RESUMO
We recently reported that in stroma-free cultures 11-33% of clonogenic cells derived from a bulk long-term culture [long-term culture-clonogenic cells (LTC-CC)] could be transduced by supernatant exposure or coculture of human CD34+ progenitors with MDR retroviral producer line A12M1. We reasoned that a stromal cell layer may generate niches in which LTC-CC could enter in the S-phase, thus becoming a more accessible target for gene delivery. In static culture studies in flasks, human engineered stromal cell line L87/4 or stromal murine M2-10B4 cells were used as feeder after irradiation, and CD34+ cells from either cord blood or peripheral blood of mobilized cancer patients were exposed to MDR supernatant for 7 consecutive days before 5-week culture for LTC-CC evaluation. In continuous flow perfusion culture studies, CD34+ cells were seeded over irradiated stromal murine M2-1OB4 cells and exposed to MDR supernatant for 7 days before LTC-CC evaluation. In mock-transduced controls, <5% of LTC-CC were found to he viable after exposure to 10 ng/ml Taxol. In cells exposed to MDR supernatant in static stroma cultures, 68 +/- 4% of seeded LTC-CC were found to be drug resistant and express MDR mRNA as evaluated by reverse transcription-PCR analysis of single colonies. The addition of cytokines did not further enhance transfer efficiency. After MDR retroviral exposure in continuous flow cultures, 88 +/- 5% of LTC-CC were found to be drug resistant (P < 0.01 versus static stroma culture). P-glycoprotein expression in CD34+ cells was evaluated using flow cytometry and found to he higher after continuous flow versus static cultures. Finally, very high levels of P-glycoprotein expression after MDR supernatant exposure in the presence of stroma were confirmed by APAAP staining of cultured cells. We conclude that engineered stromal cell layers and continuous flow culture conditions can significantly enhance retroviral-mediated gene transfer into human hematopoietic progenitor cells.
Assuntos
Resistência a Múltiplos Medicamentos , Técnicas de Transferência de Genes , Células-Tronco Hematopoéticas/citologia , Transfecção/métodos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Antígenos CD34/genética , Células Cultivadas , Expressão Gênica , Humanos , Camundongos , RNA Mensageiro/genética , Retroviridae/genética , ReologiaRESUMO
Bone marrow and/or peripheral blood of patients with chronic myeloid leukemia (CML) was investigated by the following three parameters: Ph' chromosome, bcr-abl expression in fresh blood and/or bone marrow, and bcr-abl expression in single hematopoietic progenitor colonies generated from blood and/or bone marrow. Expression of bcr-abl was proven by a reverse "nested primer" polymerase chain reaction (PCR) that is able to detect 1 pg of hybrid mRNA. We performed 108 investigations on 68 patients containing all three parameters: 12 on untreated patients, seven after interferon-alpha (IFN-alpha), seven after low-dose cytosine arabinoside (Ara-C), 22 after cyclic high-dose hydroxyurea (HU), 49 after allogeneic BMT, five before and three after stem cell mobilization, and three after autologous stem cell transplantation (ASCT). In 53 cases (49%), cytogenetics and PCR gave identical results. In 40 cases (37%), PCR from single colonies gave additional information compared to cytogenetics (e.g., mosaic in colonies when all metaphases were positive or negative). Most interesting were the results of one patient after IFN, one patient after ASCT, and 10 patients after BMT (14 investigations = 13%), showing only Ph'-negative mitoses accompanied by a negative nested primer PCR from fresh blood/bone marrow but single bcr-abl-positive progenitor colonies. False-positive results could be widely excluded by repeated insertion of negative controls into the experiments. One explanation for these results could be that CML, progenitors survive in the patient's body by being inactive and not proliferating. These cells express no or very little RNA and bcr-abl is not detectable by reverse PCR. When stimulated ex vivo in a colony assay by external growth factors, cells proliferate and produce detectable amounts of hybrid mRNA. The value of these observations is not clear. A follow-up of the patients will show if such sleeping progenitors can be activated in vivo. Concluding our observations, we can say that in special cases (therapy follow-up, detection of minimal residual disease) it could be useful to perform a PCR analysis of single progenitors in parallel with the routine investigations.
