RESUMO
OBJECTIVES: The first-passage adherent human bone marrow fibroblast-like cell population corresponds, in terms of phenotype and three-lineage differentiation capacity (assayed in bulk culture), to commonly termed "mesenchymal stem cells". Here we determine the proportion of high proliferative capacity multipotent cells present in this population in order to estimate the proportion of cells that can or cannot be considered as stem and progenitor cells. MATERIAL AND METHODS: The single-cell cultures were established starting from human bone marrow-derived first-passage fibroblast-like cells and the proliferating clones were either transferred to secondary cultures to evaluate their further clonogenicity, or split into three wells to assess differentiation into each of the three different lineages. RESULTS: The analysis of 197 single-cell cultures from three different bone marrow donors shows that onlyâ¼40% of so-called "mesenchymal stem cells" exhibit multipotency and are capable of sustained clonogenicity in secondary cultures. CONCLUSION: Even in the first ex vivo passage under favorable conditions the majority (â¼60%) of so-called "mesenchymal stem cells" are not multipotent and thus do not represent a stem cell entity.
Assuntos
Células-Tronco Mesenquimais/citologia , Antígenos CD/análise , Células da Medula Óssea/classificação , Adesão Celular , Divisão Celular , Linhagem da Célula , Autorrenovação Celular , Separação Celular , Células Cultivadas , Células Clonais/citologia , Ensaio de Unidades Formadoras de Colônias , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Análise de Célula Única , Células Estromais/citologiaRESUMO
OBJECTIVES: Since no further progress was achieved, in order to improve the long-term organ transplantation outcome, the immune tolerance appears as an interesting therapeutic goal. Dendritic cells (DCs) are specialized cells participating in the homeostasis of the immune response. Moreover, subsets of DCs, identified in humans, appear to have their respective competences in immune response modulation. Our objective is to purify from PBMC or to differentiate DC subsets from monocytes using several strategies and evaluate their IL10 secretion. METHODS: CD14+ cells were purified from peripheral blood mononuclear cell (PBMC) by affinity beads and cultured with cytokines up to 7 days. The pDCs were purified with anti-BDCA-2 beads from PBMC fraction enriched by Percoll® gradient. The moDCs, pDCs and moLCs subsets were analyzed by phenotype labelling and FACS analyses and IL10 secretion measured by ELISA. RESULTS: The moDCs were characterized by the CD209 expression and a lower expression of CD1a markers. Expression of CD207 and CD1a markers characterized moLCs and CD123+/BDCA-2+ pDCs. Variable IL-10 secretions were shown between the three DC subsets, both at basal and activated levels. CONCLUSIONS: As the several DC populations studied have different capacities of IL-10 synthesis, they might play, among others, distinct roles in the induction of immune tolerance.
Assuntos
Células Dendríticas/imunologia , Tolerância Imunológica , Adulto , Antígenos CD/análise , Moléculas de Adesão Celular/análise , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Citaferese , Células Dendríticas/classificação , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Separação Imunomagnética/métodos , Interleucina-10/metabolismo , Interleucina-4/farmacologia , Lectinas Tipo C/análise , Monócitos/citologia , Receptores de Superfície Celular/análiseRESUMO
Studying hematopoietic and mesenchymal stem cells for almost three decades revealed some similarities between the stem cell entity and the single-celled eukaryotes exhibiting the anaerobic/facultative aerobic metabolic features. A careful analysis of nowadays knowledge concerning the early eukaryotic evolution allowed us to reveal some analogies between stem cells in the metazoan tissues and the single-celled eukaryotes which existed during the first phase of eukaryotes evolution in mid-Proterozoic era. In fact, it is possible to trace the principle of the self-renewal back to the first eukaryotic common ancestor, the first undifferentiated nucleated cell possessing the primitive, mostly anaerobically-respiring mitochondria and a capacity to reproduction by a simple cell division "à l'identique". Similarly, the diversification of these single-cell eukaryotes and acquiring of complex life cycle allowed/conditioned by the increase of O2 in atmosphere (and consequently in the water environment) represents a prototype for the phenomenon of commitment/differentiation. This point of view allowed to predict the ex-vivo behavior of stem cells with respect to the O2 availability and metabolic profile which enabled to conceive the successful protocols of stem cell expansion and ex vivo conditioning based on "respecting" this relationship between the anaerobiosis and stemness. In this review, the basic elements of this paradigm and a possible application in cell engineering were discussed.
