RESUMEN
Type I interferon (IFN) is shown to control the reversible quiescence of a primitive human bone marrow mesenchymal stem cell (MSC) subpopulation. A 24 h pre-treatment of Stro1+/GlycoA- or CD45-/GlycoA- subpopulations with a monoclonal antibody (mAb) against the IFNAR1 chain of the human type I IFN receptor (64G12), or with a polyclonal anti-IFNalpha antibody, resulted in a marked increase in the number of very large colonies (CFU-F >3000 cells) obtained in the presence of low, but necessary, concentrations of bFGF. Over a 2-month culture period, this short activation promoted a faster and greater amplification of mesenchymal progenitors for adipocytes and osteoblasts. Activation correlated with inhibition of STAT1 and STAT2 phosphorylation and of STAT1 nuclear translocation. A non-neutralizing anti-IFNAR1 mAb was ineffective. We demonstrate that control and activated MSCs express ST3GAL3, a sialyltransferase necessary to produce the embryonic antigens SSEA-3 and -4. Interestingly, activated MSC progeny expressed SSEA-3 and -4 at a higher level than control cultures, but this was not correlated with a significant expression of other embryonic markers. As MSCs represent an essential tool in tissue regeneration, the use of 64G12, which rapidly recruits a higher number of primitive cells, might increase amplification safety for cell therapy.
Asunto(s)
Interferón-alfa/fisiología , Interferón beta/fisiología , Células Madre Mesenquimatosas/citología , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , División Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Cartilla de ADN , Matriz Extracelular/fisiología , Humanos , Inmunofenotipificación , Cinética , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/inmunología , Reacción en Cadena de la Polimerasa , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Human embryonic stem cells (hESCs) are derived from pre-implantation embryos given to research with the informed consent of the parents. These cells cannot give rise to a human being: they are not totipotent. They have an unlimited self-renewal capacity and they can generate the three embryonic germ layers, their respective derivatives and the extra-embryonic tissues: they are pluripotent. These cells represent an outstanding material for understanding functional genomics of not yet characterized human genes. They will be an important tool for pharmaceutical and clinical research.
Asunto(s)
Blastocisto/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Trasplante de Células Madre , División Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , InvestigaciónRESUMEN
We have previously demonstrated that purified human fibrinogen (Fg), a major plasma component removed during serum preparation, shows mitogenic properties towards lymphoma cells and normal human hematopoietic progenitors. Indeed, adding Fg with IL-3 to a serum-containing medium stimulates growth of human CD34+ progenitors. In this report, we show in serum-free medium, that this stimulating effect only occurs in the presence of IL-6. To clarify the cooperative effect between Fg and IL-6, the kinetics of IL-6 receptor (IL-6R) mRNA expression in CD34+ cells have been analyzed by semi-quantitative in situ hybridization. In the presence of both IL-3 and Fg, more cells express IL-6R mRNA, and this expression per cell is significantly greater than with each factor added separately. These results suggest that Fg does not promote the growth of normal cells by itself, but sensitizes the cells to cytokines. Fg behaves not as a "progression" factor but as a typical "competence" factor, which induces a faster and greater IL-6R expression in early human hematopoietic progenitors by cooperating with other cytokines.
Asunto(s)
Antígenos CD34/inmunología , Fibrinógeno/fisiología , Regulación de la Expresión Génica/fisiología , Células Madre Hematopoyéticas/metabolismo , Interleucina-6/fisiología , ARN Mensajero/genética , Receptores de Interleucina-6/genética , Células Madre Hematopoyéticas/inmunología , Humanos , Hibridación in SituRESUMEN
Various protocols have been described to optimize gene transfer into hematopoietic cells. However, most of these methods do not specify whether they are associated with an improved transduction of the more primitive stem/progenitor cells, the best candidates for long-term engraftment. The majority of these primitive cells remains in quiescence because of the negative control of TGF-beta1, effective on these cells at low concentrations (10 pg/ml). In this study, CD34- cells were activated by a 10 h pretreatment with anti-TGF-beta1 followed by four successive retroviral supernatant incubations of 6 h each. After 12 h (two incubations), a significant increase in TGF-beta1 mRNA in CD34+ cells was observed. We wondered whether neo-synthesized autocrine TGF-beta1 could induce reversion to quiescence of the more primitive CD34+ cells transduced after one cell cycle. This would prevent their subsequent detection in a classic clonal assay. Using the HPP-Q assay comparing a rapid mixed colony assay with or without anti-TGF-beta1, we indeed observed, that in clonal growth conditions the more primitive transduced cells were activated and detectable only with anti-TGF-beta1. Therefore, this assay represents not only a rapid means to detect quiescent multipotent stem/progenitor cells but also a necessary step for the detection of the more primitive transduced cells which have returned to quiescence after retroviral induction of TGF-beta1 secretion.