Assuntos
Proteínas de Fusão bcr-abl/genética , Células-Tronco Hematopoéticas/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Sequência de Bases , Transplante de Medula Óssea , Citarabina/uso terapêutico , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Humanos , Hidroxiureia/uso terapêutico , Interferon-alfa/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
Berardi et al. (Science 1995; 267:105) reported recently that combined cytokine stimulation and antimetabolite treatment were able to isolate cells with characteristics of hemopoietic stem cells. Bone marrow (BM) low-density cells were cultured for 1 week in the presence of Steel factor (SF), IL-3, and the antimetabolite 5-FU. Following this approach one in 10(5) BM cells were purified. These cells showed no clonogenic potential in soft gel assays but presented a striking myeloid-lymphoid potential as long-term culture-initiating cells (LTC-ICs). We investigated this new "stem cell candidate." following a similar approach we purified one in 55,000/130,000 cells from cord blood and peripheral blood in mobilized cancer patients. These cells displayed no clonogenic potential in methylcellulose assays in the presence of different cytokine combinations and generated very few or no clonogenic progenitors in liquid cultures in the presence of cytokines. When seeded on layers derived from the murine BM stromal cell line M2-10B4, 38-86% of the cells purified using this approach generated multilineage progenitors acting as LTC-lCs. In a different series of studies, after 5-week culture on human BM stromal cell line L87/4 layers, cells were forced to selected lineage differentiation by culture in the presence of low concentrations of SF and high concentrations of lineage-specific cytokines such as Flt3-ligand (myeloid and pre-B cell differentiation), Tpo (megakaryocytic differentiation). IL-7, and IL-2 (pre-B and NK differentiation). After 12-day culture under these conditions, generation of myeloid, pre-B, megakaryocytic, and NK progenitors was assessed by immunohistochemistry, flow cytometry, and mRNA expression of CD7, 14, 19, 41b, S6, and 61. We conclude that this procedure for multilineage progenitor cell purification is simple and effective and could have major implications for gene transfer and stem cell transplantation.
Assuntos
Fluoruracila , Hematopoese , Células-Tronco Hematopoéticas/citologia , Interleucina-3 , Fator de Células-Tronco , Células Estromais/citologia , Linhagem da Célula , Separação Celular , Células Cultivadas , HumanosRESUMO
Human cord blood or bone marrow cells expressing the CD34 surface antigen include a population of pluripotent progenitors. We identified and isolated a subpopulation of cells coexpressing CD34 and c-kit, a transmembrane receptor with tyrosine kinase activity. Novel monoclonal antibodies (16A6, 14A3, 3D6) directed against the extracellular domain of c-kit were used for immunofluorescence labeling and sorting of low-density mononuclear cells (MNCs) from umbilical cord blood and bone marrow. The frequency of c-kit-labeled MNCs from cord blood (mean 5.0% +/- 2.1%, n = 16) was similar to that from adult bone marrow (mean 3.7% +/- 1.3%, n = 4). On average, 1.4% of CD34-positive cells were recorded in cord blood and 2.1% in bone marrow MNCs. Roughly 60% of CD34-positive cells coexpressed c-kit. The ability of CD34+/c-kit+ cells to form multilineage colonies (CFU-GEMM) was assayed after sorting with an antibody that did not show any significant effect on c-kit ligand (RL) or granulocyte/macrophage colony-stimulating factor (GM-CSF)-induced colony formation. For CD34+/c-kit+ cells, we found a 20- to 50-fold enrichment as against total MNCs, and a 2-fold enrichment if compared with the CD34+/c-kit-population. To study expression of c-kit in lymphocytic precursors, monoclonal anti-CD7 or anti-CD10 antibodies were used simultaneously. In contrast to CD34-expressing cells, however, no consistent double-labeled subpopulation of lymphocytic cells was detected. Furthermore, coexpression of CD38 (73% +/- 14%, n = 4) or CD33 (29% +/- 12%, n = 5) on a majority of c-kit-positive cells showed their lineage commitment to erythropoiesis and granulocytopoiesis.
