Human CD34-positive hematopoietic stem cells constitute targets for carcinogenic polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are major carcinogenic environmental contaminants known to exert bone marrow toxicity and to induce leukemias, suggesting that these chemicals target hematopoietic stem cells. To investigate this hypothesis, we studied the effects of PAHs on cell proliferation and differentiation in human hematopoietic CD34+ cell cultures. Benzo(a)pyrene (BP), a prototypical PAH, was shown to markedly impair CD34+ cell expansion and to inhibit CD34+ cell differentiation into various hematological cell lineages, including erythroid, granulomacrophagic, and megakaryocytic lineages. This was associated with the induction of a caspase- and mitochondrion-related apoptosis process. CD34+ progenitor cells were found to exhibit functional expression of the aryl hydrocarbon receptor (AhR), and the use of the pure AhR antagonist 3'-methoxy-4'-nitroflavone partially counteracted the deleterious effects of BP in CD34+ cell cultures, underlining the involvement of AhR in BP toxicity. Additional events such as CYP1A1/1B1-dependent PAH metabolism and adduct formation were also required since 1) 2,3,7,8-tetrachlorodibenzo-p-dioxin, a very potent ligand of the AhR that is poorly metabolized and therefore does not generate reactive metabolites in contrast to PAHs, failed to affect CD34+ cell expansion; 2) the CYP1A1/1B1 inhibitor alpha-naphthoflavone blocked both BP adduct formation and BP toxicity; and 3) benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide, a highly reactive BP metabolite, exerted a marked toxicity toward CD34+ cell cultures. Overall, these data indicate that human hematopoietic CD34+ cells can bioactivate chemical carcinogens such as PAHs and, in this way, constitute targets for such carcinogenic environmental contaminants.     

造血干细胞移植中CD34+细胞最佳分选的应用效果评价

目的探讨联合化疗加细胞因子动员自体外周血CD34+细胞的采集及临床级磁性分选仪(CliniMACS)分选的最佳方法,评价分选后CD34+细胞在造血干细胞移植中的应用效果.方法2001-03/2002-01在南京市鼓楼医院血液科病房收治自身免疫性疾病患者9例.符合纳入标准患者9例,男3例,女6例.采用环磷酰胺[2～4 g/(m2·d)]+粒细胞集落刺激因子(G-CSF)[5～10 μg/(kg·d)]作为动员剂,CS-3000Plus血细胞分离机采集外周血单个核样细胞,CliniMACS作CD34+细胞分选,观察分选前后的细胞计数、纯度、细胞活力,流式细胞术进行细胞表型检测及粒巨细胞集落形成单位(CFU-GM)培养.计算CD34+细胞的采集得率和分选回收率.观察CD34+细胞移植后造血功能恢复的情况.结果经环磷酰胺加G-CSF动员后,采集时机多以一次采集能够获得足够数量的CD34+细胞(CliniMACS分选和冷冻保存的耗损计算在内)为原则,外周血白细胞总数＞6×109 L-1或CD34+细胞＞0.4%时开始采集,其中8例采集1次,1例采集2次.每例患者可获单个核样细胞(MNC)总数(0.924～5.360)×1010,MNC(2.6～19.5)×108/kg,CD34+细胞(2.4～37.2)×106/kg,经CliniMACS系统分选后,CD34+细胞回收率为51%～93%,平均回收率为87%,CFU-GM回收率为35%～62%.CD34+细胞纯度为94.3%～98.4%,平均96.71%.CD3+,CD19+,CD56+,CD14+细胞去除2～4个对数级.经冰冻保存后的CD34+细胞的回收率为78%～99%,CFU-GM回收率为82%～99%,实际回输CD34+细胞为(2.0～8.3)×106/kg,移植后均获造血重建.结论掌握最佳动员、采集及分选方法,CD34+细胞可获较高产率,移植后造血功能恢复较快.     