Asunto(s)
Técnicas de Transferencia de Gen , Células Madre Hematopoyéticas , Glicoproteínas de Membrana , Factor de Crecimiento Transformador beta/metabolismo , Antígenos CD/metabolismo , Antígeno CD24 , Técnicas de Cultivo de Célula , División Celular , Expresión Génica , Humanos , ARN Mensajero/genética , Transducción Genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Hematopoiesis is a remarkable cell-renewal process that leads to the continuous generation of large numbers of multiple mature cell types, starting from a relatively small stem cell compartment. A highly complex but efficient regulatory network is necessary to tightly control this production and to maintain the hematopoietic tissue in homeostasis. During the last 3 decades, constantly growing numbers of molecules involved in this regulation have been identified. They include soluble cytokines and growth factors, cell-cell interaction molecules, and extracellular matrix components, which provide a multifunctional scaffolding specific for each tissue. The cloning of numerous growth factors and their mass production have led to their possible use for both fundamental research and clinical application.
Asunto(s)
Hematopoyesis , Factor de Crecimiento Transformador beta/fisiología , Animales , Humanos , Transducción de Señal/fisiologíaRESUMEN
Transforming growth factor-beta1 (TGF-beta1) has been described as an efficient growth inhibitor that maintains the CD34(+) hematopoietic progenitor cells in quiescence. The concept of high proliferative potential-quiescent cells or HPP-Q cells has been introduced as a working model to study the effect of TGF-beta1 in maintaining the reversible quiescence of the more primitive hematopoietic stem cell compartment. HPP-Q cells are primitive quiescent stem/progenitor cells on which TGF-beta1 has downmodulated the cytokine receptors. These cells can be released from quiescence by neutralization of autocrine or endogenous TGF-beta1 with a TGF-beta1 blocking antibody or a TGF-beta1 antisense oligonucleotide. In nonhematopoietic systems, TGF-beta1 cooperates with the cyclin-dependent kinase inhibitor, p21(cip1), to induce cell cycle arrest. We therefore analyzed whether endogenous TGF-beta1 controls the expression of the p21(cip1) in the CD34(+) undifferentiated cells using a sensitive in situ hybridization method. We observed that addition of anti-TGF-beta1 is followed by a rapid decrease in the level of p21(cip1) mRNA whereas TGF-beta1 enhances p21(cip1) mRNA expression concurrently with an inhibitory effect on progenitor cell proliferation. These results suggest the involvement of p21(cip1) in the cell cycle control of early human hematopoietic quiescent stem/progenitors and not only in the differentiation of more mature myeloid cells as previously described. The modulation of p21(cip1) observed in response to TGF-beta1 allows us to further precise the working model of high proliferative potential-quiescent cells.
