RESUMEN
Physiological studies indicate that epidermal growth factor-urogastrone (EGF) acts on stomach epithelium as mitogen and modulator of acid secretion. Here, we studied the binding of 125I-EGF to gastric glands isolated from the guinea-pig fundus (acid-secreting part) and antrum. At 20 degrees C, the association of 125I-EGF to gastric glands was time-dependent (plateau at 90 min) and reversible (75-85% dissociation in 1 h). No degradation of the peptide was detected, but a time-dependent loss of binding capacity was observed. At apparent equilibrium (90 min, 20 degrees C) unlabelled EGF (80 pM to 80 nM) competed with 125I-EGF-binding in the same manner in antrum and fundus (50% inhibition, with 0.6 nM EGF). Whereas kinetics properties were similar in antrum and fundus, the binding capacity was 40-55% lower in fundus than in antrum in young animals (6-8 weeks). By contrast, in adult animals (20-30 weeks), binding was the same in both parts of stomach. Scatchard analysis showed that two orders of binding sites were present in all cases (Ki 0.34-0.47 nM, Ki 2.2-3.4 nM), and that the differences observed were only accounted for by number of binding sites. These results show that EGF possess high affinity binding sites on gastric epithelium. These sites, dependent upon the age of the animals, may be related to the modulations by EGF of gastric trophism and secretions.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Glándulas Exocrinas/metabolismo , Mucosa Gástrica/metabolismo , Receptores de Superficie Celular/metabolismo , Envejecimiento , Animales , Unión Competitiva , Receptores ErbB , Cobayas , Cinética , Masculino , Ratones , Estómago/crecimiento & desarrolloRESUMEN
Although tumor necrosis factor alpha (TNF alpha) exerts a variety of activities on hematopoietic cells, suggesting it may have some potential therapeutic applications, its long-term effects on hematopoiesis are not well defined. Therefore, we took the advantage of long-term bone marrow cultures (LTBMCs) to evaluate the long-term role of TNF alpha on both the microenvironment and the hematopoietic progenitors. LTBMCs were inoculated with 100 U/ml of recombinant human TNF alpha (rhTNF alpha) either at the onset of the cultures (d0) or at day 21 (d21) when the adherent layer (AL) was already established. Then TNF alpha was added at each weekly medium change. The cellularity and the content of progenitors in both the nonadherent layer (NAL) and AL, the formation of the AL, and the presence of various cytokines in the supernatants were examined weekly. The data showed 1) a strong and durable inhibitory effect on total nonadherent cells; 2) a rapid and transient inhibition of NA progenitors, whereas adherent progenitors were lately affected; and 3) microenvironmental changes consisting of the disappearance of adipocytes and the secretion of high levels of interleukin 6. The results suggest that the inhibitory effects of TNF alpha on the NAL are in part counterbalanced by stromal modifications that in turn lead to a faster exhaustion of hematopoiesis.
Asunto(s)
Células de la Médula Ósea , Factor de Necrosis Tumoral alfa/farmacología , Médula Ósea/química , Médula Ósea/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Factor Estimulante de Colonias de Granulocitos/análisis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/análisis , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/química , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interleucina-1/análisis , Interleucina-3/análisis , Interleucina-6/análisis , Proteínas Recombinantes/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/análisisRESUMEN
The purpose of this work was to study the effects of a tetrapeptide, acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of spleen colony-forming unit (CFU-S) entry into DNA synthesis, on human progenitor cells. Normal human mononuclear cells were incubated with concentrations of the synthetic tetrapeptide ranging from 10(-12) to 10(-7) M for 1.5 and 24 h and then plated in methylcellulose in the presence of human placenta-conditioned medium and recombinant human erythropoietin. The proportion of progenitors in DNA synthesis was determined by the thymidine suicide assay. Incubation with AcSDKP for 24 h leads to a significant inhibition of granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) growth and in some cases of erythroid colony-forming unit (CFU-E) growth. The inhibition, which was never greater than 50%, was obtained with 10(-10)-10(-9) M AcSDKP, whereas no effect was seen at higher concentrations. The percentage of CFU-GM, BFU-E, and CFU-E in DNA synthesis was significantly reduced in five consecutive patients after incubation of cells for 24 h with inhibitory doses of the peptide, indicating that it is active on cycling cells. Therefore, these studies provide the first evidence that the tetrapeptide AcSDKP, originally obtained from bovine marrow and now chemically synthesized, is able to inhibit the in vitro growth of human progenitors and to decrease their proportion in cell cycle.