The removal of human breast cancer cells from hematopoietic CD34+ stem cells by dielectrophoretic field-flow-fractionation

Dielectrophoretic field-flow-fractionation (DEP-FFF) was used to purge human breast cancer MDA-435 cells from hematopoietic CD34+ stem cells. An array of interdigitated microelectrodes lining the bottom surface of a thin chamber was used to generate dielectrophoretic forces that levitated the cell mixture in a fluid flow profile. CD34+ stem cells were levitated higher, were carried faster by the fluid flow, and exited the separation chamber earlier than the cancer cells. Using on-line flow cytometry, efficient separation of the cell mixture was observed in less than 12 min, and CD34+ stem cell fractions with a purity >99.2% were obtained. The method of DEP-FFF is potentially applicable to many biomedical cell separation problems, including microfluidic-scale diagnosis and preparative-scale purification of cell subpopulations.     

Crude collection efficiency of CD34+ hematopoietic stem cell apheresis

Understanding the apheresis principles for harvesting hematopoietic stem cells (HSCs) is critical for performing efficient procedures. However, despite significant advances in estimating the collection efficiency (CE) of aphereses, many confounding factors still need to be addressed in the classical calculations. The CE values are unrestricted, and many procedures exhibit CEs of a given cell population greater than 100%. This report introduces a simple equation that estimates the “crude” CE, which ranges from 0% to 100% and intrinsically considers the contribution of donor-related variables such as the pre-procedure mobilization and intra-apheresis recruitment of CD34⁺ cells (as a convenient marker for HSCs), as well as the performance of the apheresis system itself.     

A feasible shortcut for the mobilization outcome: Steady state CD34 positive hematopoietic stem cells

Predicting success of hematopoietic cell mobilization is an important issue for transplant physicians. We examined the steady state peripheral blood CD34+ cell count to predict ability to mobilize adequate hematopoietic progenitor cells in 63 myeloma and lymphoma patients. The median steady state CD34+ cell number was 1.56/μL (0.03-5.76). Although counting steady state CD34+ is definitely cost effective to predict the successful mobilization, we could not find a threshold steady state CD34 count of any value predicting successful mobilization.     

Monoclonal antibodies with neuroblastoma specificity: a flow cytometric analysis of cross-reactivity with CD34+ hematopoietic stem cells

The aim of this study was to evaluate the specificity of a number of monoclonal antibodies (MAbs) used for immunological in vitro purging of stem cell grafts from neuroblastoma patients. The extent of cross-reactivity of 10 neuroblastoma-specific MAbs (NB-MAb) with CD34+ stem cells from 14 leukapheresis products was analyzed. The level of cross-reactivity was analyzed on a Coulter (Fullerton, CA) flow cytometer using biotinylated NB-MAbs. There was a marked difference in the reactivity of the ten NB-MAbs with CD34+ stem cells. The antibodies could be divided into three groups with increasing levels of cross reactivity. Four antibodies (126-4, 5.1 H11, UJ127.11, and 14.G2a) all reacted with median levels of less than 2% (range 0.0 to 5.4) of CD34+ stem cells (median of 14 patients). Another three antibodies reacted with a median of 3.1-4.1% of the stem cells (UJ13A, Ab390, and Ab459) but with a wide range (0.2 to 25.6). Finally, M340, HSAN 1.2, and antiThy-1 reacted with a median of 9-16% of the stem cells (range 0.6 to 51.5). We conclude that there is a significant variation in the proportion of CD34+ stem cells reacting with each of the ten neuroblastoma antibodies investigated in this study. Therefore, to avoid a significant loss of CD34+ cells from the stem cell product, we find it important to carefully consider which antibodies to use for immunomagnetic purging.     

Transplantable hematopoietic stem cells in human fetal liver have a CD34(+) side population (SP)phenotype

Cells with a verapamil-sensitive ability to efflux Hoechst 33342 (termed side population [SP] cells) have been identified in adult marrow from several species including humans and in several tissues from adult mice. In mice, the SP phenotype appears to be a common feature of stem cells, but human SP cells have been less well characterized. We show here, for the first time to our knowledge, that SP cells are present in the second-trimester human fetal liver. They include all of the transplantable human hematopoietic stem cell activity detectable in NOD/SCID mice and also certain other, more differentiated hematopoietic cell types. Notably, the stem cell activity was confined to the CD34(+)CD38(-) SP(+) population, and isolation of these cells gave an approximately tenfold enrichment of transplantable stem cells. This subset was not, however, coenriched in hematopoietic progenitors detectable by either short- or long-term in vitro assays, indicating most of these to be distinct from transplantable stem cells. These findings suggest that the SP phenotype is an important and distinguishing property of human hematopoietic stem cells and that early in ontogeny they express CD34.     