Asunto(s)
Ciclinas/genética , Células Madre Hematopoyéticas/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/fisiología , Antígenos CD34/análisis , Ciclo Celular/fisiología , División Celular/efectos de los fármacos , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidores Enzimáticos/metabolismo , Sangre Fetal/citología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Recién Nacido , Cinética , Oligodesoxirribonucleótidos Antisentido/farmacología , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Genetic alterations of the signaling cascade of transforming growth factor-beta (TGF-beta) are often associated with neoplastic transformation of primitive cells. This demonstrates the key role for this pleiotropic factor in the control of quiescence and cell proliferation in vivo. In the high proliferative potential-quiescent cell (HPP-Q) in vitro assay, the use of TGF-beta1 blocking antibodies (anti-TGF-beta1) allows the detection within two to three weeks of primitive hematopoietic cells called HPP-Q, which otherwise would not grow. However, the possibility of triggering cell proliferation by blocking the cell-surface TGF-beta receptors has not been investigated until now. We have tested here the efficiency of a blocking antibody against TGF-betaRII (anti-TGF-betaRII) on CD34(+)CD38(-) hematopoietic cells, a subpopulation enriched in primitive stem/progenitor cells, and compared its effect with that of anti-TGF-beta1. About twice as many HPP colony-forming cells were detected in the presence of anti-TGF-beta1 or anti-TGF-betaRII, compared to the control (p < 0.02). Moreover, anti-TGF-betaRII was as efficient as anti-TGF-beta1 for activating multipotent HPP-granulocyte erythroid macrophage megakaryocyte and HPP-Mix, bipotent HPP-granulocyte-macrophage (GM) and unipotent HPP-G, HPP-M and HPP-BFU-E. We therefore propose the use of anti-TGF-betaRII to release primitive cells from quiescence in the HPP-Q assay. This strategy could be extended to nonhematopoietic tissues, as TGF-beta1 may be a pleiotropic regulator of somatic stem cell quiescence.
Asunto(s)
Receptores de Activinas Tipo I , Antígenos CD34 , Antígenos CD , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Antígenos de Diferenciación , Diferenciación Celular , División Celular , Clonación Molecular , Humanos , Glicoproteínas de Membrana , NAD+ Nucleosidasa , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Factores de Tiempo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Somatic stem cells are largely quiescent in spite of their considerable proliferative potential. Transforming growth factor-(beta)1 (TGF-(beta)1) appears to be a good candidate for controlling this quiescence. Indeed, various mutations in the TGF-beta signalling pathway are responsible for neoplasic proliferation of primitive stem/progenitor cells in human tissues of various origins. In hemopoietic single cell culture assays, blocking autocrine and endogeneous TGF-(beta)1 triggers the cell cycling of high proliferative potential undifferenciated stem/progenitor cells. However, it has never been demonstrated whether TGF-(beta)1 has an apoptotic effect or a differentiating effect on these primitive cells, as already described for more mature cells. Using single cell experiments both in liquid or semi-solid culture assays and dye tracking experiments by flow cytometry, we demonstrate that low, physiological concentrations of TGF-(beta)1, which specifically maintain primitive human hemopoietic stem/progenitor cells in quiescence, have a reversible effect and do not induce apoptosis. We moreover demonstrate that these low concentrations prevent the rapid loss of the mucin-like protein CD34, a most common marker of immature hematopoietic stem/progenitor cells, which is progressively lost during differentiation. TGF-(beta)1 not only up-modulated the CD34 antigen before S phase entry but also maintained a high level of CD34 expression on cells which had escaped cell cycle inhibition, suggesting that proliferation inhibition and differentiation control by TGF-(beta)1 may be independent. These data provide additional evidence that TGF-(beta)1 acts as a key physiological factor ensuring the maintenance of a stem cell reserve.