Assuntos
Evolução Biológica , Engenharia Celular , Células Eucarióticas/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , Aerobiose , Anaerobiose , Animais , Atmosfera , Técnicas de Cultura de Células , Divisão Celular , Células Eucarióticas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Redes e Vias Metabólicas , Mitocôndrias/fisiologia , Origem da Vida , Oxigênio/metabolismoRESUMO
The maintenance of the primitive Hematopoietic Stem Cells (HSC) in course of ex vivo expansion is a critical point to preserve the long-term reconstituting capacity of a hematopoietic graft. On the basis of the numerous experimental results, the maintenance of primitive HSC is related to their specific metabolic profile shifted towards the anaerobiosis. Hence, in addition to the exposition of the cultures to more appropriate, physiologically low O2 concentrations (usually misleadingly termed "hypoxia"), a specter of "hypoxia-mimicking" factors (cytokines, growth factors, receptor-ligands, antioxidants) can be applied to maintain this specific metabolic profile enabling an appropriate genetic and epigenetic regulation. Some factors already proved to be able to achieve this goal and "hypoxia-mimicking" ex vivo cultures were already used to produce cells for clinical trials. In this article we are discussing the approaches aimed to amplify and/or to condition the HSC, based on the manipulation of energetic metabolism properties.
Assuntos
Metabolismo Energético , Células-Tronco Hematopoéticas/metabolismo , Anaerobiose , Hipóxia Celular , Autorrenovação Celular , Células Cultivadas , Meios de Cultura/farmacologia , Citocinas/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Mitocôndrias/fisiologia , Fosforilação Oxidativa , Oxigênio/metabolismo , Oxigênio/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
A severe bacterial leaf spot was observed during June and July 2013 on commercial cultivars of sugar beet (Beta vulgaris var. saccharifera) in the Vojvodina Province of Serbia. Serbia is a major sugar beet production area in southeastern Europe, with 62,895 ha and 3 million tons of sugar beet yield in 2013. A foliar leaf spot observed in 25 commercial sugar beet fields surveyed ranged from 0.1 to 40% severity. Symptoms were characterized as circular or irregular, 5- to 20-mm diameter, white to light brown necrotic spots, each with a dark margin. Diseased leaves were rinsed in sterilized, distilled water (SDW) and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in SDW. Isolations from 48 symptomatic leaves onto nutrient agar with 5% (w/v) sucrose (NAS) produced bacterial colonies that were whitish, circular, dome-shaped, and Levan-positive. Representative isolates (n = 105) were Gram negative; aerobic; positive for catalase, fluorescence on King's medium B, and tobacco hypersensitivity; and negative for oxidase, potato rot, and arginine dehydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (2). Repetitive extragenic palindromic sequence (rep)-PCR was used for genetic fingerprinting the isolates using the REP, ERIC, and BOX primers. Twenty-five different profiles were obtained among the strains. From each profile group, one representative strain was sequenced for the gyrB gene (1). Four heterogenic groups were observed, and representative gyrB gene sequences of each group were deposited in the NCBI GenBank (Accession Nos. KJ950024 to KJ950027). The sequences were compared with those of pathotype strain P. syringae pv. aptata CFBP 1617 deposited in the PAMDB database; one strain was 100% homologous, and the other three were 99% homologous. To fulfill identification of the Serbian sugar beet isolates, gltA and rpoD partial gene sequences were determined (1), and the sequences were deposited as Accession Nos. KM386838 to KM386841 for gltA and KM386830 to KM38683033 for rpoD. The sequences were 100% homologous with those of pathotype strain CFBP 1617. Pathogenicity of each of four representative bacterial strains was tested on 3-week-old plants of the sugar beet cultivars Marinela, Serenada, and Jasmina (KWS, Belgrade, Serbia) and Lara (NS Seme, Novi Sad, Serbia) by atomizing a bacterial suspension of ~106 CFU/ml of the appropriate isolate onto the abaxial leaf surface of three plants per cultivar until water-soaking of the leaf surface was observed. Three plants of each cultivar atomized similarly with P. syringae pv. aptata CFBP 2473 and SDW served as positive and negative control treatments, respectively. Inoculated plants were kept in a clear plastic box at 80 to 100% RH and 17 ± 1°C and examined for symptom development over 3 weeks. For all test isolates and the control strain, inoculated leaves first developed water-soaked lesions 7 days after inoculation (DAI). By 10 to 14 DAI, lesions were necrotic and infection had spread to the petioles. By 21 DAI, wilting was observed on more than 50% of inoculated plants. Negative control plants were symptomless. Bacteria re-isolated onto NAS from inoculated leaves had the same colony morphology, LOPAT results, and gyrB partial gene sequences as described for the test strains. No bacteria were re-isolated from negative control plants. Based on these tests, the pathogen causing leaf spot on sugar beet in Serbia was identified as P. syringae pv. aptata. References: (1) P. Ferrente and M. Scortichini. Plant Pathol. 59:954, 2010. (2) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966.