CD34+造血干细胞在"心肌样"微环境中转化为心肌样细胞的可能性

目的一般认为CD34+细胞只能向血细胞分化,观察CD34+造血干细胞在"心肌样"微环境中转化为心肌样细胞的可能性.方法实验于2004-01/12在大连医科大学附属第二医院实验中心完成.①选用成年健康SD大鼠30只及出生1～3 d SD大鼠乳鼠.在体外分离、培养CD34+造血干细胞,并将CD34+造血干细胞与分离出的新生乳鼠心肌细胞共培养.②将新生乳鼠心肌细胞的培养环境模拟为在体心肌细胞的生活环境即心肌样微环境.③在细胞培养瓶内加入5-溴脱氧尿嘧啶核苷标记CD34+细胞.在倒置显微镜下观察CD34+造血干细胞形态学变化,用免疫细胞化学法检测心肌特异性肌钙蛋白T.结果①CD34+造血干细胞与心肌细胞共培养3 d,CD34+细胞变成长杆状,即呈现较为一致的极性排列,靠近心肌细胞的CD34+细胞形态学改变更早、更明显.②共培养的细胞经免疫细胞化学染色,发现部分5-溴脱氧尿嘧啶核苷标记的CD34+细胞核在荧光显微镜下呈现红色,同时其细胞浆心肌特异性肌钙蛋白T表现阳性,说明确实有部分CD34+造血干细胞转化成幼稚心肌细胞.结论将CD34+造血干细胞与心肌细胞共培养,CD34+造血干细胞可以分化为心肌样细胞,从而验证了体外"心肌样"微环境可以促使CD34+造血干细胞向心肌样细胞转化的潜能.     

腺相关病毒载体介导的人凝血因子Ⅸ基因在CD34+造血干/祖细胞中的表达

目的 研究2型重组腺相关病毒（rAAV-2）介导的人凝血因子Ⅸ（hFⅨ）基因在脐血CD34^＋细胞及其子代细胞巾的表达。方法 采用rAAV-2／hFⅨ转导经预刺激的人脐血CD34^＋细胞，分别向粒单系、巨核系和红系分化培养21d，从转录水平、蛋白质水平和其功能活性检测hFⅨ的表达，同时检测子代细胞的活力、增殖倍数、各系标志分化抗原的表达及集落产率来评估rAAV-2对其增殖分化能力的影响。结果 经测序证实转导组子代细胞的RNA可扩增出hFⅨ cDNA片段，上清液中可检测到hFⅨ抗原的表达，每24h分泌量达14.10ng／10^6细胞．转导组和未转导组细胞培养21d后其活力、增殖倍数、标志性分化抗原的阳性率及集落产率差异均无统计学意义（P值均〉0．05）。结论 rAAV-2／hFⅨ能有效地转导人脐血CD34^＋细胞并在其子代细胞中表达具有凝血活性的hFⅨ，且对其体外培养21d的细胞增殖分化能力无明显影响.     

Increased immature hematopoietic progenitor cells CD34+/CD38dim in myelodysplasia

BACKGROUND: Myelodysplastic syndromes (MDS) are clonal disorders affecting hematopoietic progenitor cells (HPC). Despite the relevance of clonal CD34+ cells in developing MDS, only few studies analyze the phenotype of this cell population. The aim of this study was to evaluate phenotypic changes on HPC in MDS that could reflect abnormalities in the differentiation process of stem cells. METHODS: We analyzed the expression of CD38 and HLA-DR on CD34+ cells by flow cytometry in 36 patients with MDS, as well as in healthy donors (n = 12) and patients with other hematological disorders: non-Hodgkin lymphomas and multiple myeloma, both in complete remission (CR) (n = 32); acute lymphoblastic leukemia in CR (n = 17); de novo acute myeloblastic leukemia (AML) at diagnosis (n = 22) and in CR (n = 37); and AML secondary to MDS at diagnosis (n = 19). Cases with available karyotype were grouped according to the International Prognostic Scoring System (IPSS). RESULTS: Compared to normal BM, the fraction of immature HPC, characterized as CD34+bright, intermediate FSC/SSC, and CD38dim, was significantly increased in high risk MDS and secondary AML, but not in low risk MDS, (P < or = 0.001, P = 0.03, and P = 0.7). De novo AML showed decreased immature HPC. High numbers of immature HPC correlated with higher IPSS risk groups (P = 0.05) and showed significant impact on disease progression (P = 0.03). CONCLUSION: Our study confirms that evaluation of CD38 expression pattern on HPC is an easy and reproducible test that allows evaluating the immature subset of progenitor cells. Increased immature HPC in high risk MDS and secondary AML may reflect blocked differentiation of CD34+ cells in these diseases.     

