explanation for this, cells cultured in simulated micro-g were found to exit G0/G1 phase of cell cycle at a slower rate than 1-g controls. When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial micro-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in micro-g and 1-g-cultured cells suggested similar growth patterns in the two settings. These data begin to elucidate the effects of micro-g on proliferation of human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.
Assuntos
Células da Medula Óssea/citologia , Divisão Celular , Células-Tronco Hematopoéticas/citologia , Simulação de Ausência de Peso , Antígenos CD34/análise , Apoptose , Células da Medula Óssea/imunologia , Moléculas de Adesão Celular/análise , Ciclo Celular , Células Cultivadas , Hematopoese , Células-Tronco Hematopoéticas/imunologia , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Fator de Células-Tronco/farmacologia , Trombopoetina/farmacologiaRESUMO
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
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
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
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.
Assuntos
Apoptose , Hematopoese , Células-Tronco Hematopoéticas/patologia , Antígenos CD34 , Contagem de Células Sanguíneas , Divisão Celular , Células Cultivadas , Células-Tronco Hematopoéticas/imunologia , HumanosRESUMO
Flt3-Ligand (Flt3-L) is a stimulatory cytokine for a variety of hematopoietic lineages, including dendritic cells and B cells. The antitumor properties of Flt3-L were evaluated in C3H/HeN mice challenged with the syngeneic C3L5 murine breast cancer cell line. Eighty % of animals receiving 500 microg/kg/day of Chinese hamster ovary-derived human Flt3-L for 10 days were protected from tumor growth, whether the tumor challenge was administered on the first or fourth days of Flt3-L administration. The protection provided by soluble Flt3-L was transient. All tumor-free animals rechallenged 4 weeks after the primary challenge developed tumor. Transduction of C3L5 with retroviral vectors expressing human or murine Flt3-L did not influence in vitro growth or MHC expression but decreased in vivo tumor development to 0 and 10% of mice, respectively. This compares with tumor growth of 52% with interleukin-2 transduced C3L5 and over 85% with untransduced and control vector-transduced C3L5. Unlike animals treated with soluble Flt3-L, administration of Flt3-L as a tumor vaccine protected mice from a subsequent challenge with untransduced C3L5 in 60-78% of mice, compared to 0% of controls. Our initial work used the most common Flt3-L isoform, which is membrane bound but can undergo proteolytic cleavage to generate a soluble form. To evaluate the role of the various Flt3-L isoforms in preventing tumor formation, retroviral vectors encoding only the membrane-bound form or only the soluble isoform were evaluated in the C3L5 model. Tumor formation was similar with either isoform, preventing tumor formation in 80-90% of mice after the primary challenge and 88-89% after the secondary challenge. Splenocytes obtained 4 weeks after the secondary challenge conferred adoptive immunity to naive mice in 60% of animals. This initial report of antitumor activity by Flt3-L is consistent with its known stimulatory effect on antigen-presenting cells and suggests it may enhance the development of tumor vaccines.
Assuntos
Vacinas Anticâncer/farmacologia , Neoplasias Mamárias Experimentais/prevenção & controle , Proteínas de Membrana/farmacologia , Transferência Adotiva , Animais , Células CHO , Vacinas Anticâncer/imunologia , Cricetinae , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Citometria de Fluxo , Vetores Genéticos , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Retroviridae/genética , TransfecçãoRESUMO
Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with <20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0/G1, unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.