RESUMO
During the spring of 2014, a severe leaf spot disease was observed on carrot (Daucus carota), parsley (Petroselinum crispum), and parsnip (Pastinaca sativa) on a 0.5-ha vegetable farm in Vojvodina Province, Serbia. The disease appeared under wet and cool conditions with 5 to 25% of plants infected for each of the three crops. Symptoms were characterized as brown angular leaf spots, ~2 mm in diameter, often limited by veins. Collected symptomatic leaves were rinsed and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in sterile phosphate buffer and streaked onto nutrient agar with 5% (w/v) sucrose (NAS). After isolation, whitish, circular, dome-shaped, Levan-positive colonies consistently formed. Five strains from each host (carrot, parsley, and parsnip) were used for further study. Strains were gram-negative, aerobic, and positive for catalase and tobacco hypersensitive reaction but negative for oxidase, rot of potato slices, and arginine dihydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (3). Repetitive extragenic palindromic sequence (Rep)-PCR fingerprint profiles using the REP, ERIC, and BOX primers (4) were identical for all strains. Sequence typing of the housekeeping genes gyrB and rpoD (1) was performed for three representative strains (one from each host). Sequences were deposited in the NCBI GenBank database as accessions KM979434 to KM979436 (strains from carrot, parsnip, and parsley, respectively) for the gyrB gene and KM979437 to KM979439 (strains from parsnip, parsley and carrot, respectively) for the rpoD gene. Sequences were compared with pathotype strain Pseudomonas syringae pv. coriandricola ICMP12471 deposited in the Plant Associated and Environmental Microbes Database ( http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl ). BLAST analysis revealed 100% homology for gyrB and 99% homology for rpoD. Pathogenicity was tested with five representative strains from each host on four-week-old plants of carrot (cv. Nantes), parsley (cv. NS Molski), and parsnip (cv. Dugi beli glatki) using two methods: spraying the bacterial suspension (108 CFU ml-1) on the leaves until runoff (5) and injecting the bacterial suspension into leaves with a hypodermic syringe (2). Four plants were used per strain and method. Sterile distilled water was applied as a negative control treatment for each plant species. All plants were kept in a mist room with 100% humidity for 4 h, then transferred to a greenhouse at 25°C and 80% relative humidity and examined for symptom development over a period of three weeks. For all strains, inoculated leaves first developed water-soaked lesions on the leaves 5 to 7 days after inoculation (DAI); 14 DAI lesions became dark brown, often surrounded by haloes. No symptoms were observed on control plants inoculated with sterile distilled water. For fulfillment of Koch's postulates, re-isolations were done onto NAS. Re-isolated bacteria were obtained from each inoculated host and confirmed to be identical to the original isolates using the LOPAT tests and Rep-PCR fingerprinting profiles. Based on the pathogenicity test accompanied by completion of Koch's postulates, sequence analysis, and bacteriological tests, the strains were identified as P. s. pv. coriandricola. To our knowledge, this is the first report of bacterial leaf spot of carrot, parsley, and parsnip in Serbia. It may present a threat to production due to quality requirements for fresh market. References: (1) P. Ferrente and M. Scortichini. Plant Pathol. 59:954, 2010. (2) M. Gupta et al. Plant Dis. 97:418, 2013. (3) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (4) F. J. Louws et al. Appl. Environ. Microb. 60:2286, 1994. (5) X. Xu and S. A. Miller. Plant Dis. 97:988, 2013.