A clinical-scale expansion of mobilized CD 34+ hematopoietic stem and progenitor cells by use of a new serum-free medium

BACKGROUND: The autologous transplantation of CD 34+ cells expanded ex vivo in serum-free conditions dramatically reduces post-myeloablative neutropenia in myeloma patients. In our cell therapy unit, cells for this clinical assay have been expanded under GMP with serum-free Irvine Scientific (IS) medium with stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF; 100 ng/mL, respectively). Because this clinical-grade IS medium is no longer available, a new serum-free medium, Maco Biotech HP 01 (Macopharma), was evaluated. STUDY DESIGN AND METHODS: Purified CD 34+ cells (Isolex 300i, Baxter) from mobilized peripheral blood samples of myeloma patients were thawed, washed, and cultured, as for previous clinical assays. Twenty million CD 34+ cells were resuspended per 1 L of SCF-, G-CSF-, and MGDF-supplemented medium (HP 01 or IS), introduced into 3-L culture bags (AFC), and cultured for 10 days in 5 percent CO(2), at 37 degrees C, and at 100 percent humidity. RESULTS: A higher amplification of total nucleated cells (NCs) and colony-forming cells (CFCs) was obtained with HP 01 medium than with IS medium (42+/-16.6-fold vs. 20.5+/-5.9-fold for NCs and 26.7+/-7.4-fold vs. 15.5+/-2.5-fold for CFCs, respectively), whereas an increase in CD 34+ cells (3.5+/- 1.2-fold for HP 01 vs. 2.7+/- 1.5-fold for IS) was not significant. IS medium partially maintained SCID-repopulating cells (SRC), whereas the culture in HP 01 medium fully maintained the stem cell activity for 10 days. A higher frequency of CD 41+ cells after expansion in HP 01 than in IS medium was also observed. CONCLUSION: Maco Biotech HP 01 medium is suitable for clinical-scale expansion of CD 34+ cells with the SCF, G-CSF, and MGDF cytokine cocktail, permitting an intensive amplification of CFCs and maintenance of SRCs.     

Homing of lin/CD117 hematopoietic stem cells

In this report, we describe the homing of hematopoietic stem cells (HSCs) to non-hematopoietic tissues in lethally irradiated (9 Gy) hybrid mice transplanted intravenously with lin⁻/CD117⁺ bone marrow cells from ROSA26 mice. The numbers of CFU-GM in spleen of irradiated transplanted mice were well above the levels found in non-irradiated group as early as day 8 after transplant. On 12th day regeneration of lymphocytes was observed, an increase in granulocytes was detected as late as on 33rd day. Transplanted cells containing lacZ gene were detected in recipient mice by histochemistry and their location in the thymus, liver, stomach and ileum was followed during 33 days post-transplantation. On 8 and 33 days post-transplantation, we found massive presence of donor (lacZ⁺) cells in the thymic cortex. Hematopoietic stem cell transplantation led not only to recovery of hematopoietic and lymphoid tissues but also facilitated recovery of the small intestinal mucosa, which was significantly damaged by ionizing radiation.     

关于CD34-造血干细胞的新观点

最近的研究表明CD34-造血干细胞的存在,改变了传统的造血干细胞必须表达CD34抗原的观点.近来大量资料对其功能、与CD34+细胞的关系、表面标志以及基因表达等作了报道,现对此做一综述.     