Assuntos
Antígenos CD/análise , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas/análise , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Anticorpos Monoclonais , Antígenos CD34 , Antígenos de Diferenciação/análise , Antígenos de Diferenciação Mielomonocítica/análise , Linfócitos B/metabolismo , Células da Medula Óssea , Separação Celular , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Granulócitos/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Macrófagos/citologia , Glicoproteínas de Membrana , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-kit , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Linfócitos T/metabolismoRESUMO
We present a detailed analysis of cytokine expression patterns of the two permanent human bone marrow stromal cell lines, L87/4 and L88/5. These cell lines, previously established in our laboratory, are highly radiotolerant without cell detachment and support long-term cultures of CD(34+)-enriched human cord blood cells. RT-PCR analysis of 22 different cytokines or cytokine receptor mRNAs showed an almost identical expression pattern in the two stromal cell lines compared to primary human Dexter-type stroma. Since stromal feeder lines employed in long-term cultures usually are irradiated and grown in media containing corticosteroids, we analyzed the impact of irradiation and dexamethasone on cytokine production in the two cell lines by RT-PCR, Northern blot analysis, bioassays, and RIAs. By RT-PCR analysis, constitutive mRNA expression of c-kit, G-CSF, GM-CSF, IL-1 beta, IL-6, IL-7, IL-8, IL-11, Kit ligand (KL), LIF, M-CSF, MIP-1 alpha, TGF-beta, and TNF-alpha was demonstrated in both cell lines, with L87/4 a more potent cytokine producer than L88/5. Northern blot data showed an increase in mRNA levels for GM-CSF, IL-1 beta, and LIF by irradiation and IL-1 alpha treatment in both cell lines. IL-1 alpha-induced GM-CSF, IL-1 beta, IL-6, IL-11, and LIF mRNA levels were reduced by the addition of dexamethasone, whereas dexamethasone had no influence on the amounts of IL-1 alpha-induced G-CSF mRNA. L87/4 and, to a lower extent, L88/5 cells showed dexamethasone-dependent increases in KL mRNA, while KL mRNA levels were not stimulated by IL-1 alpha.
Assuntos
Medula Óssea/metabolismo , Citocinas/genética , Expressão Gênica , Células Estromais/metabolismo , Sequência de Bases , Northern Blotting , Medula Óssea/efeitos da radiação , Células da Medula Óssea , Linhagem Celular , Dexametasona/farmacologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Inibidores do Crescimento/genética , Humanos , Interleucina-1/genética , Interleucina-1/farmacologia , Interleucina-11/genética , Interleucina-6/genética , Fator Inibidor de Leucemia , Linfocinas/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , DNA Polimerase Dirigida por RNA , Células Estromais/efeitos da radiaçãoRESUMO
Chronic myelogenous leukemia is a clonal proliferative disorder of pluripotent hematopoietic stem cells. Cure may be achieved by myeloablative conditioning treatment and marrow transplantation. In addition, allogeneic marrow can exert a graft-versus-leukemia effect. The graft-versus-leukemia effect may be directed against leukemia-specific antigens or against antigens on all hematopoietic cells, or it can be part of a graft-versus-host reaction. We report an informative post-transplant course of a patient with yet another leukemia-specific effect. This patient was transplanted with marrow from his HLA-identical sister in an advanced phase of CML and developed acute and chronic GVHD. After a severe pneumonia a high proportion of his metaphases in the bone marrow were male and Philadelphia chromosome negative. Later all metaphases were again female and leukemic cells could not be detected by reverse transcriptase polymerase chain reaction analysis (RT-PCR) for BCR/ABL. This course indicates that normal hematopoietic stem cells may survive intensive chemotherapy, bone marrow transplantation and GVHD. They may be recruited from a dormant state into proliferation during severe infections. In contrast, CML may be eliminated by the graft-versus-host reaction that recognizes recruited cells and spares dormant cells.