RESUMO
In September 2012, soft rot symptoms on broccoli (Brassica oleracea L. var. italica Plenck) were observed in several commercial fields in the western part of Serbia. Following the first harvest, water-soaked areas developed on broccoli stem tissue and progressed into soft rot decay of entire plants. The incidence of disease was approximately 30%. In Serbia, broccoli is grown on smaller fields compared to other vegetables, but its production and consumption increased significantly in recent years. From the diseased tissue, shiny, grayish white, round colonies were isolated on nutrient agar. Six non-fluorescent, gram-negative, facultative anaerobic, oxidase-negative, and catalase-positive bacterial strains were chosen for further identification. All strains caused soft rot on potato and carrot slices and did not induce hypersensitive reaction on tobacco leaves. They grew at 37°C and in yeast salts broth medium containing 5% NaCl (2), did not produce acid from α-methyl glucoside, but utilized lactose and trehalose, and did not produce indole or lecitinase. Investigated strains formed light red, 1.5-mm-diameter colonies on Logan's medium (2), and did not produce blue pigmented indigoidine on glucose yeast calcium carbonate agar (2) nor "fried egg" colonies on potato dextrose agar. Based on biochemical and physiological characteristics (1) and ITS-PCR and ITS-RFLP analysis (4), the strains were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rRNA gene sequence from two strains (GenBank KC527051 and KC527052) showed 100% identity with sequences of P. carotovorum subsp. carotovorum previously deposited in GenBank (3). Pathogenicity of the strains was confirmed by inoculation of broccoli head tissue fragments. Three florets per strain were inoculated by pricking the petals with a syringe and hypodermic needle and depositing a droplet of bacterial suspension (approx. 1 × 108 CFU/ml) at the point of inoculation. Sterile distilled water was used as a negative control. Inoculated florets were placed in a sealed plastic container and incubated in high humidity conditions at 28°C. Tissue discoloration and soft rot developed around the inoculation point within 48 to 72 h. No symptoms developed on control florets. Identity of bacterial strains reisolated from inoculated plant tissues was confirmed by ITS-PCR using G1/L1 primers followed by digestion of PCR products with Rsa I restriction enzyme (4). In Serbia, P. carotovorum subsp. carotovorum has been isolated from potato, some vegetable crops, and ornamentals, but not from broccoli until now. References: (1) S. H. De Boer and A. Kelman. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (2) P. C. Fahy and A. C. Hayward. Page 337 in: Plant Bacterial Diseases: A Diagnostic Guide. P. C. Fahy and G. J. Persley eds. Academic Press, New York, 1983. (3) S. Nabhan et al. J. Appl. Microbiol. 113: 904, 2012. (4) I. K. Toth et. al. Appl. Environ. Microbiol. 67:4070, 2001.
RESUMO
In late summer 2011, shallow, irregular cankers were observed on trunks and branches of non-chemically-treated walnut trees (Juglans regia L.) on a 30-year-old orchard in the region of Fruska Gora (Vojvodina, Serbia). Disease incidence was ~80% and yield loss was ~50%. For pathogen isolation, small pieces (~5 mm diameter) of wood tissue collected at the edge of the cankers were macerated in sterile distilled water and streaked onto nutrient agar with 5% sucrose. Plates were then incubated at 28°C for 2 days. The prevalent bacterial colonies and those similar in appearance to Brenneria nigrifluens (Wilson et al.) Hauben et al. were purified on nutrient agar (NA). Eight gram-negative, oxidasenegative, catalase-positive strains, showing oxidative and fermentative metabolism, were selected for further characterization. To identify the bacteria on a molecular basis, we analyzed the 16S rDNA and gyr B gene sequences. The 16S rDNA partial sequences of analyzed strains were amplified using the primers P0 (5'-GAGAGTTTGATCCTGGCTCAG-3') and P6 (5'-CTACGGCTACCTTGTTACGA-3') (3). Additionally, the gyr B gene sequences were generated with primers GyrB-F (5'-MGGCGGYAAGTTCGATGACAAYTC-3') and GyrB-R (5'-TRATBKCAGTCARACCTTCRCGSGC-3') (2). All amplicons were purified using the QIAquick PCR purification kit (QIAGEN) according to the manufacturer's instructions and sequenced by Macrogen Inc. (Seoul, South Korea) using the same primers used for amplification. The sequences were edited using FinchTV v.1.4.0, assembled using the Clustal W program integrated into MEGA5 software (4), and deposited in NCBI GenBank under accessions JX484738 to 40 for the 16S rDNA gene and KC571240 to 47 for the gyr B gene. The 1,359-bp 16S rDNA sequences obtained for the eight strains were compared to the reference 16S rDNA sequences retrieved from GenBank. BLAST analysis revealed 100% homology of Serbian strains with sequences of B. nigrifluens (Z96095 and FJ611884). The gyr B gene sequences of our strains were 100% homologous to the sequences of B. nigrifluens deposited in GenBank (JF311612 to 15). Pathogenicity of all strains was confirmed on young fruits by infiltration of bacterial suspensions (108 CFU ml-1 from a 48 h NA culture) with syringe into the mesocarp of walnut fruits and by stem infiltration with syringes without needles