P-Selectin coated microtube for enrichment of CD34+ hematopoietic stem and progenitor cells from human bone marrow

BACKGROUND: Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematologic disorders and in basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion on selectins than mature CD34- mononuclear cells (MNCs). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow by perfusing MNCs through selectin-coated microtubes. METHODS: Bone marrow MNCs were perfused through the cell-capture microtubes coated with adhesion molecules. We washed the device lumen and visualized and estimated captured cells by video microscopy. Adherent cells were eluted by high shear, calcium-free buffer, and air embolism. We used immunofluorescence staining followed by flow cytometry to analyze CD34+ HSPCs. RESULTS: CD34+ HSPC purity of cells captured in adhesion molecule-coated devices was significantly higher than the fraction of CD34+ cells found in bone marrow MNCs [mean (SE) 2.5% (0.8%)]. P-selectin-coated surfaces yielded 16% to 20% CD34+ cell purity, whereas antibody-coated surfaces yielded 12% to 18%. Although CD34+ cell purity was comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (approximately 5.7 x 10(4) to 7.1 x 10(4)) than antibody devices (approximately 1.74 x 10(4) to 2.61 x 10(4)). CONCLUSIONS: P-selectin can be used in a compact flow device to capture HSPCs. Selectin-mediated capture of CD34+ HSPCs resulted in enrichment approximately 8-fold higher than the CD34+ cell population from bone marrow MNCs. This study supports the hypothesis that flow-based, adhesion molecule-mediated capture may be a viable alternative approach to the capture and purification of HSPCs.     

Generation of interferon alpha-producing predendritic cell (Pre-DC)2 from human CD34(+) hematopoietic stem cells

Upon viral stimulation, the natural interferon (IFN)-alpha/beta-producing cells (IPCs; also known as pre-dendritic cells (DCs 2) in human blood and peripheral lymphoid tissues rapidly produce huge amounts of IFN-alpha/beta. After performing this innate antiviral immune response, IPCs can differentiate into DCs and strongly stimulate T cell-mediated adaptive immune responses. Using four-color immunofluorescence flow cytometry, we have mapped the developmental pathway of pre-DC2/IPCs from CD34(+) hematopoietic stem cells in human fetal liver, bone marrow, and cord blood. At least four developmental stages were identified, including CD34(++)CD45RA(-) early progenitor cells, CD34(++)CD45RA(+) late progenitor cells, CD34(+)CD45RA(++)CD4(+)interleukin (IL)-3Ralpha(++) pro-DC2, and CD34(-)CD45RA(++) CD4(+)IL-3Ralpha(++) pre-DC2/IPCs. Pro-DC2s have already acquired the capacity to produce large amounts of IFN-alpha/beta upon viral stimulation and to differentiate into DCs in culture with IL-3 and CD40 ligand. CD34(++)CD45RA(-) early progenitor cells did not have the capacity to produce large amounts of IFN-alpha/beta in response to viral stimulation; however, they can be induced to undergo proliferation and differentiation into IPCs/pre-DC2 in culture with FLT3 ligand.     

CD133+ cell selection is an alternative to CD34+ cell selection for ex vivo expansion of hematopoietic stem cells

CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. This study compared the expansion capacities of CD133(+) and CD34(+) cells isolated from the same cord blood (CB) samples. After 14 days culture in stroma-free, serum-free medium in the presence of stem cell factor (SCF), Flt3-1, megakaryocyte growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF), the CD133(+) and CD34(+) fractions displayed comparable expansion of the myeloid compartment (CFC, LTC-IC, and E-LTC-IC). The expansion of CD133(+) CB cells was up to 1262-fold for total cells, 99-fold for CD34(+) cells, 109-fold for CD34(+) CD133(+) cells, 133-fold for CFU-GM, 14.5-fold for LTC-IC, and 7.5-fold for E-LTC-IC. Moreover, the expanded population was able to generate lymphoid B (CD19(+)), NK (CD56(+)), and T (CD4(+) CD8(+)) cells in liquid or fetal thymic organ cultures, while expression of the homing antigen CXCR4 was similar on expanded and nonexpanded CD133(+) or CD34(+) cells. Thus, the CD133(+) subset could be expanded in the same manner as the CD34(+) subset and conserved its multilineage capacity, which would support the relevance of CD133 for clinical hematopoietic selection.