Long-term culture of chronic myeloid leukemia (CML) bone marrow cells.

BACKGROUND. Clonal Ph1+ hematopoiesis is not allowed to proliferate under a long-term culture system: in 3,4 weeks residual normal (Phl-) hematopoiesis emerges. This culture system has recently been proposed as a method for purging autografts. METHODS. In our study we evaluated for cytogenetic conversion cells harvested from the non-adherent (NA) fraction of LTBMCs from CML patients. Moreover we investigated the effects of prior therapy (busulfan or hydroxyurea) on CML hematopoiesis maintained under long-term culture. RESULTS. Time-course analysis of a large number of metaphases of NA cells from LTBMCs showed that the disappearance of Ph1+ cells is fortuitous. Although most of the analyzed cells were more mature cells, a complete cytogenetic conversion at the level of the early hematopoietic compartment, located within the adherent stromal layers, seems unlikely, at least for the first 3,4 weeks of culture. Thus the possibility exists of reinfusing an indefinite number of Ph1+ progenitor or stem cells, which renders proper evaluation of the clinical benefits of this purging method difficult. Moreover we found that prior chemotherapy (busulfan or hydroxyurea) significantly affected CML hematopoiesis, reducing time-course recovery of clonogenic cells from LTBMCs. CONCLUSIONS. Overall data suggest caution in the reinfusion of bone marrow cells maintained under long-term culture for previously treated CML patients.     

Changes in the growth properties of CD34+, CD38- bone marrow progenitors during human fetal development

We have previously described the isolation of separate populations of CD34+, CD38- stromal and hematopoietic progenitors cells within fetal bone marrow. The CD34+, CD38-, CD50+, HLA-DR+ population contained the majority of primitive hematopoietic progenitor cells, whereas stromal progenitors were contained within the CD34+, CD38-, CD50-, HLA-DR- population. In this study, we compared the frequencies and total numbers of clonogenic CD34+, CD38- stromal and hematopoietic cells as a function of fetal gestational age using single-cell fluorescent- activated cell sorting (FACS). At 14 weeks of gestation, 1/500 fetal bone marrow mononuclear cells were primitive hematopoietic CD34+, CD38- , HLA-DR+ progenitor cells, whereas 1/1,000 were stromal progenitors with the CD34+, CD38-, HLA-DR- phenotype. During fetal ontogeny there was a continuous, age-dependent decrease in the frequency of stromal progenitors, such that, at 24 weeks of gestation, only 1/100,000 of bone marrow cells had the CD34+, CD38-, HLA-DR- phenotype and were clonogenic stromal cells when isolated by FACS. In contrast, 1/250 bone marrow cells in a 24-week fetus had the CD34+, CD38-, HLA-DR+ phenotype and were clonogenic hematopoietic progenitors. The decrease in the frequency of stromal progenitors was a function of both a decreased frequency of cells with the CD34+, CD38-, HLA-DR- phenotype and a decrease in the growth potential of individual with this phenotype. The total numbers of mononuclear cells and the total numbers of hematopoietic progenitors in two fetal femurs increased in parallel, 100-fold, between 14 and 24 weeks of gestation. In contrast, the total numbers of clonogenic CD34+, CD38-, HLA-DR- stromal progenitor cells remained constant during this period. Although adult bone marrow samples contained stromal progenitor cells at a frequency of approximately 1/7,000 mononuclear cells, clonogenic stromal cells with the CD34+, CD38-, HLA-DR- phenotype could not be isolated by single- cell FACS from these samples. Thus, there are significant differences between the frequencies and biologic characteristics of stromal and hematopoietic stem cells during fetal and postnatal ontogeny.     

造血干细胞移植后白血病细胞染色体核型和bcr/abl融合基因表达观察

目的：探讨造血干细胞移植(HSCT)后白血病细胞染色体核型和基因表达观察的意义。方法：用骨髓细胞短期培养法和直接法G显带分析染色体；双色荧光原位杂交检测bcr／abl融合基因。结果：2例供者为女性的男性急性髓细胞白血病(AML)患者异体外周血干细胞移植(allo-PBSCT)后染色体检测持续46，XX。最长生存50个月。4例慢性髓细胞白血病(CML)经allo-PBSCT后，Ph染色体和bcr／abl融合基因阳性1例，经供者淋巴细胞输注加干扰素治疗，已生存70个月。Ph染色体阴性bcr／abl融合基因阳性2例，最长生存27个月。Ph染色体阳性bcr／abl融合基因阴性1例，已生存14个月。2例AML自体造血干细胞移植后检测出非特征性染色体畸变，复发后再化疗达完全缓解，最长生存期达81个月。结论：造血干细胞移植后白血病细胞遗传学检测可观察近期疗效，指导后续治疗选择。     

Blast crisis in a murine model of chronic myelogenous leukemia.