into branch wounds (1). Inoculated fruits were incubated in plastic boxes for 8 days at 20°C, 80 to 100% RH, with a 12-h photoperiod. Inoculated plants were maintained for 3 months at 22 to 28°C with continuous light and at 70 to 80% RH in plastic tunnels. Inoculated fruits developed bark canker symptoms at the inoculation sites, which became necrotic and released a reddish brown exudate. Necrotic lesions were observed on inoculated branches. B. nigrifluens was reisolated from the margins of necrotic fruit and stem tissue. Physiological and biochemical tests showed that strains grew at 36°C and did not produce arginine dihydrolase, H2S, indole, nitrate, nor a fluorescent pigment on King's B medium. They did not induce a hypersensitive reaction on tobacco leaves and did not hydrolyse gelatin and starch. They produced acid without gas from glucose, inositol, sorbitol, arabinose, and sucrose, but not from maltose and lactose (1). Results of pathogenicity and biochemical tests were also the same for reisolated strains. This is the first report of B. nigrifluens as the causal agent of shallow-bark canker on walnut trees in Serbia. References: (1) E. G. Biosca and M. M. López. J. Plant Pathol. 94:105, 2012. (2) P. Ferrente and M. Scotrichini. Plant Pathol. 59:954, 2010. (3) A. Grifoni et al. FEMS Microbiol. Lett. 127:85, 1995. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.
RESUMO
The most globally recognized and economically important nematode on wheat is the cereal cyst nematode (CCN) complex (1). One of the most important species of this group is Heterodera filipjevi (Madzidov, 1981) Mulvey and Golden, 1983. During regular soil quarantine control in September 2010, Heterodera sp. cysts were found in soil samples originating from a wheat field in Gunaros, Vojvodina Province, in northern Serbia. The wheat was a winter crop grown in a dryland production system and had an average cyst density of 2.50/100 cm3 of soil. Morphologically, the cysts were golden brown and lemon shaped with a posterior protuberance. The vulval cone was bifenestrate with horseshoe-shaped semifenestra, bullae, and underbridge. Cyst measurements (n = 30) ranged as follows: cyst length (without neck): 511.50 to 899.00 µm, cyst width: 201.50 to 682.00 µm, fenestral length: 44.80 to 65.60 µm, fenestral width: 24.00 to 40.00 µm, vulval bridge length: 12.80 to 20.80 µm, vulval bridge width: 6.40 to 14.40 µm, vulval slit: 6.00 to 12.80 µm, and underbridge length: 60.00 to 112.00 µm. The second-stage juveniles had an offset head, stylet with characteristic anchor-shaped basal knobs, four incisures, and a conical tail with a rounded tip. The J2 morphometrics (n = 30) were: length: 447.30 to 611.10 µm, width: 22.40 to 25.60 µm, stylet: 20.80 to 24.00 µm, tail length: 56.00 to 68.80 µm, tail width: 14.40 to 19.20 µm, and hyaline length: 35.20 to 44.80 µm. The ITS region was used for molecular analysis. Each DNA sample was extracted from a single cyst. Sequencing was done with primers TW81 and AB28 (2). In comparison with other H. filipjevi populations, the obtained sequence (GenBank Accession No. JX235959) revealed 99 to 100% similarity. Morphological and molecular data confirmed the existence of H. filipjevi. This is, to our knowledge, the first report of H. filipjevi from Serbia. Since wheat has important socioeconomic value for Serbia, after extensive surveys, additional phytosanitary measures may be necessary to prevent the spread of this parasite. References: (1) J. M. Nicol et al. Current Nematode Threats to World Agriculture. Genomics and Molecular Genetics of Plant-Nematode Interactions, Springer, New York, 2011. (2) A. M. Skantar et al. J. Nematol. 39:133, 2007.
RESUMO
High-dose chemotherapy with stem cells support has largely improved in terms of hematopoietic stem and progenitor cells harvest procedures as well as in those, which target or manipulate the cellular composition of autologous graft. Optimal preparative regimens and supportive care had lead to better use of autologous transplantation procedure. For other patients assigned to hematopoietic transplantation, availability of allogeneic donors appears to be an interesting alternative source of hematopoietic stem cells. Since three decades, hematopoietic growth factors development has allowed mobilization optimization and collection of peripheral hematopoietic stem cells leading to reduced days of hospitalization and less blood products requirements, being more cost-effective for patients in autologous transplantation settings and for stem cell collection facilities in allogeneic ones. New perspectives include, besides ex vivo manipulation of graft, development of mobilizing drugs in order to perform transplantation even in poor mobilizers patients. An important goal is achieved with the description of genetic polymorphisms related to optimal mobilization of stem cells. New approach using more promising and selective agents called chemokines, such as plerixafor the main leader among these agents are now available and appear complementary for alternative approach using cytokines alone (G-CSF, GM-CSF, SCF). The aim of this review is to assess the evolution of theses biotechnologies and their role in different steps of autologous transplantation and allogeneic stem cells collection.