Assuntos
Doença Enxerto-Hospedeiro/fisiopatologia , Células-Tronco Hematopoéticas/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/fisiopatologia , Adulto , Feminino , Humanos , Cariotipagem , Masculino , Indução de Remissão , Doadores de TecidosRESUMO
Human erythroid progenitor cells grown in a suspension culture system were used to study possible interactions between different guanine nucleotide-binding protein (G-protein)-coupled receptor-effector systems during normal cell differentiation. Agonist-stimulated adenylyl cyclase was not inhibited by any one of a panel of ligands (ADP, UTP, platelet-activating factor, thrombin, alpha2-adrenoceptor agonists, interleukin 8, lysophosphatidic acid) most of which are known, in other cells, to reduce cAMP formation by a Gi-mediated, pertussis toxin-sensitive mechanism. The first four of these ligands are also known to cause transient changes in intracellular [Ca2+] in erythroid cells. Rather than inhibiting, thrombin (but not ADP, UTP or PAF) specifically caused a fivefold increase in the maximum adenosine- or prostaglandin E1-stimulated cAMP formation, without any shift of the concentration/response curves. Thrombin did not enhance forskolin- and AlF4-stimulated cyclase activity and had only a marginal effect on isoprenaline-dependent stimulation. The effect of thrombin seemed to be unrelated to intracellular Ca2+ release but could be partially mimicked by phorbol ester (PMA)-induced stimulation of protein kinase C (PKC) and was inhibited by staurosporin or by inactivation of PKC after long-term incubation with PMA. The activity of thrombin was restricted to proliferating, colony-forming progenitor cells while proerythroblasts were completely unresponsive. Our results suggest that the interaction of thrombin with Gs-linked receptors requires phosphorylation of a target protein that is different from adenylyl cyclase, Gs or Gi but may be involved in the regulation of receptor desensitization.
Assuntos
Adenosina/farmacologia , Adenilil Ciclases/metabolismo , Células Precursoras Eritroides/metabolismo , Trombina/farmacologia , Cálcio/metabolismo , Divisão Celular , AMP Cíclico/biossíntese , Células Precursoras Eritroides/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Humanos , Proteína Quinase C/fisiologia , Transdução de SinaisRESUMO
Human erythroid progenitor cells were isolated from peripheral blood of healthy donors and amplified in a suspension culture system using recombinant growth factors (stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin) as well as conditioned medium from a human bone marrow stroma cell line to support cell proliferation. After 6-8 days of culture, the cell population consisted mainly of erythroid colony-forming cells (burst-forming units, BFU-Es and colony-forming units, CFU-Es). In these cells, we studied ligand-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and cAMP formation as the primary effector systems of guanine nucleotide-binding protein (G protein)-coupled receptors. The results confirmed the functional expression of receptors for adenosine (type A2B), prostaglandin E1 and isoprenaline (beta-adrenoceptor), all of which stimulated adenylyl cyclase, as well as for ADP (purinoceptor types P2T and P2U), platelet-activating factor and thrombin all of which caused a transient increase in [Ca2+]i. The efficacy of adenosine and prostaglandin E1 in stimulating cAMP formation was more than 5 times higher than that of isoprenaline, suggesting a low beta-adrenoceptor density. The response to adenosine and isoprenaline decreased by 80 and 55% respectively during maturation into the proerythroblast stage. Similarly, thapsigargin-sensitive intracellular Ca2+ stores and ligand-induced Ca2+ release declined by about 60% during the CFU-E-to-erythroblast transition. The overall functional expression pattern of G protein-coupled receptors differed from that in human erythroleukaemia cell lines or from that in platelets. Primary culture systems for nontransformed cells, such as the one presented here, thus will be indispensable for the study of the functional role of G protein-dependent signalling during haematopoiesis.