Asunto(s)
Células de la Médula Ósea , Células Precursoras Eritroides/citología , Células Madre Hematopoyéticas/citología , Oligopéptidos/farmacología , Secuencia de Aminoácidos , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Granulocitos/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Macrófagos/citología , Datos de Secuencia MolecularRESUMEN
To examine mechanisms of cytopenia in acute nonlymphoblastic leukemia (ANLL), we determined whether leukemic plasma (LP) contains growth-promoting factors that support mammalian erythroid progenitor and pluripotential stem cell proliferation in vitro. When added to serum-free cultures of human bone marrow and peripheral blood cells, LP from anemic patients with ANLL stimulated erythroid burst formation to greater levels than did normal human plasma (p less than 0.05 for each). While LP also enhanced erythroid burst development in murine bone marrow cells, preincubation of marrow cells with LP did not alter the formation of splenic colonies (CFU-S-derived colonies) in irradiated mice (p greater than 0.10). To determine whether erythropoietin or other growth factors (functionally similar to burst-promoting activity, BPA) are important in mediating the erythropoietic effects observed in vitro, LP was preabsorbed with monospecific IgG raised against human erythropoietin or human BPA. Although elevated erythropoietin levels were found in each LP, preabsorption with antierythropoietin IgG did not alter its capacity to enhance human burst formation. In contrast, preabsorption with antimembrane IgG capable of recognizing human BPA abrogated the stimulatory effects of LP (p less than 0.05). In addition, LP was found to increase the percentage of murine CFU-S that are synthesizing DNA by the (3H) Tdr suicide technique, an effect which was not abrogated by preabsorption of LP with monospecific IgG raised against human BPA. We conclude that both erythropoietin and BPA are appropriately increased in ANLL. In addition, a factor is present in LP which induces DNA replication in murine pluripotential stem cells.
Asunto(s)
Eritrocitos/fisiología , Células Madre Hematopoyéticas/citología , Leucemia/sangre , Absorción , Enfermedad Aguda , División Celular , Eritrocitos/citología , Hematopoyesis , HumanosRESUMEN
OBJECTIVE: In previous work, we showed that CD34+ bone marrow cells can be successfully expanded along the myeloid pathway in stroma- and serum-free conditions in the presence of SCF+IL-3+IL-6+Flt3-l+G-CSF+MGDF. Due to the lack of phenotypically detectable lymphoid cells, it was necessary to address the question of the lymphoid potential of the expanded populations under these conditions. MATERIALS AND METHODS: The present report describes a long-term culture system that supports human B- and NK-cell differentiation from the day 14 fraction without further selection of the more primitive cells. In NK proliferation assays, the cells were maintained over stroma cells in the presence of IL-2 for 4-5 weeks. NK initiating cells (NK-IC) were determined by a limiting dilution assay. In B-cell cultures, the expanded cells were maintained over MS5 in the presence of Flt3-l for 4-8 weeks. RESULTS: NK cells rose from 0.2%+/-0.04% at culture initiation to 71%+/-6% at week 5. These cells displayed cytolytic activity. NK-IC evaluation showed a mean 18-fold expansion in the day 14 expanded fraction as compared to the initial day 0 fraction. Similarly, CD19+ cells rose from 0.1% at culture initiation to 30%+/-1% at week 6. Cells produced under these B-LTC conditions were CD34-CD19+CD10+. We also demonstrated that the CD34+/Lin- sorted cells from the day 14 fraction gave rise to NK and B cells. CONCLUSION: This culture system permits the revelation of a population that, although poorly represented in terms of phenotypically detectable cells, nevertheless retains high levels of lymphoid NK and B potential after 14 days expansion. Such data suggest the persistence, or expansion, of lymphoid progenitors and, hence, the multipotentiality of the expanded progenitor/stem cells.