Assuntos
Mobilização de Células-Tronco Hematopoéticas , Benzilaminas , Remoção de Componentes Sanguíneos , Moléculas de Adesão Celular/antagonistas & inibidores , Quimiocinas/farmacologia , Ciclamos , Filgrastim , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Hormônio do Crescimento/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Mobilização de Células-Tronco Hematopoéticas/tendências , Transplante de Células-Tronco Hematopoéticas , Compostos Heterocíclicos/farmacologia , Humanos , Hormônio Paratireóideo/farmacologia , Polietilenoglicóis , Receptores de Quimiocinas/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/farmacologia , Transplante AutólogoRESUMO
Oxygen (O(2)) concentrations in bone marrow vary from 4% in capillaries to <0.1% in subendosteum, in which hematopoietic stem cells reside in specific niches. Culture at low O(2) concentrations (3, 1 and 0.1%) influences hematopoietic stem and progenitor cells survival, proliferation and differentiation, depending on their level of differentiation. Culture of human CD34(+) cells at low O(2) concentrations (O(2) ≤3%) maintains stem cell engraftment potential better than at 20% O(2) (NOD/Scid xenograft model). In contrast, progenitors disappear from cultures at/or <1% O(2) concentrations. A very low O(2) concentration (0.1%) induces CD34(+) quiescence in G(0). The exploration of molecules and mechanisms involved in hematopoietic stem and progenitor cells' quiescence and differentiation related to low O(2) concentrations is unfeasible with primary CD34(+) cells. Therefore, we performed it using murine hematopoietic nonleukemic factor-dependent cell Paterson (FDCP)-Mix progenitor cell line. The culture of the FDCP-Mix line at 0.1% O(2) induced in parallel G(0) quiescence and granulo-monocytic differentiation of most cells, whereas a minority of undifferentiated self-renewing cells remained in active cell cycle. Hypoxia also induced hypophosphorylation of pRb and increased the expression of p27(KIP1), the two proteins that have a major role in the control of G(0) and G(1) to S-phase transition.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células-Tronco Hematopoéticas/citologia , Oxigênio/metabolismo , Células-Tronco/citologia , Animais , Antígenos CD34/metabolismo , Diferenciação Celular , Hipóxia Celular , Fase G1 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Fosforilação , Receptores de Quimiocinas/metabolismo , Fase de Repouso do Ciclo Celular , Proteína do Retinoblastoma/metabolismo , Fase S , Células-Tronco/metabolismoRESUMO
A new discipline was born and grew up over the last 4 decades of 20th century: Experimental Hematology. In addition to yield the concept of Stemness, a paradigm later applied for the other tissues than hematopoietic one, it provided the results allowing a preclinical development and a therapeutic exploitation. The concept of ex vivo expansion of hematopoietic cells for transplantation is directly issued from this knowledge. It enabled us to realize that a critical quantity of different sub-populations of stem and progenitor cells are necessary to obtain a rapid and sustained hematopoietic reconstitution. These principles, transposed to human cells (originating from: bone marrow, peripheral blood, cord blood) required some important technological innovations (conception of the specific media, recombinant technology of cytokine production...), to achieve, after several attempts, the first efficient clinical trials (at the moment for cells mobilized in peripheral blood). This goal remains to be achieved for cord blood cells too. The developments in this field as well as its actual state are the subjects of this review.
Assuntos
Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Animais , Células Cultivadas , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Células-TroncoAssuntos
Doadores de Sangue , Infecções por HIV/genética , HIV-1/isolamento & purificação , Programas de Rastreamento/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Adulto , Infecções por HIV/diagnóstico , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Masculino , Reação Transfusional , Carga ViralRESUMO
The influence of lipoxygenase metabolites of arachidonic acid on proliferation and differentiation of CD34(+) cells was studied. Their effects on the CFU-GM and BFU-E progenitors were investigated by culture of CD34(+) cells in liquid or semisolid medium. Only 12-HETE (1 microM) stimulated the [(3)H]thymidine as well as BrdU incorporation and increased the number of cell divisions (PKH2 tracking). Addition of 12-HETE and 15-HETE but not of LXA(4), LXB(4), LTB(4), and LTC(4) to liquid cultures of CD34(+) cells for 3 and 8 days reduced in a time-dependent manner the number of CFU-GM and BFU-E. Both HETEs also increased the percentage of glycophorin A(+) cells while they reduced the percentage of CD34(-)/CD33(+) cells after 3 and 5 days of liquid cultures. These results show that HETE treatment stimulates proliferation and accelerates the differentiation of CD34(+) cells, mostly toward the erythroid lineage.