Assuntos
Células da Medula Óssea , Citocinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Adulto , Antígenos CD34/análise , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/análise , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , RNA/análise , Fase de Repouso do Ciclo Celular , Fator de Células-Tronco/farmacologia , Fatores de TempoRESUMO
It is believed that long-term cultures of CML marrow cells favor the outgrowth of BCR/ABL negative hematopoietic progenitor cells (HPC) and that this phenomenon may be enhanced with negative hematopoietic regulators which can maintain primitive HPC in a quiescent state. Proliferation of CML marrow CD34+ cells in primary short-term cultures, maintained in the presence or absence of macrophage inhibitory protein-1 alpha (MIP-1 alpha), was tracked with the membrane dye PKH2. After 7 to 10 days it was possible to distinguish between cytokine responsive (CR) CD34+ cells (cells which had divided thus becoming PKH2dim) and cytokine nonresponsive (CNR) CD34+ cells (cells which had not divided and had therefore remained PKH2bright). CR and CNR CD34+ cells were isolated by flow cytometric cell sorting, seeded in secondary long-term cultures, and their progeny cells assayed weekly for their clonogenic progenitor cell content and expression of BCR/ABL by reverse transcriptase polymerase chain reaction (RT-PCR). Whereas CNR cells isolated from control primary cultures (control/CNR) sustained in vitro hematopoiesis, similar cells from cultures treated with MIP-1 alpha (MIP-1 alpha/CNR) supported a higher and, in some patients, a more extended production of clonogenic HPC, indicating that MIP-1 alpha was able to maintain primitive HPC in a quiescent state. Predominance of BCR/ABL negative progenitors in vitro was more evident in secondary cultures initiated with CNR cells than in those initiated with CR cells, especially those established with MIP-1 alpha/CNR cells. Of interest is the observed decline in the percentage of BCR/ABL+ progenitors in these cultures with time. Whereas up to 100% of progenitors were BCR/ABL+ on day 0, by day 14, only 46% of progenitors in MIP-1 alpha/CNR secondary cultures were BCR/ABL+ and by day 28 and beyond, the percentage of BCR/ABL+ progenitors dropped to below 20%. These results suggest that the quiescent nature of normal HPC present in CML marrow may favor their identification via cell tracking and, subsequently, their isolation from the more actively cycling leukemic cells. These studies also confirm the feasibility of employing negative hematopoietic regulators to augment the sequestration of normal HPC among the cytokine nonresponsive fraction of CD34+ cells, an approach that may be clinically feasible for autotransplantation.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Adulto , Antígenos CD34/metabolismo , Sequência de Bases , Purging da Medula Óssea , Divisão Celular , Células Cultivadas , Quimiocina CCL4 , Citocinas/farmacologia , Primers do DNA/genética , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Técnicas In Vitro , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Proteínas Inflamatórias de Macrófagos/farmacologia , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Transplante AutólogoRESUMO
We have previously shown by reverse transcriptase-PCR (rtPCR) that CML CD34+ HLA-DR- cells are enriched for BCR/ABL(-) hematopoietic progenitor cells (HPC) while leukemic HPC reside predominately within CML CD34+ HLA-DR+ cells. We investigated whether the 30/35 kDa fragment of fibronectin (FN) could be used to enhance retroviral-mediated gene transfer (RMGT) in chronic phase CML marrow HPC. CML CD34+ HLA-DR- and CD34+ HLA-DR+ cells were transduced with vector supernate containing the neomycin resistance gene on plates coated with either FN or bovine serum albumin (BSA) as control, then assayed for transduced HPC in progenitor cell assays in the presence or absence of G418. Transduction efficiency of CML CD34+ HLA-DR- cells over BSA ranged from 0.09 to 7.2% (mean 3.3 +/- 1.5%), while that over FN plates ranged from 3.8 to 23% (mean 11.0 +/- 4.5%) (n = 4). Transduction efficiencies of CML CD34+ HLA-DR+ cells ranged from 0.4 to 9.8% (mean 3.7 +/- 1.7%) and 6.0 to 26% (mean 17.3 +/- 4.5%) (n = 5) over BSA and FN, respectively. rtPCR analysis for BCR/ABL mRNA of individual G418-resistant HPC generated from CD34+ HLA-DR- cells revealed that normal BCR/ABL(-) HPC were successfully transduced under these experimental conditions. These results demonstrate the feasibility of transducing normal CML primitive HPC, and illustrate the potential clinical use of FN in the setting of gene therapy for CML, as well as other diseases.