Asunto(s)
Linfocitos B/citología , Células de la Médula Ósea/citología , Células Madre Hematopoyéticas/citología , Células Asesinas Naturales/citología , Linfocitos/citología , Antígenos CD34/biosíntesis , Complejo CD3/biosíntesis , Antígeno CD56/biosíntesis , Técnicas de Cultivo de Célula , Diferenciación Celular , División Celular , Células Cultivadas , Citometría de Flujo , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Asesinas Naturales/metabolismo , Linfocitos/metabolismoRESUMEN
The disappearance of normal hematopoiesis during acute nonlymphoblastic leukemia (ANLL) is poorly understood. Several reports indicate that conditioned medium obtained from leukemic cells might inhibit the formation of normal hematopoietic progenitors. However, these blast-conditioned medium (BCM) inhibitory activities are not well characterized. In order to evaluate whether BCM might contain an activity inhibiting the growth of normal marrow progenitors, BCM from 13 consecutive patients with ANLL were tested on normal bone marrow in methylcellulose assays. In all the cases, a significant inhibition of the growth of granulocyte-macrophage colony-forming unit (CFU-GM) progenitors was observed, whereas erythroid burst-forming unit (BFU-E) progenitors were not affected. Further characterization of the BCM inhibitory activity showed using both a biological assay and RIA, the presence of tumor necrosis factor-alpha (TNF-alpha) in 10 out of 13 BCM. Northern blot analysis performed in six patients showed a correlation between the expression of TNF-alpha mRNA by leukemic cells and the presence of TNF-alpha in BCM. Moreover, the BCM inhibitory activity could be neutralized with an anti-TNF-alpha antiserum. These data indicate that leukemic cells express and release frequently TNF-alpha, which may therefore play an important role in the inhibition of granulopoiesis during leukemia.
Asunto(s)
Granulocitos/patología , Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/metabolismo , Macrófagos/patología , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos/farmacología , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo , Femenino , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Hibridación de Ácido Nucleico , ARN/análisis , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.
Asunto(s)
Antígenos CD34/análisis , Diferenciación Celular , Sangre Fetal/citología , Granulocitos/citología , Células Madre Hematopoyéticas/citología , Linfocitos/citología , Animales , Linfocitos B/citología , Células Cultivadas , Supervivencia de Injerto , Factor Estimulante de Colonias de Granulocitos/farmacología , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Humanos , Células Asesinas Naturales/citología , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Factor de Células Madre/farmacología , Linfocitos T/citología , Trombopoyetina/farmacologíaRESUMEN
The aim of the study is to define the ability of Flt3 ligand, MGDF, Epo and G-CSF to modulate the expansion of different hematopoietic compartments in association with a basic cocktail of SCF + IL-3 + IL-6 (S36). CD34+ cells from normal bone marrow were cultured in stroma-free, serum-free medium for 10 days. Using various concentrations of cytokines, total cells could be expanded up to 5200-fold, CD34+ cells up to 78-fold, CFU-GM up to 143-fold, BFU-E up to 46-fold, CFU-MK up to six-fold and LTC-IC up to four-fold. The results were assessed by multiparametric analysis of variance. Three factors had a significant stimulatory effect on the late precursor compartment: Epo (P < 10(-5)), G-CSF (P=5 x 10(-3)) and FL (P=10(-5)). Two were critical for CD34+ cell expansion: FL (P=4 x 10(-5)) and Epo (P=6 x 10(-5)), while two were critical for BFU-E expansion: MGDF (P=8 x 10(-4)) and FL (P=0.017). FL strongly stimulated CFU-GM expansion (P < 10(-5)), whereas none of the growth factors studied had any effect on CFU-MK. FL (P=10(-4)) and MGDF (P=0.002) were essential to obtain high levels of expansion of LTC-IC as determined in limiting dilution assays. In the light of the above results showing a preferential effect on the expansion of precursor cells (3080-fold), CD34+ cells (53-fold), CFU-GM (134-fold), BFU-E (46-fold) and LTC-IC (five-fold), the combination SCF, IL-3, IL-6, FL, MGDF, Epo and G-CSF was chosen as a putative cytokine cocktail for further studies on long-term culture. Sustained production of precursor cells, progenitor cells, LTC-IC and E-LTC-IC for up to 100 days reflects the persistence of very primitive stem cells. This suggests that these populations are probably able to undergo self-renewal divisions. The above combination of cytokines meets the required criterion for potential clinical application, which may be defined as an effective capacity to expand all cell compartments, using as the starting material high concentrations of low purity CD34+ cells.