Assuntos
Células Precursoras Eritroides/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Adenilil Ciclases/metabolismo , Cálcio/metabolismo , Células Cultivadas , Humanos , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores Purinérgicos P2/fisiologia , Receptores de Trombina/fisiologiaRESUMO
BACKGROUND: CD34(-) stem cells are apparently the earliest progenitors of hematopoiesis and mesenchymal tissues. The majority of those progeny rests in the BM as fibroblast-like cells, but can also circulate the peripheral blood. Nevertheless, CD34(-), fibroblast-like cells can be isolated from BM aspirates and PBMC, mediated by their ability to adhere to the plastic surface of tissue culture flasks. In standard colony assays, CD34(-), fibroblast-like cells produce a significant number of colony-forming-units (CFUs), mainly CFU-F (fibroblast). METHODS: Despite advanced cell-culture techniques and the application of various growth factors, the life span of those multipotent stem cells is limited. Therefore, we immortalized and cloned fibroblast-like, CD34(-) stem cells and used retroviral constructs containing the green-fluorescence protein (GFP) to determine the gene-transfer efficiency and their use for gene marking prior to transplantation into NOD/SCID mice. RESULTS: We could demonstrate a highly efficient retroviral gene transfer into those immortalized CD34(-), fibroblast-like hematopoietic cells (up to 95% transduced cells), maintaining their ability to produce CFUs, as well as a distinct organ distribution after transplantation into the recipient animals, functioning as SCID-repopulating cells (SRC). Transplanted cells could be detected in the BM, as well as other parenchymal organs, such as the lung, liver, skin, small intestine and brain. DISCUSSION: CD34(-), fibroblast-like progenitor cells can give rise to hematopoietic progeny, but also home to mesenchymal organ sites in recipient animals. There is increasing evidence that pluripotent CD34(-) stem cells can be isolated from various sources and still maintain their capabilities to generate progeny of different tissues. This could be a promising approach to using peripheral-blood derived stem cells for cellreplacement therapy and tissue engineering.
Assuntos
Antígenos CD34/imunologia , Técnicas de Transferência de Genes/tendências , Vetores Genéticos/uso terapêutico , Sobrevivência de Enxerto/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/imunologia , Retroviridae/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Linhagem Celular Transformada , Cães , Feminino , Fibroblastos/citologia , Fibroblastos/imunologia , Proteínas de Fluorescência Verde , Células-Tronco Hematopoéticas/citologia , Indicadores e Reagentes , Proteínas Luminescentes , Pulmão/citologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Baço/citologia , Baço/imunologia , Transdução Genética/métodos , Vísceras/citologia , Vísceras/imunologiaRESUMO
Transcription from one of the two initiation sites, P1 and P2, of the dual human MYC promoter seems to be essential in all proliferating cells. To identify proteins and target structures for MYC regulation, a DNA region was analyzed that is critical for P2 promoter activity. Here, we show that a nuclear factor binds to a DNA element within P2, which is conserved perfectly between mouse and man and displays a striking homology to the E1a-inducible E2 promoter of adenovirus type 5 (Ad5). We demonstrate that the same transcription factor, defined recently as E2F, which plays an essential role in the activation of adenovirus early promoters and enhancers, also interacts as a dominant nuclear factor with the MYC promoter. The presence of an intact E2F binding site is required for basic expression and for trans-activation of the P2 promoter by E1a proteins. The human MYC promoter is the first cellular target described for E2F. The results suggest that expression of MYC might be regulated via modulation of E2F by cellular 'E1a-like' factors.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Transcrição , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cloranfenicol O-Acetiltransferase/metabolismo , Elementos Facilitadores Genéticos , Células HeLa , Humanos , Metilação , Dados de Sequência Molecular , Fatores de Transcrição NFI , Proteínas Nucleares , Mapeamento de Nucleotídeos , Sondas de Oligonucleotídeos , Plasmídeos , Transfecção , Proteína 1 de Ligação a Y-BoxRESUMO
Two human stromal cell lines were established previously from bone marrow-derived primary long-term cultures by immortalization using the SV40 large T antigen and cellular cloning. After irradiation, the fibroblast-like cell lines L87/4 and L88/5 support hematopoietic differentiation of allogeneic cord blood cells in vitro. The stromal cells do not express CD34 and CD50, but some adhesion molecules and integrins, such as CD44, CD54 and CD58. Their expression profiles on RNA and protein levels are suggestive of their osteogenic potency. The quality and quantity of osteocalcin and osteopontin protein expression depended on the culture conditions. Expression of the osteogenic markers increased over time in culture, especially in cells growing in clusters. The stromal cells also expressed collagens I and V, but did not show any expression of collagens II and III. The potentially osteoblastic stromal cells were transplanted into NOD/ SCID recipient mice by intravenous injection and were found in various mesenchymal organs up to 10 weeks after transplantation. Osteocalcin-positive human stromal cells could be detected in the bone marrow, thymus, liver, brain and gut of the recipient animals. In summary, there is evidence that human bone-marrow-derived stromal cells have to be considered mesenchymal progenitors, persistently expressing osteogenic markers in vitro and in vivo.