Asunto(s)
Antígenos CD34/biosíntesis , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Compuestos Orgánicos , Factor de Crecimiento Transformador beta/fisiología , Antígenos CD34/genética , Biomarcadores , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Diferenciación Celular/efectos de los fármacos , División Celular , Células Cultivadas , Colorantes/análisis , Relación Dosis-Respuesta a Droga , Sangre Fetal/citología , Citometría de Flujo , Colorantes Fluorescentes/análisis , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Interleucina-6/farmacología , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
INTRODUCTION: Transforming Growth Factor-beta1 is known to maintain primitive human hematopoietic stem/progenitor cells in a quiescent state. However, its specific role in the control of distinct progenitor cell types needs to be further elucidated. In this study, we have investigated the dose-response effect of TGF-beta1 on progenitors ranging from primitive high proliferative potential (HPP)-Mix, -GM or -BFU-E to later BFU-E, CFU-G or CFU-M. MATERIALS AND METHODS: A clonal semi-solid assay has been used to analyze the effects of a TGF-beta1 blocking antibody (anti-TGF-beta1) or that of active TGF-beta1 added to the medium at concentrations from 10-3000 pg/ml, on these different hematopoietic stem/progenitor cell types. RESULTS AND CONCLUSION: A preferential growth inhibitory effect on the earlier progenitors was observed when low concentrations of TGF-beta1 (10-300 pg/ml) were used. Concentrations of 10-30 pg/ml TGF-beta1 were sufficient to inhibit 90% of the primitive multipotent HPP-Mix, while 100-300 pg/ml TGF-beta1 were required to inhibit 70% of the bipotent HPP-GM and early HPP-BFU-E. TGF-beta1 did not significantly inhibit or activate the growth of later CFU-G and CFU-M, even when added at concentrations 10-100 fold higher. In contrast, a significant growth-inducing effect of very low TGF-beta1 concentrations (< or =30 pg/ml) on a subset of later BFU-E was observed and cannot be explained by a switch of early into later BFU-E. These results emphasize the polyfunctional role of TGF-beta1 in the regulation of hematopoiesis and the need for low, physiological concentrations of TGF-beta1, when studying both the stem cell compartment and more mature progenitor cell subpopulations.
Asunto(s)
Sangre Fetal/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Parto Obstétrico , Femenino , Sustancias de Crecimiento/farmacología , Células Madre Hematopoyéticas/citología , Humanos , Recién Nacido , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta1RESUMEN
We investigated the effect of a new fusion protein of IL-6 and the soluble IL-6R, H-IL-6, on the long-term ex vivo expansion of hematopoietic progenitors derived from AC133+cord blood cells. H-IL-6, which acts on both IL-6Ralpha-positive and IL-6Ralpha-negative cells, effectively synergized with FL and TPO with or without SCF for the propagation of primitive progenitors. However, IL-6 showed a greater synergistic effect with FL and TPO than H-IL-6 for long-term progenitor propagation. During the first 6 weeks of culture under stroma-free serum-containing conditions, IL-6 induced a 1.96 +/- 0.64-fold higher expansion of nucleated cells, a 2.28 +/- 0.33-fold higher expansion of CD34+ cells and a 2.74 +/- 0. 28-fold higher expansion of CD34+ AC133+ cells than H-IL-6 in combination with FL and TPO. The propagation of week 6 CAFC was up to four-fold higher in the presence of IL-6 than with H-IL-6. While the expansion of CD34+ and CD34+ AC133+ cells dropped after 5-7 weeks in the stroma-free cultures with FL, TPO and H-IL-6, a sustained expansion for 12 weeks was obtained in the presence of FL, TPO and IL-6. Stroma-contact greatly enhanced the progenitor expansion induced by FL and TPO or FL, TPO and H-IL-6 although the highest proliferation was again obtained in the presence of IL-6. In contrast, the presence of SCF resulted in increased differentiation. Since the majority of primitive progenitors are proposed to be IL-6Ralpha-negative, the results suggest that the synergistic effect of IL-6 is mediated by accessory cells, which have been more effectively stimulated by IL-6 than by the fusion peptide, H-IL-6, in this culture system.