The P210bcr/abl protein is produced in cells from patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML). Retroviral transfer of the gene encoding P210bcr/abl into murine bone marrow induces a granulocytic leukemia that models the chronic phase of human CML. We have transferred the leukemic clone to syngeneic animals, albeit with surprising inefficiency, and have observed CML and clonally related acute leukemias of lymphoid or myeloid phenotype in some transplant recipients. These data show that murine CML can result from retroviral transfer of the bcr/abl gene into pluripotent hematopoietic stem cells, that infected clones repopulate poorly after adoptive transfer, and that these clones can give rise to acute leukemia, reflecting evolution to a phase resembling blast crisis in the human disease.     

Benign hematopoietic progenitors in chronic myeloid leukemia: Current status and future prospects

Summary Many patients with chronic myeloid leukemia (CML) retain a certain degree of normal hematopoiesis at disease presentation. This fact, suspected on the basis of cytogenetic findings, has been confirmed by long-term bone marrow cultures (LTBMC) and the combined use of phenotypic and molecular studies. Based on the lack of HLA-DR expression, it has been possible to recognize a benign subpopulation within the stem-cell compartment in CML. Different in vitro techniques have been developed for the selection of these benign progenitors, including LTBMC, marrow incubation with cytolytic drugs or interferon, positive selection based on their phenotypic characteristics, and exposure to synthetic antisense oligodeoxynucleotides. In vivo selection with interferon or intensive chemotherapy is also possible. The primary goal of the selection of benign hematopoietic progenitors is their use for autotransplantation. To date, a few hundred CML patients have been submitted to the latter procedure using bone marrow or peripheral blood. The fact that the majority of them show evidence of persistent disease emphasizes the necessity for better selection methods of the benign progenitors, for intensifying the conditioning regimen to reduce the tumor burden as much as possible, and for the use of adjuvant therapy post-transplantation. Future trends include the refinement of positive selection methods, negative selection by taking advantage of the different stromal adhesiveness of the benign and malignant progenitors, or the use of autologous natural killer cells, antisense oligodeoxynucleotides, or specific antibodies to the bcr/abl junction region, and retroviral marking to determine the origin of relapse in autologous transplantation.     

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.     

Detection of breakpoint cluster region- negative and nonclonal hematopoiesis in vitro and in vivo after transplantation of cells selected in cultures of chronic myeloid leukemia marrow

Philadelphia (Ph1) chromosome-positive clonogenic progenitors usually disappear within 4 to 6 weeks in long-term cultures established from the marrow of patients with chronic myeloid leukemia (CML). In contrast, coexisting chromosomally normal hematopoietic cells are relatively well maintained. Thus, even though normal cells are initially undetectable, they may become the predominant population. Recently, we have begun to explore the potential of such cultures as a strategy for preparing CML marrow for autografting, and based on cytogenetic studies of the differential kinetics of Ph1-positive and Ph1-negative clonogenic cells, have chosen a 10-day period in culture to obtain maximal numbers of selectively enriched normal stem cells. Here we present the results of molecular analyses of the cells regenerated in vivo for the initial three CML patients to be treated using this approach by comparison with the differentiated cells generated by continued maintenance of an aliquot of the autograft in vitro (using a slightly modified culture feeding procedure to enhance the production and release of cells into the nonadherent fraction after 4 weeks) for the one patient whose genotype made molecular analysis of clonality status also possible. These analyses showed that cells with a rearranged breakpoint cluster region (BCR) gene were not detectable by Southern blotting in either in vitro or in vivo populations of mature cells that might be assumed to represent the progeny of primitive cells present at the end of the initial 10 days in culture. Production of BCR-negative cells was also shown to be temporally correlated with the appearance of nonclonal hematopoietic cells both in culture and in vivo. These findings provide support for the view that prolonged maintenance of CML marrow cells in long-term culture may allow molecular characterization of both the BCR-genotype and clonality status of cells with in vivo regenerative potential.     

成人骨髓间充质干细胞与造血细胞的发育级系的关系

目的：探讨骨髓间充质干细胞在细胞发育级系中与造血细胞的关系。方法：分离培养成年慢性髓性白血病患者骨髓的间充质干细胞，用FCM鉴定其纯度。应用RT－PCR检测细胞是否携带白血病特异性基因，结合流式细胞术观察多种造血调控因子刺激后是否具有造血细胞的表面分子标志及基因标志，结果：骨髓间充质干细胞不表达造血细胞特异性白血病基因，细胞因子作用2周后未出现血细胞表型，CD34及CD45均为阴性，结论：造血干细胞与间充质干细胞之间似乎不存在交叉分化现象，为解释其他细胞间的相互转化提出了新的线索。     