Asunto(s)
Eritropoyetina/farmacología , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-3/farmacología , Interleucina-6/farmacología , Proteínas de la Membrana/farmacología , Factor de Células Madre/farmacología , Trombopoyetina/farmacología , Células Madre Hematopoyéticas/fisiología , HumanosRESUMEN
Peripheral blood CD34+ cell samples from eight children with advanced neuroblastoma and from 10 healthy adult donors were seeded at 5 x 10(4) cells/ml in stroma-free, serum-free medium with FL, SCF, MGDF (100 ng/ml each), G-CSF, IL6 (10 ng/ml each) and IL3 (5 ng/ml), and incubated for 10 days. The levels of expansion of PBCD34+ cells observed in neuroblastoma patients, with up to 214-fold expansion for total nucleated cells, 39-fold for CD34+ cells, 79-fold for CFU-GM and nine-fold for LTC-IC were identical to those obtained with PBCD34+ cells of healthy donors (P>/=0.5). All samples from patients with neuroblastoma and five donor's PBCD34+ cell samples contaminated with IMR-32 neuroblasts, were screened for the number of tyrosine hydroxylase (TH) mRNA transcript using LightCycler software. In all samples, progressive 1.9-4.4 log decreases in the number of TH transcripts were observed between days 0 and 10 of expansion. Our results show that in extensively pretreated children with neuroblastoma, the culture conditions that were effective for BM and CB cell expansion can generate an expansion of PBCD34+ cells and provide a purge of tumour cells.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Neoplásicas Circulantes/patología , Neuroblastoma/terapia , Trasplante de Células Madre de Sangre Periférica/métodos , Antígenos CD34/análisis , Técnicas de Cultivo de Célula/métodos , División Celular , Niño , Preescolar , Humanos , Lactante , Neoplasia Residual/diagnóstico , Neuroblastoma/patología , ARN Mensajero/análisis , ARN Neoplásico/análisis , Trasplante Autólogo , Trasplante Homólogo , Tirosina 3-Monooxigenasa/genéticaRESUMEN
We tested two positive selection techniques for separation of CD34+ cells from bone marrow and analyzed the yields of CD34+ cells, BFU-E, CFU-GM, CFU-MK and LTC-IC after selection and expansion. An immunoadsorption procedure (CellPro) and an immunomagnetic (Baxter) CD34+ cell separation method were employed to purify the same bone marrow samples from seven normal subjects. Mean yields of CFU-GM and CFU-MK and absolute numbers of LTC-ICs were not different in the two purified cell populations. In contrast, the mean recovery of BFU-E was significantly lower for the immunoadsorption (21 +/- 14%) than for the immunomagnetic technique (44 +/- 27%). After separation, CD34+ cells were evaluated in 10-day liquid cultures for their expansion capacity in terms of total cells and progenitors. The expansion capacity of progenitors such as CFU-GM, CFU-MK and especially BFU-E selected by immunoadsorption was higher than the capacity of progenitors obtained by immunomagnetism, although final total and progenitor cell numbers are similar. Our results suggest that the populations separated by the two techniques differ mainly in the expansion capacity of progenitors and in the recovery of BFU-E after the selection procedure. These differences between two methods, which already are widely employed in research and in clinical transplantation, should be taken into account when considering the aims of the experiments.
Asunto(s)
Antígenos CD34/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Separación Celular/métodos , Separación Inmunomagnética/métodos , Técnicas de Inmunoadsorción , Ensayo de Unidades Formadoras de Colonias , Estudios de Evaluación como Asunto , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , HumanosRESUMEN
The aim of this study was to evaluate the ex vivo expansion of normal CD34+ cells in gas-permeable polypropylene bags suitable for clinical use. Cells were cultured for 14 days in serum-free medium supplemented with SCF, IL3, IL6, FLT3-1, G-CSF + MGDF or Epo. The bags supported the expansion of hematopoietic cells in a similar manner to small scale well or flask systems, allowing mean expansions of up to 2193-fold for total nucleated cells, 140-fold for CFU-GM and 66-fold for LTC-IC. Increasing the initial cell concentration from 5 x 10(3) to 1 x 10(5)CD34+ cells/ml induced the production of granulocytic cells with terminal differentiation while simultaneously decreasing the overall extent of expansion of the white blood cells produced. We tested the phagocytic activity and oxidative metabolism of the white blood cells produced. The percentage of phagocytic cells was 39+/-0.5% in expanded cultures derived from fractions initiated at 5 x 10(3), 10(4) or 10(5) cells/ml and 45+/-6% in cultured cells obtained from starting fractions containing 5 x 10(4) cells/ml, as compared to 58+/-4% in normal controls. A study of the potential for oxygen-dependent microbe killing showed that the expanded cells produced H2O2, although in lesser quantities than control cells. We subsequently investigated the possibility of freezing expanded cells. Total cell recovery after thawing was 45+/-4%, while recoveries of progenitors and stem cells ranged from 65 to 90%, without any influence of the initial cell concentration. This new approach could be of major interest for clinical practice, as it would allow evaluation of the quality of a graft prior to its infusion and employs experimental conditions which meet the criteria for potential clinical use.