Asunto(s)
Antígenos CD/fisiología , Sangre Fetal/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/farmacología , Trombopoyetina/farmacología , Receptor gp130 de Citocinas , Sangre Fetal/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Recién Nacido , Factor de Células Madre/farmacología , Células del Estroma/fisiologíaRESUMEN
Human adult hematopoietic stem cells are mostly quiescent or slow cycling. We have previously demonstrated that blocking of transforming growth factor-beta1 (TGF-beta1) is able to activate, in the presence of cytokines, primitive quiescent hematopoietic multipotent progenitors which could not grow in a two week semi-solid culture assay (short term culture). We have also shown that anti-TGF-beta1 can up-modulate c-KIT, the receptor of the stem cell factor (steel factor). To elucidate whether TGF-beta1 plays a central role in controlling the quiescence of hematopoietic primitive cells, it was necessary to demonstrate, as detailed in this study, that: (1) whatever the cytokine combination tested, addition of anti-TGF-beta1 releases from quiescence multipotent progenitors with a significantly higher hematopoietic potential than those activated by cytokines alone. (2) Other important cytokine receptors controlling the most primitive hematopoietic cells such as FLT3 and the IL6 receptor (IL6-R) are down-modulated by TGF-beta1 but rapidly up-modulated by anti-TGF-beta1. (3) Anti-TGF-beta1-sensitive multipotent and high proliferative potential progenitors express these cytokine receptors at a low level (FLT3(low) and IL6-Rlow). According to these results, we propose the working model of 'High Proliferative Potential-Quiescent cells' to refer to these primitive hematopoietic multipotent progenitors that are highly sensitive to the growth inhibitory effect of TGF-beta1. This model could be valid not only to study the human hematopoietic quiescent progenitors but also for other somatic stem cell systems.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Modelos Biológicos , Anticuerpos/farmacología , Antígenos CD34/análisis , Antígenos CD34/biosíntesis , División Celular/efectos de los fármacos , Células Cultivadas , Sangre Fetal , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Inmunofenotipificación , Interfase/efectos de los fármacos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/farmacología , Receptores de Interleucina-6/análisis , Receptores de Interleucina-6/biosíntesis , Receptores de Interleucina-6/metabolismo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
In addition to its well-known functions in blood clotting and cell adhesion, fibrinogen has been reported to be a mitogen for lymphoid cell lines and for human hematopoietic progenitors. Two specific receptors, the mitogenic fibrinogen receptor (MFR) and intercellular adhesion molecule-1 (ICAM-1/CD54), have been identified as possible candidates for the mediation of the mitogenic effect of fibrinogen. However, it has been questioned whether the MFR and ICAM-1 are truly distinct molecules. Using an antiserum specific for the MFR, we demonstrate that the MFR is a cell surface molecule clearly distinct from ICAM-1. Both receptors can be expressed separately or coexpressed on different cell types. Moreover, they are regulated differently: ICAM-1 is calcium-dependent whereas the MFR is not and the MFR is down-regulated by fibrinogen whereas ICAM-1 is not. The inhibition by an anti-MFR serum of the mitogenic effect of fibrinogen confirms the mitogenic function of the MFR.
Asunto(s)
Fibrinógeno/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Mitógenos/farmacología , Receptores de Péptidos/metabolismo , Anticuerpos/farmacología , División Celular/efectos de los fármacos , Línea Celular , Fibrinógeno/metabolismo , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Humanos , Técnicas In Vitro , Mitógenos/metabolismo , Receptores de Péptidos/antagonistas & inhibidoresRESUMEN
We have previously shown that a low concentration of CSF-1 (1 U/ml) can trigger human immature monocytic progenitor proliferation in the presence of low concentrations of IL3 (1.7 U/ml). No c-fms down-regulation was observed during this early cell activation. In contrast, 20 U/ml of CSF-1, active on late monocytic cell growth, down-regulated c-fms mRNA expression in immature progenitors and monocytes derived from bone marrow CD34+ cells in culture. We have now extended this study to include the effects of various concentrations of GM-CSF, IL3 and G-CSF on c-fms expression. We observed that high doses of GM-CSF or IL3 down-modulated c-fms mRNA, whereas low doses of GM-CSF or IL3, which were active on early monocytic growth, had no such effect. Similar results were observed at the protein level. In contrast, whatever the concentration, G-CSF had no effect on c-fms mRNA or protein levels. We further observed that the more immature the c-fms expressing progenitors, the faster the down-modulation of this receptor. This was observed within less than 1 hour for immature bone marrow cells, 6 hours for peripheral blood monocytes and even longer for transformed monocytic cells. These results suggest that oncogene expression can be regulated much more rapidly in immature progenitors than was previously observed in mature cells or transformed cell lines.