Assuntos
Diferenciação Celular , Linhagem Celular , Mesoderma/citologia , Células Estromais/citologia , Animais , Biomarcadores/análise , Colágeno/metabolismo , Humanos , Imuno-Histoquímica , Imunofenotipagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Especificidade de Órgãos , Osteocalcina/genética , Osteogênese , Osteopontina , Reação em Cadeia da Polimerase , Sialoglicoproteínas/genética , Células-Tronco/citologia , Células Estromais/químicaRESUMO
The hematopoietic system of vertebrates can be completely reconstituted with hematopoietic stem cells derived from the bone marrow, fetal liver, or cord blood, or even from peripheral-blood-derived cells. A cellular marker to identify those cells is the proteoglycan CD34, although we have shown that the earliest identifiable hematopoietic stem cell is a CD34(-) fibroblast-like cell which can differentiate into CD34(+) hematopoietic precursors. Peripheral blood mononuclear cells were isolated from the heparinized blood of a dog and incubated in tissue culture in the presence of interleukin 6. After 10-14 days, an adherent layer of fibroblast-like cells had developed and cells were immortalized using the SV-40 large T antigen. Cells were cloned and subcloned by measures of limiting dilution, and various fibroblast-like clones were established. These fibroblast-like cells either do not express the CD34 antigen or express CD34 on a low level, although transcribing CD34. The CD34(-/low) cells express osteocalcin as a mesenchymal cell marker. The fibroblast-like cells eventually differentiate spontaneously in vitro into CD34(+) precursors and show colony formation. Prior to autologous stem cell transplantation, one clone of choice (IIIG7) was transfected with a retroviral construct containing the green-fluorescence protein (GFP). The recipient dog was totally irradiated with 300 cGy and received a stem cell transplant with GFP-containing, immortalized, fibroblast-like monoclonal autologous stem cells (0.5 x 10(8)/kg dog). No additional growth factors were applied. The peripheral blood counts recovered after 23 days (WBC >500; platelets >10,000). A peripheral blood smear showed some dim but definite, although timely, limited expression of the GFP protein in nucleated peripheral blood cells just five weeks after transplantation. A bone marrow biopsy showed GFP-positive cells in the marrow cavity predominantly as "bone-lining cells."
Assuntos
Antígenos CD34 , Células-Tronco Hematopoéticas/fisiologia , Animais , Antígenos CD34/genética , Células Cultivadas , Células Clonais , Cães , Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Leucócitos Mononucleares/imunologia , Mesoderma , Osteocalcina/biossíntese , RNA Mensageiro , Transcrição GênicaRESUMO
We describe the establishment of two permanent Simian virus 40-transformed human stromal cell lines, designated L87/4 and L88/5, derived from the bone marrow of a hematologically normal male patient. Both cell lines show a fibroblastoid morphology and do not express hematopoietic cell markers. L87/4 but not L88/5 expresses the macrophage marker CD68. The most remarkable feature of these new stromal cell lines is their ability to persist as growth-arrested adherent feeder cells after ionizing-irradiation at doses up to, and exceeding 20 Gy (L87/4). This renders them particularly useful for studying aspects of feeder dependence of hematopoietic cell development in long-term culture. Both cell lines are able to function as feeder cells, supporting the long-term proliferation of CD34+ human cord blood cells as well as the clonogenic growth of the human Burkitt lymphoma B-cell line BL70.