Asunto(s)
Conservación de la Sangre , Criopreservación , Movilización de Célula Madre Hematopoyética/métodos , Células Madre Hematopoyéticas , Técnicas de Cultivo de Célula/métodos , Trasplante de Células Madre Hematopoyéticas , Humanos , Factores de TiempoAsunto(s)
Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Leucemia/fisiopatología , Enfermedad Aguda , Médula Ósea/patología , Médula Ósea/fisiopatología , Diferenciación Celular , Células Cultivadas , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/fisiología , Células Madre Hematopoyéticas/patología , Humanos , Leucemia/patología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
The binding of 125I-insulin was studied in epithelial cells isolated from human gallbladder. Kinetic studies at 22 degrees C showed that binding of 125I-insulin to gallbladder cells was rapid (maximum 1 h.) and reversible. In the presence of added excess of unlabelled insulin, 50% of initially bound insulin is dissociated in 10-15 min. At apparent equilibrium (1 h at 22 degrees C) unlabelled insulin (10(-10) to 10(-7) M) inhibited competitively tracer (5 X 10(-11) M) binding, with 50% inhibition at about 2 X 10(-9) M. Scatchard analysis gave curvilinear plots, that may be attributed either to negative cooperativity, or to two orders of binding sites: In the first case, the extreme average affinities are Ke = 1.8 X 10(8) M-1, and Kf = 3.2 X 10(7) M-1 for 49,000 sites/cell. In the latter case, gallbladder cells present 8,000 sites/cell of high affinity (Kd = 1.16 X 10(-9) M) and 42,000 sites/cell of low affinity (Kd = 3.3 X 10(-8) M). In addition, in the same cellular preparation at 37 degrees C, insulin at a concentration of 10(-8) M significantly stimulated (p less than 0.01) 3H-leucine incorporation into acid-insoluble fraction by 1.4 +/- 0.1 fold at 30 min. A maximal effect is obtained at 10(-7) M insulin (2.11 +/- 0.06 fold). Our results suggest a stimulatory effect of insulin or related "insulin-like" peptides on growth in human gut.
Asunto(s)
Vesícula Biliar/citología , Insulina/metabolismo , Receptor de Insulina/metabolismo , Aminoácidos/metabolismo , Cicloheximida/farmacología , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Masculino , Proteínas/metabolismoRESUMEN
As an approach for characterizing the molecules involved in the proliferation and differentiation of hematopoietic stem cells we have compared the ability of four murine stromal cell lines, MS-5, MS-K, both derived from Dexter cultures, BMS1 and BMS2 both derived from Whitlock-Witte cultures, to sustain murine long term hematopoiesis and to express the major hematopoietic cytokine genes. As opposed to the three other cell lines, MS-5 supports the maintenance of stem cells for up to 4-5 weeks. However, reconstituting stem cell output was reduced while clonogenic cell (day 12 and day 8 spleen colony-forming units, granulo-macrophagic, and erythroid progenitor cells) output was markedly increased. This hematopoietic-promoting activity is at least in part mediated by soluble molecules since medium conditioned with MS-5 cells was able to partially complement the nonsupportive cell line BMS1. The comparative study of the cytokine gene expression in MS-5 and in the nonsupportive cell lines included Northern blot and reverse transcriptase-polymerase chain reaction analysis of messenger RNA for interleukin-1, -3, -6, granulo-macrophage-colony-stimulating factor (GM-CSF), granulocyte-CSF, macrophage-CSF, stem cell factor, transforming growth factor-beta, tumor necrosis factor-alpha, macrophage inflammatory protein-1 alpha, and leukemia inhibitory factor. None of these molecules or their association were found to clearly confer to the MS-5 cell line its hematopoietic-promoting activity raising the possibility that uncharacterized molecule(s) would be involved in the proliferation and differentiation of stem cells.