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Genes fms , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-3/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Monocitos/metabolismo , ARN Mensajero/biosíntesis , Diferenciación Celular , Células HL-60 , Hematopoyesis/genética , Humanos , Monocitos/citología , ARN Mensajero/genéticaRESUMEN
Growth factor receptors in human hematopoietic progenitor cells have become the focus of intense interest, because they may provide tools for the monitoring, enrichment, and expansion of stem cells. We have shown earlier that the Tie receptor tyrosine kinase is expressed in erythroid and megakaryoblastic human leukemia cell lines, in the blood islands of the yolk sac, and in endothelial cells starting from day 8.0 of mouse development. Here, the expression of Tie was studied in human hematopoietic cells of various sources. Peripheral blood mononuclear cells were Tie-. However, a large fraction of CD34+ cells from umbilical cord blood (UCB) and bone marrow (BM) expressed tie protein and mRNA. On average, 64% of the fluorescence-activated cell sorting-gated UCB CD34+ cells including CD38- cells and a fraction of cells expressing low levels of c-Kit were Tie+. Also, 30% to 60% of BM CD34+ cells were Tie+, including most of the BM CD34+CD38-, CD34+Thy-1+, and CD34+HLA-DR- cells. Under culture conditions allowing myeloid, erythroid, and/or megakaryocytic differentiation, purified UCB CD34+ cells lost Tie mRNA and protein expression concomitantly with that of CD34; however, a significant fraction of cells expressed Tie during megakaryocytic differentiation. These data suggest that, in humans, the Tie receptor and presumably its ligand may function at an early stage of hematopoietic cell differentiation.
Asunto(s)
Células Madre Hematopoyéticas/enzimología , Megacariocitos/enzimología , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores de Superficie Celular/biosíntesis , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD34 , Células de la Médula Ósea , Diferenciación Celular , Línea Celular Transformada , Células Cultivadas , Chlorocebus aethiops , Medios de Cultivo Condicionados , Endotelio Vascular/enzimología , Inducción Enzimática , Sangre Fetal/citología , Humanos , Leucemia/enzimología , Leucemia/patología , Antígenos Comunes de Leucocito , Megacariocitos/química , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores TIE , Proteínas Recombinantes/farmacología , Trombopoyetina/farmacología , Células Tumorales CultivadasRESUMEN
It has been previously demonstrated that prestimulation with cytokines could improve gene transfer in hematopoietic progenitors. However, we have shown that no combination of cytokines so far tested is able to release rapidly in vitro the stem cell compartment from quiescence unless an autocrine transforming growth factor-beta 1 (TGF-beta 1) is blocked by specific oligonucleotide antisense or antiserum (Hatzfeld et al., 1991, J. Exp. Med., 174, 925). We now report that a 10-hr cytokine prestimulation of SBA-CD34high human umbilical cord blood progenitors increases retrovirally mediated transfer of the nls-lacZ reporter gene from 1% to 23.8% and addition of anti-TGF-beta serum doubles this increase (47.3%). Interestingly, the effect of anti-TGF-beta preincubation on gene transfer is most effective on the most immature progenitors, which develop into high proliferative potential mixed colonies with 1-2 x 10(5) cells. Anti-TGF-beta serum pretreatment increases gene transfer in these early colony-forming units granulocyte-erythroid-megakaryocyte-macrophage (CFU-GEMM) from 54.1% to 93.3%. It augments significantly the stability of gene expression in all subpopulations of mixed colonies. Colonies obtained after pretreatment with anti-TGF-beta serum are larger and the expression of the stably integrated recombinant provirus does not reduce their size. This prestimulation method provides a substantial improvement for gene transfer efficiency within the quiescent stem cell compartment that is responsible for long-term engraftment.