Assuntos
Antígenos CD34/análise , Ácidos Araquidônicos/farmacologia , Divisão Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Lipoxinas , Lipoxigenase/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/efeitos dos fármacos , Ácidos Araquidônicos/metabolismo , Contagem de Células , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura/farmacologia , Relação Dose-Resposta a Droga , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoetina/farmacologia , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/citologia , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacologia , Leucotrieno B4/farmacologia , Metilcelulose , Soluções , Timidina/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Serum alpha 1-antitrypsin (alpha 1AT) antigen concentration is elevated in malignancies as the result of acute phase reaction. In the present study, we examined whether the alpha 1AT elevation in monoclonal gammopathies was accompanied by an adequate increase of its functional activity. METHODS: In this case-control study, serum alpha 1AT concentration was measured in 187 ambulatory patients with monoclonal gammopathies and 320 healthy blood donors matched according to sex and age. The alpha 1AT antigen concentration was assayed by immunonephelometry, whereas its functional activity was measured as trypsin inhibitory capacity (TIC). The specific alpha 1AT inhibitory activity (SIA) was calculated, defined as the TIC/antigen concentration ratio. RESULTS: The alpha 1AT antigen concentrations obtained in the patients' samples were very significantly higher as compared with the corresponding values in the control group (mean +/- SD = 134 +/- 41.9% of normal, p < 0.001). However, the TIC values were higher in the patients than in the healthy controls only by 4% (104 +/- 23.8%, p < 0.05). The specific alpha 1AT activity was very significantly lower in the patients, as compared with the controls (p < 0.001), indicating that serum alpha 1AT in monoclonal gammopathies was partially inactive. CONCLUSIONS: As poor correlation between the TIC values and the antigen concentrations was obtained in the patient group as well as the decreased specific alpha 1AT activities, the TIC values in patients with monoclonal gammopathies should be interpreted with caution.
Assuntos
Paraproteinemias/sangue , alfa 1-Antitripsina/análise , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Concentração Osmolar , Valores de Referência , Tripsina/metabolismoRESUMO
The in vivo effects of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) administration on endogenous IL-1 levels in the circulation and conditioned media (CM) from different immunohematopoietic organ/tissues were studied in CBA mice under steady state and postirradiation conditions. In normal mice, constitutive IL-1 levels were demonstrated in the plasma, CM of peritoneal exudate cells and full-thickness skin explants with low or undetectable levels in CM of splenic and bone marrow cell suspensions. In irradiated mice (2 Gy, X rays) on day 3 post exposure a significant increase of IL-1 levels was seen in the circulation and CM of peritoneal exudate cells, with no significantly different levels in postirradiation bone marrow, spleen and skin. After rhIL-1Ra treatment of the animals (2 x 50 microg/mouse, i.p.), significantly elevated IL-1 levels were observed in the skin and CM of peritoneal exudate cells in normal mice, whereas slightly increased levels were detected in CM of splenic cells. The rhIL-1Ra administration in irradiated mice led to decreased IL-1 concentrations in the circulation, and CM of peritoneal exudate cells and skin. The results pointed out the importance of IL-1 secretion and receptor expression in the maintenance of homeostasis in steady state, as well as during recovery after irradiation. Modulatory effects of IL-1Ra on IL-1 production were dependent on basic endogenous IL-1 concentration.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/efeitos da radiação , Interleucina-1/sangue , Sialoglicoproteínas/farmacologia , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Meios de Cultivo Condicionados/química , Homeostase/efeitos dos fármacos , Homeostase/efeitos da radiação , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/análise , Masculino , Camundongos , Camundongos Endogâmicos CBA , Pele/citologia , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Baço/citologia , Baço/efeitos dos fármacos , Baço/efeitos da radiaçãoRESUMO
In liquid cultures of murine bone marrow cells stimulated with interleukin-3 and granulocyte/macrophage colony-stimulating factor, hypoxia (1% oxygen) induced a reversible block of hematopoiesis, maintaining the progenitors' expansion potential unreduced. Progenitors repopulating day-14 hypoxic cultures with cells or granulocyte/macrophage colony-forming units (CFU-GM) were found, on the basis of their maintenance in hypoxia (12% and 76%, respectively), to belong to different subsets, the latter being much more efficiently maintained. The maintenance in hypoxic cultures of progenitors detectable by marrow-repopulating ability (MRA) assay was 18% for MRAcell progenitors and 69% for MRACFU progenitors. Thus, the repopulation of hypoxic cultures with cells or CFU-GM closely reflected the presence of progenitors capable of repopulating, with cells or CFU-GM, the bone marrow of lethally irradiated syngeneic animals. Progenitors repopulating hypoxic cultures were, like MRA progenitors, significantly resistant to 5-fluorouracil, progenitors repopulating cultures with CFU-GM being two-fold more resistant than those repopulating cultures with cells. We concluded that the repopulation of day-14 hypoxic cultures occurring after their transfer to air is to be considered an indicator of the maintenance of MRA progenitors in hypoxia. The relevance of these results to stem cell biology and their potential practical applications are discussed.