Assuntos
Antígenos CD34 , Fibronectinas/farmacologia , Técnicas de Transferência de Genes , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Fragmentos de Peptídeos/farmacologia , Adulto , Quimotripsina , Resistência a Medicamentos/genética , Proteínas de Fusão bcr-abl/análise , Genes Reporter , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/imunologia , Humanos , Separação Imunomagnética/métodos , Interleucina-2/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Pessoa de Meia-Idade , Neomicina , Reação em Cadeia da Polimerase , RNA , Retroviridae , Soroalbumina Bovina/farmacologiaRESUMO
Mutations in the enzyme dihydrofolate reductase (DHFR) can confer resistance to the inhibitory effects of folate analogs such as methotrexate (Mtx) and trimetrexate (Ttx). Retroviral vectors expressing the DHFR-Arg22 mutants and the newly described DHFR-Tyr22 mutant were used to transduce the hematopoietic cell line K562. In vitro selection of vector-containing cells was documented via polymerase chain reaction and Southern analysis. When proliferation of selected vector-containing cells was evaluated over a range of Mtx concentrations (0.01 to 10 micromol/L), both Arg22 and Tyr22 provided protection from Mtx, but Tyr22 proved superior to Arg22 in conferring Mtx resistance at low concentrations. Ttx proved to be 10- to 100-fold more potent than Mtx in inhibiting proliferation of nontransduced K562, but the relative effectiveness of individual mutants in conferring drug resistance was similar to that of Mtx. Decreasing the amount of folate in the culture medium to more physiological concentrations increased the potency of administered Mtx and Ttx. Drug resistance in retrovirally transduced K562 cells is consistent with the enzymatic characteristics of the individual mutants. Our findings suggest that the new Tyr22 form of DHFR may prove better in conferring drug resistance than the previously reported Arg22 mutant.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Medula Óssea/metabolismo , Técnicas de Transferência de Genes , Metotrexato/farmacologia , Retroviridae/genética , Tetra-Hidrofolato Desidrogenase/genética , Arginina/química , Células da Medula Óssea , Divisão Celular , Linhagem Celular , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Tetra-Hidrofolato Desidrogenase/química , Tirosina/químicaRESUMO
The objective of ex vivo expansion of primitive hematopoietic progenitor cells (HPC) is to increase the number of progeny cells possessing hematopoietic potential similar to the original HPC. In the context of bone marrow (BM) transplantation in mice, this implies that expanding a number of HPC sufficient for long-term rescue of one lethally irradiated animal should generate enough cells to rescue more than one lethally irradiated recipient. In the present study, Sca-1+Lin- cells from male C57Bl/6 mice were expanded in vitro with stem cell factor (SCF), interleukin-1alpha (IL-1alpha), IL-3, and IL-6 and used to transplant lethally irradiated syngeneic female recipients. Expanded cells were tracked in vitro with the fluorescent membrane dye PKH2, which becomes evenly distributed among dividing daughter cells, and fractionated on day 7 into Sca-1+ cells which did not divide (Sca-1+PKH2bright), those which had divided 1 to 2 times (Sca-1+PKH2moderate), or those which had divided four or more times (Sca-1+PKH2dim). Grafts of expanded cells consisted of either the same number of fresh cells proven to rescue lethally irradiated animals [3X10(3) cells; referred to as one repopulating dose (1 RD)] or the expansion equivalent (EE) of these cells. One EE of cells represented 3X10(3) multiplied by the fold increase in the number of cultured cells on day 7. All animals transplanted with 3X10(3) freshly isolated Sca-1+Lin- cells survived long-term. Only 53% of animals receiving 1 EE of all cultured day-7 cells survived. One RD from all three PKH2 fractions (bright, moderate, and dim) of day-7 cultured Sca-1+ cells failed to rescue more than 30% of lethally irradiated recipients. Comparable survival rates were obtained when 1 EE of Sca-1+PKH2dim or only 4 RD of Sca-1+PKH2bright cells were used as grafts, suggesting that a larger frequency of long-term repopulating cells may have been retained within the fraction of Sca-1+ cells undergoing minimal or no proliferation in culture. Engraftment of male ex vivo expanded cells in recipients was confirmed by polymerase chain reaction (PCR) analysis with Y chromosome-specific primers. When analyzed for their cell cycle status, Sca-1+PKH2bright cells were mostly quiescent, whereas a higher percentage of Sca-1+PKH2dim cells were in active phases of cell cycle. These data suggest that ex vivo expansion does not augment the number of BM repopulating HPC and that ex vivo expansion generates classes of progenitor cells with different BM repopulating potentials depending on their proliferative history. These studies also suggest that the cell cycle status of graft cells may affect the ability of these cells to engraft in myeloablated hosts.