Effect of recombinant gamma interferon on chronic myelogenous leukemia bone marrow progenitors

In order to understand its mechanism of action and explore its potential as a therapeutic agent, we studied the effect of recombinant gamma interferon (IFN) on in vitro proliferation and on karyotype of bone marrow-derived hematopoietic stem cell progenitors (BFUe, CFUmix) obtained from patients with Ph1-positive chronic myelogenous leukemia (CML). Addition of IFN to culture resulted in a dose-dependent inhibition of both normal and CML BFUe and CFUmix. The maximum dose-dependent suppression of CML BFUe (92% +/- 4%) and CML CFUmix (100%) exceeded the maximum suppression of normal BFUe (40% +/- 4%) and normal CFU mix (68% +/- 6%) (p less than 0.001 and p = 0.008). In parallel studies, CML BFUe and CFUmix were cultured with and without IFN, and cells recovered from culture were examined cytogenetically. Treatment of CML bone marrow cells (BMC) with IFN resulted in an increase in the proportion (p less than 0.001) of Ph1-negative metaphases when compared to control cells grown in the absence of IFN. Recombinant gamma interferon has a significant antiproliferative effect against CML bone marrow-derived stem cell progenitors in vitro, and the addition of this agent to culture increases our ability to identify a cell population derived from a Ph1-negative progenitor pool. Recombinant gamma interferon may selectively spare Ph1-negative hematopoietic progenitors, and may be an active agent in the treatment of CML.     

Activation-dependent alpha5beta1 integrin-mediated adhesion to fibronectin decreases proliferation of chronic myelogenous leukemia progenitors and K562 cells

Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell characterized by abnormal circulation and proliferation of malignant progenitors. In contrast to their normal counterparts, CML progenitors adhere poorly to bone marrow stroma or fibronectin (FN). Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal progenitors is also associated with inhibition of their proliferation. As the beta1 integrin expression on CML progenitors is normal, we hypothesized that decreased integrin affinity may underlie the abnormal adhesive and proliferative characteristics of CML progenitors. We examined the effect of affinity modulation by the activating antibody 8A2 on the adhesion and proliferation of CML progenitors and the CML cell line, K562. 8A2 induced alpha5Beta1-dependent adhesion of Philadelphia chromosome-positive (Ph+) CD34+/HLA-DR+ cells and K562 cells to FN. Increased adhesion was 8A2- and FN concentration- dependent, time-dependent, and energy-dependent. Further, 8A2-induced adhesion to FN significantly inhibited the proliferation of malignant CML progenitors as well as K562 cells independent of cell differentiation, necrosis, or apoptosis. These studies demonstrate that affinity modulation of the alpha5Beta1 integrin on CML progenitors and K562 cells by 8A2 results in increased adhesion to FN with subsequent decreased proliferation, suggesting that decreased beta1 integrin affinity contributes to the abnormal circulation and proliferation of malignant progenitors in CML.     

Phenotypic heterogeneity of primitive leukemic hematopoietic cells in patients with chronic myeloid leukemia.

The peripheral blood of chronic myeloid leukemia (CML) patients with chronic-phase disease and elevated white blood cell (WBC) counts typically contains markedly increased numbers of a variety of neoplastic pluripotent and lineage-restricted hematopoietic progenitors. These include cells detected in standard colony assays as well as their more primitive precursors. The latter are referred to as long-term culture-initiating cells (LTC-IC) because of their ability to generate clonogenic cell progeny detectable after a minimum of 5 weeks incubation on competent fibroblast feeder layers. In this study, we have investigated a number of the properties of the LTC-IC and clonogenic cells present in the blood of such CML patients with high WBC counts. This included an analysis of the light scattering properties of these progenitors, as well as their expression of CD34 and HLA-DR, Rhodamine-123 staining, and in vitro sensitivity to 4-hydroperoxycyclophosphamide. In the case of LTC-IC, the production of different types of lineage-restricted and multipotent progeny was also analyzed. Most of the circulating LTC-IC and clonogenic cells in the CML patients studied (on average approximately 70% and approximately 90%, respectively) showed features of proliferating or activated cells. This is in marked contrast to the majority of progenitors in the blood of normal individuals and most of the LTC-IC in normal marrow, all of which exhibit a phenotype expected of quiescent cells. Interestingly, a significant proportion of the circulating clonogenic cells and LTC-IC in the CML samples studied (on average approximately 10% and approximately 30%, respectively) appeared to be phenotypically similar to normal circulating progenitors, although their absolute numbers were indicative of a neoplastic origin. Both phenotypes of circulating CML clonogenic cells and LTC-IC could be obtained at approximately 10% to 20% purity by differential multiparameter sorting. These findings suggest that expansion of the Philadelphia chromosome-positive clone at the level of the earliest types of hematopoietic cells results from the activation of mechanisms that enable some, but not all, signals that block the cycling of normal stem cells to be bypassed or overcome. In addition, they provide strategies for purifying these primitive leukemic cells that should facilitate further analysis of the mechanisms underlying their abnormal proliferative behavior.     