Assuntos
Células da Medula Óssea , Linhagem Celular , Sangue Fetal/citologia , Hematopoese , Humanos , Masculino , Tolerância a Radiação , Células Estromais/fisiologia , Células Estromais/efeitos da radiaçãoRESUMO
Previous work had revealed that a CD34- fibroblast-like cell is the earliest hematopoietic progenitor population. This cell type is able to differentiate into hematopoietic progeny of all lineages and circulates in the peripheral blood, from where it can be isolated by IL-6-mediated plastic adherence. We isolated peripheral blood-derived mononuclear cells (MNC) from male CBA mice and established in vitro a fibroblast-like, adherent growing cell layer. Cells were immortalized by SV-40 transfection for cellular cloning. Monoclonal fibroblast-like cell clones were established, and the surface expression of early stem cell markers was determined by flow cytometry. Clones were CD34-, Sca-1+, Thy-1(low), and c-kit+. Lethally irradiated female CBA mice were successfully transplanted with a fibroblast-like cell clone, R-M26/2-1. After syngeneic transplantation, peripheral blood counts were back to normal in transplanted mice on days 15-20, and fluorescence in situ hybridization (FISH) revealed the sole presence of male hematopoietic cells in the BM of female recipients at weeks 7, 9, 11, and 16 after transplantation. Immunohistochemistry for the expression of CD34, Sca-1, Thy-1, and c-kit showed the presence of the phenotype of the transplanted stem cell clone along the bone spicules in the marrow cavity, giving rise to HPC of all lineages. In summary, we have shown that a CD34-, Sca-1+, Thy-1(low), and c-kit+ fibroblast-like cell is consistent with the phenotype of the earliest hematopoietic and repopulating stem cell and can be isolated from peripheral blood cells.
Assuntos
Linhagem da Célula , Fibroblastos/patologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Animais , Antígenos CD34 , Antígenos Ly , Diferenciação Celular , Feminino , Fibroblastos/transplante , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos CBA , Proteínas Proto-Oncogênicas c-kit , Antígenos Thy-1 , Transplante IsogênicoRESUMO
The CD34-negative, adherent growing, fibroblast-like canine haematopoietic stem cell line D064 was recently identified as the earliest progenitor population in the bone marrow. D064 cells are predominately quiescent. Quiescence is mediated by the accumulation of the cyclin-dependent kinase inhibitor p27(kip-1)and in parallel, by the downregulation of Cyclin B, leading to an accumulation of quiescent cells in the G(0)/G(1)-phase of the cell cycle. Stem cell factor (SCF), the ligand for the tyrosine kinase receptor c-kit, usually induces differentiation of the CD34-negative stem cells into CD34-positive haematopoietic precursors. SCF also suppresses the expression of c-myc-dependent Cyclin E, which is not transcribed initially, but expression occurs later on. Interleukin 6 (IL-6) instead rather promotes proliferation, but fails to induce proliferation in the majority of CD34-negative stem cells due to no STAT activation in quiescent cells. Nevertheless, the potential of quiescent D064 cells to proliferate eventually, becomes apparent by the low-level expression of IL-6 dependent STAT factors. D064 cells also spontaneously start to express Bax, while Bcl-2 is downregulated in parallel. In summary, CD34-negative haematopoietic stem cells dwell in the marrow or other niches as quiescent cells, until they can respond to autocrine or paracrine growth factor-mediated signals.
Assuntos
Ciclina B/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Transativadores/metabolismo , Animais , Antígenos CD34/metabolismo , Células COS , Ciclo Celular , Proteínas de Ciclo Celular/biossíntese , Divisão Celular , Células Cultivadas , Cães , Regulação para Baixo , Células-Tronco Hematopoéticas/citologia , Humanos , Interleucina-6/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Fator de Transcrição STAT1 , Fator de Células-Tronco/fisiologia , Transcrição Gênica , Proteína X Associada a bcl-2RESUMO
The molecular basis and functional significance of interactions between haemopoietic progenitor cells and the stromal microenvironment is still poorly understood. Here we investigated a broad panel of surface adhesion molecules for their involvement. For this purpose, the colony-forming capacity of stroma-adherent Bl-CEC, BFU-E and GM-CFC was studied. Both mononuclear bone marrow cells (BMC) and bone marrow-derived stromal cells (BMSC) express a wide variety of adhesion molecules. However, only antibodies against beta 1-, alpha 4-integrin (both chains of the very late activation antigen-4 (VLA-4)) and vascular cell adhesion molecule (VCAM-1) inhibited colony formation from stroma-adherent Bl-CFC by 50% or more. Antibodies against a panel of other adhesion molecules, including the alpha 5-integrin chain, were without effect. Subsequent pretreatment experiments revealed that VLA-4 on progenitors interacted with stromal VCAM-1. The inhibitory antibodies did not interfere with the clonogenic capacity of but with adhesion of BFU-E and GM-CFC. Whether the inhibitory antibodies act similarly on progenitors which depend on BMSC for growth and/or differentiation, such as BI-CFC, remains to be determined.