Asunto(s)
Técnicas de Transferencia de Gen , Células Madre Hematopoyéticas/fisiología , Retroviridae/genética , Antígenos CD34/análisis , Sangre Fetal , Expresión Génica , Genes Reporteros/genética , Factores de Crecimiento de Célula Hematopoyética/farmacología , Humanos , Sueros Inmunes , Interleucina-6/farmacología , Factor de Crecimiento Transformador beta/fisiología , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
We have previously shown that early human CD34high hematopoietic progenitors are maintained quiescent in part through autocrine transforming growth factor-beta 1 (TGF-beta 1). We also demonstrated that, in the presence of interleukin-3, interleukin-6, granulocyte colony-stimulating factor, and erythropoietin, TGF-beta 1 antisense oligonucleotides or anti-TGF-beta serum have an additive effect with KIT ligand (Steel factor [SF]), which suggests that they control different pathways of regulation in these conditions. This finding also suggests that autocrine TGF-beta 1 might suppress c-kit expression in primitive human hematopoietic progenitors. We have now distinguished two subpopulations of CD34high cells. One subpopulation expresses a c-kit mRNA that can be downmodulated by exogenous TGF-beta 1 within 6 hours. Another subpopulation of early CD34high cells expresses a low or undetectable level of c-kit mRNA, but its expression can be upmodulated within 6 hours by anti-TGF-beta. These effects disappear 48 hours after induction and cannot be maintained longer than 72 hours, even if TGF-beta 1 or anti-TGF-beta serum are added every day. Similar kinetics, although delayed, are observed with KIT protein expression. On the contrary, no specific effect of TGF-beta 1 was observed on c-fms, GAPDH, and transferrin receptor gene expression in these early progenitors. These results clarify the complex interaction between TGF-beta 1 and SF in normal early hematopoietic progenitors. SF does not switch off the TGF-beta 1 inhibitory pathway. Autocrine TGF-beta 1 appears to maintain these cells in a quiescent state, suppressing cell division by downmodulating the receptor of SF, a key cytokine costimulator of early progenitors.
Asunto(s)
Antígenos CD/análisis , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/citología , Proteínas Proto-Oncogénicas/análisis , Proteínas Tirosina Quinasas Receptoras/análisis , Receptores del Factor Estimulante de Colonias/análisis , Factor de Crecimiento Transformador beta/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos CD34 , Biomarcadores de Tumor/análisis , Separación Celular/métodos , Eritropoyetina/farmacología , Sangre Fetal/citología , Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/biosíntesis , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Recién Nacido , Interleucina-3/farmacología , Interleucina-6/farmacología , Cinética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-kit , Proto-Oncogenes , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Receptores del Factor Estimulante de Colonias/biosíntesis , Receptores de Transferrina/biosíntesis , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
The human hematopoietic pluripotent repopulating "stem cell" is thought to be present within a minor subpopulation of CD34+ cells. This has not been definitively shown, although the more primitive CD34+ cell subset contains precursors for all lymphoid and nonlymphoid cell lineages. When purifying CD34+ cells, it is important to recover these early progenitors, which are more strongly immunoadsorbent to the separation devices. Using a commercialized panning system (AIS CELLector flasks), we observed that a high degree of purity requires a thorough washing procedure so that cells not binding or weakly binding to CD34 antibodies are removed. High recoveries can be obtained if the adherent cells are then efficiently detached by a 2-hour incubation in culture medium without added cytokines. In this way, we can routinely obtain 93.5 +/- 3.4% purity of CD34+ cells with a 74% yield of the multipotent colony-forming units (CFU-GEMM). Complete recovery of the putative "stem cell," or at least the early progenitor cell compartment (CD34+ CD38low/- CD34+ Thy-1+ cells), is also obtained. More than 30% of these cells can generate day-14 colonies in vitro. Comparable results were obtained when the separation was scaled up for clinical application using appropriate large-scale devices. The various incubation times of the procedure can be easily adjusted to the work schedule. This renders the procedure easy to handle, efficient, safe, and, because the cells can be observed under light microscopy, easy to control.