Assuntos
Células da Medula Óssea/citologia , Hipóxia Celular , Animais , Antimetabólitos/farmacologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Resistência a Medicamentos , Fluoruracila/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos CBA , Quimera por RadiaçãoRESUMO
We developed previously a hypoxic culture system in which progenitors endowed with marrow-repopulating ability (MRA), unlike committed progenitors, were selected and maintained better than in air. We report here an improvement to this system targeted at combining the maintenance of progenitors sustaining MRA with the numerical expansion of multipotent and committed progenitors. Murine bone marrow cells were incubated at 1% oxygen in liquid medium supplemented with stem cell factor, granulocyte colony-stimulating factor, interleukin-6 and interleukin-3. In day 8 hypoxic cultures, the numbers of high proliferative potential and granulocyte/macrophage colony-forming cells (HPP-CFC and CFU-GM) were increased with respect to time zero. Colonies generated by HPP-CFC derived from hypoxic cultures exhibited a high replating ability, whereas colonies generated by HPP-CFC derived from control cultures exhibited a low replating ability. MRA was fully maintained in hypoxia and markedly reduced in air. Thus, severe hypoxia is able to ensure a full maintenance of progenitors sustaining MRA, together with a significant expansion of in vitro-detectable clonogenic progenitors, including those endowed with replating ability. This system could contribute to the improvement of current techniques for the in vitro treatment of human haematopoietic cell populations before transplantation.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Hematopoéticas/citologia , Oxigênio , Animais , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos CBARESUMO
BACKGROUND: The liquid culture of murine bone marrow cells at 1-percent oxygen maintains the balance between primative progenitor cell renewal and clonogenic progenitor expansion better than that at 20-percent oxygen. These results are of potential interest for the ex vivo expansion of human progenitor cells, as low O(2) tension could preserve the engraftment potential of cultured apheresis products. STUDY DESIGN AND METHODS: G-CSF-mobilized blood cells collected by apheresis, now the main source of progenitor cells for autologous transplantation, were cultured at 1-percent and 20-percent O(2) for 7 days in serum-free liquid cultures in the presence of IL-3 and SCF (5 ng/mL). The growth of the clonogenic progenitors (CFU-GM, BFU-E, CFU-Mix) and of the more primitive human HPCs that are capable of generating clongenic progenitors in secondary liquid culture, as well as the proliferation and differentiation of total and CD34+ cells, was analyzed. RESULTS: The expansion of CD34+ cells and of clonogenic progenitors was significantly lower in liquid cultures at 1-percent O(2) than at 20-percent O(2). On the contrary, the primitive human HPCs were better maintained and expanded at 1-percent O(2), although the number of CD34+ cells remaining quiescent was lower. After 7 days of liquid culture at 1-percent or 20-percent O(2) the percentage of CD34+ cells was similar. However, the CD34+ cells that divided more than four times (PKH2 staining) were more numerous in liquid cultures incubated at 1-percent O(2). CONCLUSION: When cultured at 1-percent O(2) for 7 days in presence of IL-3 and SCF, the CD34+ cells present in apheresis components underwent more cell divisions and better maintained their primitive progenitor cell potential. As suggested by previous results in mice, our data on human cells emphasize the potential interest of cultures at low O(2) tension (1%) for cell therapy protocols aimed at expanding primitive HPCs in autografts.