Asunto(s)
Citocinas/metabolismo , Hematopoyesis/fisiología , Células del Estroma/metabolismo , Animales , Secuencia de Bases , Línea Celular , Medios de Cultivo Condicionados/farmacología , Sustancias de Crecimiento/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Ratones , Datos de Secuencia Molecular , Células del Estroma/fisiologíaRESUMEN
CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. This study compared the expansion capacities of CD133(+) and CD34(+) cells isolated from the same cord blood (CB) samples. After 14 days culture in stroma-free, serum-free medium in the presence of stem cell factor (SCF), Flt3-1, megakaryocyte growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF), the CD133(+) and CD34(+) fractions displayed comparable expansion of the myeloid compartment (CFC, LTC-IC, and E-LTC-IC). The expansion of CD133(+) CB cells was up to 1262-fold for total cells, 99-fold for CD34(+) cells, 109-fold for CD34(+) CD133(+) cells, 133-fold for CFU-GM, 14.5-fold for LTC-IC, and 7.5-fold for E-LTC-IC. Moreover, the expanded population was able to generate lymphoid B (CD19(+)), NK (CD56(+)), and T (CD4(+) CD8(+)) cells in liquid or fetal thymic organ cultures, while expression of the homing antigen CXCR4 was similar on expanded and nonexpanded CD133(+) or CD34(+) cells. Thus, the CD133(+) subset could be expanded in the same manner as the CD34(+) subset and conserved its multilineage capacity, which would support the relevance of CD133 for clinical hematopoietic selection.
Asunto(s)
Antígenos CD34/análisis , Antígenos de Superficie/análisis , Diferenciación Celular/fisiología , Sangre Fetal/citología , Glicoproteínas/análisis , Células Madre Hematopoyéticas/citología , Péptidos/análisis , Antígeno AC133 , Antígenos CD , Linfocitos B/citología , Linfocitos B/inmunología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , División Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Humanos , Separación Inmunomagnética/métodos , Recién Nacido , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas/farmacología , Proteínas Tirosina Quinasas Receptoras/farmacología , Proteínas Recombinantes/farmacología , Factor de Células Madre/farmacología , Linfocitos T/citología , Linfocitos T/inmunología , Trombopoyetina/farmacología , Timo/embriología , Timo/inmunología , Tirosina Quinasa 3 Similar a fmsRESUMEN
The present study was conducted to establish defined culture conditions for ex vivo expansion of normal human bone marrow cells. We investigated the role of three experimental expansion parameters: the cell concentration in the initial culture medium, the role of animal serum, human plasma and serum-free substitute, and the expansion potential of mononucleated cells (MNC) versus CD34+ cells. Cells were cultured in suspension with stem cell factor (SCF), IL3, IL6 and Erythropoietin (Epo) for 10 days. 1) Reducing the cell concentration from 3 x 10(4) to 1.5 x 10(3)/ml increased total cell expansion almost 20 fold, progenitor expansion more than 3 fold, and the maintenance of long term culture-initiating cells (LTC-IC). 2) In medium containing a serum-free substitute, total and CD34+ cell expansion was 3 times greater than in medium containing 1-10% human AB plasma or 25% animal serum. 3) The expansion potential of selected CD34+ cells was significantly greater than that of the total MNC population. However, taking into account the cell loss due to CD34+ selection, the overall results for quantitative expansion in relation to the initial number of MNCS favor the use of non-selected MNCS. 4) SCF + IL3 + IL6 was clearly the best combination of early cytokines for LTC-IC maintenance, with or without lineage-restricted cytokines, whereas the presence of IL1 beta in any combination augmented the decrease in LTC-IC. Addition of G-CSF to the medium resulted in 1 log increase in total cell expansion and a 2-fold increase in CFU-GM expansion. Addition of Epo always induced a dramatic proliferation of erythroid cells (up to 2000 fold) as well as of CFU-GM (up to 4 fold), without exhausting the LTC-IC pool. We concluded that the expansion of hemopoietic cells for clinical purposes needs establishment of controlled, reproducible and reliable culture conditions.