Asunto(s)
Antígenos CD , Células de la Médula Ósea , Separación Celular/métodos , Células Madre Hematopoyéticas , Técnicas de Inmunoadsorción , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Antígenos CD34 , Antígenos de Diferenciación/análisis , Ensayo de Unidades Formadoras de Colonias , Factores Estimulantes de Colonias/farmacología , Eritropoyetina/farmacología , Antígenos HLA-DR/análisis , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Humanos , Inmunofenotipificación , Interleucina-3/farmacología , Interleucina-6/farmacología , Glicoproteínas de Membrana , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-kit , Proteínas Tirosina Quinasas Receptoras/análisis , Receptores del Factor Estimulante de Colonias/análisis , Proteínas Recombinantes/farmacología , Antígenos Thy-1/análisisRESUMEN
We previously demonstrated that TGF-beta 1 antisense oligodeoxynucleotides can release early CD34+ bone marrow progenitors from quiescence, and increase the numbers of mixed and large erythroid colonies. As Steel Factor (SF) has a similar effect on colony formation by CD34+ cells, we tested whether this factor acts by blocking the inhibitory effects of TGF-beta. That this is not generally the case was demonstrated by the finding that the combination of TGF-beta 1 antisense with SF in cultures of CD34+ bone marrow cells yielded enhanced colony formation that was more than additive when compared to cultures containing the single agents. This combination also yielded enhanced colony formation by CD34+ umbilical cord blood cells, but in this case, the effect was slightly less than additive. Thus in cord blood, some, but not all, of the progenitors that are maintained in quiescence by TGF-beta can be triggered into cycle by SF. However, the absolute number of CFU-GEMMs found in the antisense TGF-beta plus SF cultures of cord blood was 4-fold higher than that found in comparable bone marrow cultures. These data correlate well with our previous observations that umbilical cord blood contains 4-fold more CD34+ CD38- cells, the population found to respond to TGF-beta 1 antisense oligodeoxynucleotides.
Asunto(s)
Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Factor de Crecimiento Transformador beta/farmacología , Antígenos CD , Antígenos CD34 , Sinergismo Farmacológico , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Fenotipo , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-kit , Proteínas Tirosina Quinasas Receptoras/análisis , Receptores del Factor Estimulante de Colonias/análisis , Factor de Células MadreRESUMEN
Steel factor (SF) increases the frequency of colony formation by CD34+ CD38- cycling cells, but it does not reverse the effect of an autocrine production of transforming growth factor (TGF)-beta 1 by early progenitors of the stem cell compartment. We have used optimal culture conditions supplemented with SF and anti-TGF-beta serum to estimate the proliferative capacity and ability to generate early progenitors in long-term cultures of bone marrow and umbilical cord blood cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU), twice as many granulocyte-macrophage (GM) CFU, and three times as many erythroid burst-forming units as the same population from an average bone marrow sample used in adult transplantation. These results suggest that umbilical cord blood is a suitable source of cells for adult transplantation.
Asunto(s)
Sangre Fetal/citología , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Adulto , Antígenos CD/análisis , Antígenos CD34 , Antígenos de Diferenciación/análisis , Células de la Médula Ósea , Recuento de Células , División Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Sueros Inmunes/farmacología , Glicoproteínas de Membrana , Factor de Células Madre , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
The effect of human fibrinogen on the proliferation of purified SBA- CD34+ human bone marrow progenitors was investigated in clonal cultures. Fibrinogen alone or in combination with erythropoietin had no significant effect. However, in the presence of recombinant human interleukin-3 (IL-3), fibrinogen increased significantly in a dose-dependent manner the number of mixed and burst-forming unit-ethrocyte--derived colonies, whereas the number of other colonies did not significantly change. In the presence of fibrinogen, low concentrations of IL-3 (0.17 U/mL) produced three times more mixed colonies than without fibrinogen, reaching the number of colonies obtained with optimal concentrations of IL-3 (1.67 U/mL). Fibrinogen fragment D had the same effect in the presence of IL-3 as intact fibrinogen, whereas fibrinogen fragment E and human collagen IV did not. This effect was not mediated by integrins, because peptides or monoclonal antibodies that block fibrinogen binding on integrins alpha IIb beta 3, alpha v beta 3 (RGD-peptides), alpha m beta 2 (OKM-1), and alpha x beta 2 (HC1/1) did not affect the observed mitogenic effect. The mitogenic effect of fibrinogen and its D fragment was not mediated by induction of IL-6 or granulocyte--colony-stimulating factor secretion, because it was not inhibited by blocking antisera against these two growth factors. Our results indicate that fibrinogen potentiates the effect of IL-3 on primitive hematopoietic progenitors and suggest that the mitogenic effect of fibrinogen could be mediated via a specific mitogenic receptor that does not belong to the integrin family.