Assuntos
Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Sequência de Bases , Divisão Celular , Linhagem da Célula , Células Cultivadas/transplante , Feminino , Corantes Fluorescentes , Genótipo , Glucose-6-Fosfato Isomerase/genética , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Compostos Orgânicos , Quimera por RadiaçãoRESUMO
Mobilized peripheral blood hematopoietic progenitor cells obtained from cancer patients treated with high-dose cyclophosphamide (7g/m2) followed by G-CSF, GM-CSF, IL-3, PIXY321, or combinations of these cytokines have been successfully used for autologous stem cell transplantation. We investigated the ability of hematopoietic progenitor cells (HPC) derived from mobilized peripheral blood (PB) to undergo ex vivo expansion in short term cultures by enumerating numbers of de novo generated CD34+ cells, assayable progenitor cells, and the frequency of long-term hematopoietic culture-initiating cells (LTHC-IC). These parameters were examined in CD34+ cells generated in culture through the use of cell tracking with the membrane dye PKH2. Fresh isolated mobilized CD34+ cells contained 0.49 +/- 0.36% LTHC-IC. However, due to the high number of total CD34+ cells in mobilized PB, the absolute number of LTHC-IC was higher than that contained in a bone marrow (BM) harvest. Mobilized CD34+ cells were stained with PKH2 and incubated with SCF, IL-3, and IL-6. After 5 to 6 days, numbers of total CD34+ cells and clonogenic progenitors increased 1.4- and 2.2-fold, respectively. Numbers of total progenitors continued to increase such that 10 to 12 days after the initiation of cultures a 6.4-fold increase was demonstrable. However, between days 5 and 7 of culture, the frequency of LTHC-IC in CD34+PKH2bright cells (cells which did not divide) was less than 50% of that determined for fresh cells, while the frequency among CD34+PKH2dim cells (cells that had divided) was very low or undetectable. However, moderately higher frequencies of LTHC-IC were detected following expansion for 48 hours only. In similar assays, both BM and cord blood cells were capable of generating LTHC-IC in CD34+PKH2dim cells but not to expand the overall number of these progenitors. These observations suggest that although mobilized PB CD34+ cells contain large numbers of LTHC-IC, these cells might not be capable of further ex vivo expansion and generation of additional LTHC-IC in vitro. Furthermore, these data indicate that mobilized PB CD34+ cells may have undergone maximal "in vivo expansion" such that additional ex vivo expansion of primitive progenitor cells may not be possible.
Assuntos
Medula Óssea/patologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Neoplasias/patologia , Antígenos CD34/imunologia , Medula Óssea/imunologia , Técnicas de Cultura de Células , Divisão Celular , Separação Celular , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/imunologia , Humanos , Neoplasias/imunologia , Condicionamento Pré-TransplanteRESUMO
Cord blood (CB) has been used recently for stem cell transplantation. We have investigated two different approaches to deplete CB samples of T cells capable of mounting graft-vs.-host disease (GVHD). The methods used were selection of CD34+ cells using avidin-biotin columns (CellPro) and T cell immunodepletion with T10B9 monoclonal antibody (mAb) plus complement. Using the avidin-biotin columns, 10.3% of the original CD34+ cells were recovered. Although this technique yielded a population containing 60 +/- 5.5% CD34+ cells, about 1 log of CFU-GM progenitors were lost. In contrast, after the T10B9 mAb and complement immunodepletion, 75 +/- 19% and 62 +/- 7% of the CD34+ cells and CFU-GM were recovered, respectively. T cell depletion was 3.6 logs using the CellPro columns and 2.2 logs after immunodepletion. To investigate whether cell losses following T cell depletion could be overcome by ex vivo expansion, cells were cultured in the presence of recombinant human interleukin-3 (rhIL-3) and recombinant human c-kit ligand (stem cell factor [rhSCF]) for 7 days. There were 14- and six-fold expansions in the number of progenitors recovered after CellPro and immunodepletion, respectively. To asses the engraftment potential of expanded cells, we used a murine transplantation model in which the presence of human cells was identified by the anti-CD45 mAb. Cells expanded in vitro engrafted in irradiated BNXid mice as efficiently as nonexpanded cells, suggesting that expansion did not affect their transplantability. This study shows that both techniques resulted in significant T cell depletion of CB. Furthermore, in vitro expansion could overcome cell losses sustained during the separation techniques without impairing the engraftment potential of the expanded cells.