Asunto(s)
Antígenos CD34/metabolismo , Células de la Médula Ósea , Médula Ósea/metabolismo , Técnicas de Cultivo de Célula/métodos , Animales , Antígenos CD34/fisiología , Bovinos , Recuento de Células , División Celular , Medios de Cultivo/normas , Medio de Cultivo Libre de Suero/farmacología , Sangre Fetal/fisiología , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/citología , Humanos , Células del Estroma/citologíaRESUMEN
We have previously defined the experimental conditions for hematopoietic cell expansion. CD34+ human marrow cells were maintained in a serum-free, stroma-free liquid culture system, at a concentration of 10(3) cells/ml, for 10 days at 37 degrees C, in the presence of various cytokine combinations. The basic combination of early cytokines SCF (100 ng/ml), IL3 (5 ng/ml), IL6 (10 ng/ml), has a modest stimulating effect on all compartments: the number of total cells increased 56-fold and CD34+ cells 1-fold; CFU-GM, BFU-E and CFU-MK, increased 6-fold, 5-fold and 3-fold respectively. As far as CD34+ cells are concerned, the subpopulation CD34+/CD38- was only maintained. Interestingly, the addition of 100 ng/ml of Flt3 ligand (FL) significantly enhanced the amplification of total cells (276-fold), CFU-GM (54-fold) and BFU-E (15-fold). The number of CD34+ cells and the subpopulation CD34+/38- increased to 7-fold and 22-fold respectively. Moreover, long term culture-initiating cells (LTC-ICs) in limiting dilution assay (LDA) were found to increase 3-fold. Further addition of MGDF (10 ng/ml), G-CSF (10 ng/ml) and Epo (0.5 U/ml), in various combinations, acted synergically with the previous cytokine combination to support the formation of multiple types of hematopoietic colonies. As expected, the addition of MGDF increased the number of CFU-MK up to 5-fold expansion. Interestingly, MGDF addition was synergistic also for BFU-E and CFU-GM expansion. In the combination of SCF+ IL3+ IL6+ FL + MGDF, CFU-GM expanded to 73-fold and BFU-E to 17-fold. G-CSF in SCF + IL3 + IL6 + FL conditions stressed the expansion of the granulopoietic compartment doubling the number of CFU-GM and CD33+ cells, with no consequence on LTC-IC or BFU-E. Surprisingly, G-CSF induced the expansion of the megakaryocytic lineage up to 6-fold, in a similar way as MGDF. Epo in presence of SCF+ IL3+ IL6+/-FL dramatically increased total cell expansion (2300-2800-fold), mainly erythroblastic (70% glycoA) without exhaustion of all other compartments. The simultaneous use of these three cytokines (MGDF + G-CSF + Epo) in presence of four early cytokines (SCF + IL3 + IL6 + FL) clearly allows a significant expansion of all hematopoietic compartments, precursors, progenitors, and primitive stem cells. In conclusion, these data show the ability of a stroma-free, serum-free liquid system to expand all myeloid lineages, including CFU-MK and LTC-IC which are critical for clinical application of ex vivo expanded cells.
Asunto(s)
Células Madre Hematopoyéticas/citología , Proteínas de la Membrana/fisiología , Ensayo de Unidades Formadoras de Colonias , Citocinas/administración & dosificación , Sinergismo Farmacológico , Hematopoyesis/efectos de los fármacos , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Técnicas In Vitro , Proteínas de la Membrana/administración & dosificaciónRESUMEN
Hematopoiesis depends on a pool of quiescent hematopoietic stem/progenitor cells. When exposed to specific cytokines, a portion of these cells enters the cell cycle to generate an amplified progeny. Myeloblastin (MBN) initially was described as involved in proliferation of human leukemia cells. The granulocyte colony-stimulating factor (G-CSF), which stimulates the proliferation of granulocytic precursors, up-regulates MBN expression. Here we show that constitutive overexpression of MBN confers factor-independent growth to murine bone marrow-derived Ba/F3/G-CSFR cells. Our results point to MBN as a G-CSF responsive gene critical to factor-independent growth and indicate that expression of the G-CSF receptor is a prerequisite to this process. A 91-bp MBN promoter region containing PU.1, C/EBP, and c-Myb binding sites is responsive to G-CSF treatment. Although PU.1, C/EBP, and c-Myb transcription factors all were critical for expression of MBN, its up-regulation by G-CSF was associated mainly with PU.1. These findings suggest that MBN is an important target of PU.1 and a key protease for factor-independent growth of hematopoietic cells.