Assuntos
Medula Óssea/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Integrinas/fisiologia , Receptores de Retorno de Linfócitos/fisiologia , Molécula 1 de Adesão de Célula Vascular/fisiologia , Anticorpos Monoclonais/fisiologia , Células da Medula Óssea , Células Cultivadas , Humanos , Integrina alfa4beta1 , Células Estromais/fisiologiaRESUMO
Chronic myelogenous leukemia is a disease of the pluripotent stem cell that involves the myeloid and, to a varying degree, the lymphoid compartment. We studied the involvement of B cells in chronic myelogenous leukemia at diagnosis and during treatment. B lymphocytes were immortalized by infection with Epstein-Barr virus. B-lymphoid cell lines could be established from 25 patients suffering from Philadelphia-chromosome (Ph1)-positive chronic myelogenous leukemia. The cell lines were tested for expression of the typical 210-kDa fusion protein, p210, using Western-blot analysis, and/or for mRNA expression of bcr-abl fusion genes, using reverse transcriptase polymerase chain reaction analysis. At diagnosis, mosaicism of B cells was demonstrated in every patient. During treatment with interferon alpha, p210-expressing B-lymphoid cell lines could not be established from 8 of 8 patients. Following discontinuation of IFN-alpha therapy, p210-positive cell lines were found early, even before cytogenetic recurrence. Resistance to IFN-alpha therapy and progression of the disease were both associated with the appearance of p210-positive cell lines. Cell lines established from 3 healthy individuals and from patients suffering from Ph1-negative diseases did not show p210 expression in Western blots. Our data suggest that B lymphocytes are involved early in the disease, and that B-cell mosaicism may be a sensitive marker for resistance to IFN-alpha therapy and disease progression.
Assuntos
Linfócitos B/patologia , Proteínas de Fusão bcr-abl/biossíntese , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , RNA Mensageiro/biossíntese , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
Bone marrow (BM) failure associated with cytomegalovirus (CMV) infection is a feared complication after clinical BM transplantation. Experiments in long-term BM cultures have indicated that BM stromal cells (BMSC) are targets of productive CMV infection, but an in situ infection of BM stroma remained to be documented, and the pathomechanism is open to question. Here we describe a murine in vivo model of lethal CMV aplastic anemia (CMV-AA). The reconstitution of hematopoietic progenitor cells expressing stem cell factor (SCF) receptor was found to be defective in CMV-AA. While murine CMV replication in permissive parenchymal tissues is cytolytic, the hematopoietic cord was found to be a site of very limited virus production with foci of reticular BMSC expressing the intranuclear viral IE1 protein, but with only a few BMSC positive for viral genome in the in situ hybridization. XX-XY BM chimeras were established in order to quantitate Y-chromosome-tagged BMSC by a PCR specific for the male-sex-determining gene Tdy. This approach revealed that murine CMV infection is not associated with a significant loss of BMSC. Despite the physical integrity of the stromal network, the functional integrity of the stroma was impaired. While housekeeping genes were expressed normally in BMSC of infected mice, the expression of genes encoding the essential hemopoietins SCF, granulocyte colony-stimulating factor, and interleukin-6 was markedly reduced. In conclusion, the mechanism of BM failure is not a stromal lesion but an insufficient stromal function. These findings explain CMV-AA as a manifestation of multiple hemopoietin deficiency.