应用RT-nest-PCR法检测慢性髓细胞白血病非亲缘异基因骨髓移植后微小残留病变

目的：应用筑巢式RT—PCR（RT—nest-PCR）法检测慢性髓细胞性白血病（CML）患者非亲缘异基因骨髓移植（URD）后微小残留病变（MRD），并探讨它与复发的相关性。方法：分别采用RT-nest—PCR法和常规细胞遗传学方法检测bcr／abl融合基因和Ph染色体。结果：20例CML行URD患者术前Ph染色体和bcr／abl融合基因检测均为阳性，移植术后30dPh染色体皆转为阴性，bcr／abl融合基因完全转阴时间为1～3个月，中位时间2个月；在随防的18例CML患者中，有16例患者bcr／abl融合基因完全转阴后没再转为阳性。在1例复发的CML患者中可见到血型、Ph染色体和bcr／abl融合基因动态变化，另1例CML患者在移植成功后的21个月和36个月检测到bcr／abl融合基因，经随访未见临床和血液学复发征象。结论：检测bcr／abl融合基因是目前观察CML患者非亲缘异基因骨髓移植后微小残留病变最敏感的方法之一，非亲缘异基因骨髓移植能最大限度地消除CML患者体内残留病变，患者能长期无病生存。     

Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro

To develop a purification strategy for isolating the most primitive hematopoietic stem cells present in normal human marrow we have combined cell separation techniques with an assay for cells that initiate sustained hematopoiesis in vitro in the presence of irradiated human marrow adherent cells. These "feeders" were established by subculturing 2- to 6-week-old primary long-term marrow culture adherent layers at a density of 3 x 10(4) irradiated cells per square centimeter. Test "long-term culture (LTC)-initiating cells" were plated on top of the feeders and the cocultures then maintained as standard long-term marrow cultures with half-media changes and removal of half of the nonadherent cells each week. The total number of myeloid, erythroid, and multilineage clonogenic progenitors present after 5 weeks was used to provide a quantitative assessment of the number of LTC-initiating cells originally added. Using this assay, the density, light scatter, and two cell surface antigen properties of LTC- initiating cells have been defined and compared with cells capable of directly forming colonies in methylcellulose. While the majority of the clonogenic cells were found in the high forward light scatter (FLS) "blast" window, LTC-initiating cells had significantly lower FLS properties and in this respect were more similar to lymphocytes. LTC- initiating cells also expressed less HLA-DR antigen than clonogenic cells. The majority of LTC-initiating cells were found in the top 2% of the CD34 (My10) fluorescence profile, whereas clonogenic cells were found throughout the top 5% of the CD34 fluorescence profile. By combining low FLS, low orthogonal light scatter (OLS), low HLA-DR expression, and high CD34 expression, a population could be obtained that was enriched for LTC-initiating cells approximately 800-fold over unseparated marrow. This population contains only 0.06% of the marrow cells and 2% of the total clonogenic cells, but retains 50% to 60% of the LTC-initiating cells present in the original marrow. The ability to purify these two populations independently shows that the LTC and clonogenic assays identify distinct, although not necessarily nonoverlapping cell types in human marrow. Since clonogenic cells are derived from LTC-initiating cells, the LTC assay clearly detects a more primitive population. The availability of a simple approach that allows the purification of such cells by three orders of magnitude in high yield should be useful for the investigation of early events in hematopoiesis as well as for the definitive isolation of human hematopoietic stem cells with long-term in vivo repopulating potential.     

实时定量RT-PCR检测慢性粒细胞白血病bcr/abl融合基因的临床意义

目的：建立实时定量RT-PCR方法检测慢性粒细胞白血病(CML)bcr／abl mRNA的方法；探讨CML bcr／abl融合基因的表达水平与疗效的关系。方法：可同时检测b2a2和b3a2两种亚型．GAPDH的mRNA作为内参照。检测14例CML患者的22个外周血和骨髓样本中bcr／abl融合基因表达水平．并对2例异基因造血干细胞移植后复发的患者进行动态监测。结果：bcr／abl及GAPDH标准曲线的相关系数均为0．999；灵敏度为10^-6；14例初诊患者的mRNA表达水平范围为2．81～145；2例异基因造血干细胞移植后复发患者的bcr／abl mRNA表达水平随临床治疗而变化。结论：实时定量PCR检测CML患者的bcr／abl融合基因，敏感可靠，重复性好；其融合基因表达水平的改变与临床疾病进展关系相一致，有助于反映白血病细胞负荷、评价疗效及判断疾病预后。