不同来源的CD34^＋细胞归巢相关分子的表达

目的 研究不同来源CD34^＋细胞归巢相关分子（HRM）的表达情况。方法采用免疫磁珠法（MACS）分选不同来源的CD34^＋细胞，免疫荧光标记流式细胞仪测定HRM的表达。结果骨髓（BM）、动员后的外周血（mPB）及脐血（UCB）来源的CD34^＋细胞均高表达细胞黏附分子CD49d、CD49e、CD54、CD11a、CD62L、CD44、CD31。UCB来源的CD34^＋细胞表面表达的细胞黏附分子中，CD49e表达显著低于BM和mPB来源的CD34^＋细胞（P〈0．05），CD54表达显著低于mPB来源者（P〈0．05），CXCR4表达显著低于BM来源者（P〈0．05）。结论UCB来源的造血干／祖细胞归巢能力低可能是UCB移植造血重建延迟的原因之一。     

骨髓间充质干细胞的黏附分子表达及其对脐血造血干细胞扩增的支持作用

目的:探讨骨髓间充质干细胞(MSC)的黏附分子表达及其对脐血造血干/祖细胞体外增殖的支持作用。方法:取正常骨髓单个核细胞贴壁培养,梭形细胞完全融合后传代,用流式细胞仪检测免疫表型;将脐血CD34+细胞接种到MSC或其他培养液上,比较不同培养条件对造血细胞扩增能力及黏附分子表达的影响。结果:MSC在体外培养中分为成纤维样成熟MSC(mMSC)和体积很小的圆形RS(rapidlyselfrenewingcells)细胞;两群细胞的CD34、CD45、CD3、CD19、CD33、HLADR、CD38均为阴性,而CD90、CD105、CD166、CD29、CD44、CD49e、CD54、CD13呈阳性,但RS细胞表面抗原表达的阳性率和平均荧光强度都明显低于mMSC,而CD117的表达高于mMSC;脐血CD34+细胞在MSC和细胞因子作用下,扩增8d后有核细胞、CD34+、CD34+CD38-细胞和CFUCs分别扩增(145.57±17.89)、(37.47±13.78)、(69.78±50.07)、(10.74±5.89)和(20.73±5.54)倍,均显著高于对照组,扩增后CD34+细胞的ALCAM、VLAα4、VLAα5、VLAβ1、HCAM、PECAM和LFA1表达较扩增前无显著变化。结论:MSC是一个异质性的细胞群体,可为造血干细胞(HSC)体外扩增提供适宜的微环境,有助于抑制HSC分化并保持其归巢能力。     

体外扩增脐血巨核细胞生物学特性的研究

目的:研究脐血巨核细胞的体外扩增及扩增后脐血巨核细胞的生物学特性和功能,为脐血巨核细胞的扩增和临床应用提供依据。方法:收集16份足月妊娠健康新生儿的脐带血,免疫磁珠法分离出其中的CD34 细胞。采用细胞因子组合(1)(TPO加SCF加IL-3加IL-6)、细胞因子组合(2)(TPO加SCF)2种细胞因子组合．将富集的脐血CD34 接种于无血清无基质细胞的悬浮培养体系中,分别在3、7、10、14 d收集扩增产物。运用流式细胞术检测巨核细胞的表型和黏附分子的表达;血浆块法检测巨核细胞集落(CFU-MK)的形成;对巨核细胞进行DNA含量检测以评价巨核细胞的成熟程度。结果:不同细胞因子组合和培养时间扩增后,巨核细胞的数量和生物学特性不同。随着培养时间的延长,巨核细胞(CD41 )的数量逐渐增加,但培养至14 d时增势减缓。因子组合(1)各时间段的扩增能力(分别扩增5．2、40．7、121．2、149．7倍)均比因子组合(2)(分别扩增3．8、27．4、85．9、106．5倍)强(P<0．05),但因子组合(2)的扩增能力仍能满足临床的需要。巨核祖细胞(CD34 ／CD41 )的数量在7 d时最多,这也被CFU-MK所证实。DNA含量检测发现．随着培养天数的增加,多倍体细胞所占的百分比增加。扩增后巨核细胞黏附分子CD49d和CD11a的表达维持在高水平,而CD54降低(P<0．05)。结论:通过对扩增后巨核细胞的生物学特性研究,有助于寻找有效、简便、易于植入受者体内的扩增方法。     

脐血造血细胞中AC133抗原表达及其功能特征研究

目的 探讨AC133抗原在正常人造血细胞中表达的意义。方法 采用双色直接免疫荧光标记法,使用流式细胞仪测定脐血中造血细胞的免疫表型。通过祖细胞集落形成单位（CFC）和长期培养启动细胞（LTC-IC）的测定对脐血中AC133^ ,CD34^ 和AC133^-CD34^ 细胞的造血潜能进行了研究。结果 脐血单个核细胞（MNCs)中AC133^ 细胞比例为0.67%,AC133^ 细胞中93.4%表达CD34抗原,94.2%为PKH26阳性,虽然三群细胞产生的CFC总数差异无显著性。但AC133^ 细胞中50%以上为粒-单集落形成单位,CD34^ ,AC133^-CD34^ 细胞中60%和80%以上为红系集落形成单位,AC133^ 细胞产生的LTC-IC显著高于CD34^ 和AC133^-CD34^ 细胞。体外培养7d,AC133^ 细胞组细胞总数和CFC分别扩增28.2和7.1倍,6.8%细胞仍保持PKH26阳性。结论 脐血中早期造血干/祖细胞富集在AC133^ 细胞群中,AC133^ 细胞在体外具有较强的增殖能力,既能产生大量的早期定向祖细胞,又能保持一定数量的早期干/祖细胞,AC133抗原可作为临床分选造血干/祖细胞的有效标志。     

乙型肝炎病毒感染对造血干细胞活性的影响

目的 了解HBV感染对脐血来源的造血干细胞活性的影响.方法 将分离纯化的健康脐血CD34+细胞,在含有干细胞生长因子(SCF)、酪氨酸激酶受体家族Ⅲ配体(FL)、促血小板生成素(TPO)、IL-3和10%FBS的IMDM培养基中扩增,同时加入高拷贝HBV,观察干细胞扩增与病毒复制规律;干细胞扩增后,在巨细胞集落刺激因子(GM-CSF)和IL-4作用下诱生树突状细胞,对干细胞及树突细胞进行形态学观察,并检测其表面分子的表达.两组间比较采用独立t检验,多组间比较采用方差分析.结果 感染HBV的造血干细胞自然增殖能力明显低于正常干细胞(P<0.01);加入细胞因子后细胞增殖增加(P<0.01),细胞内HBV DNA复制也增加(P<0.01),但其干细胞增殖仍低于正常干细胞加细胞因子组(P<0.05).电子显微镜观察发现HBV感染干细胞后胞质内出现Dane颗粒;HBV感染的干细胞经诱导为树突状细胞后,其免疫表型CD80、CD86、CD1a、HLA-DR的表达均低于未感染组(P<0.01).结论 HBV可以感染CD34+造血干细胞,并随干细胞增殖而复制增加,HBV不仅抑制造血干细胞增殖,而且下调干细胞分化来源的树突状细胞免疫表型的表达.     

低浓度增植细胞核抗原反义核酸对脐血CD34+ 造血干/祖细胞体外扩增的影响

目的研究增植细胞核抗原反义核酸(PCNA-ASODN)对脐血造血干/祖细胞体外扩增的调节作用.方法采用免疫磁珠分离技术从新鲜脐带血中分离CD34+细胞,使用低浓度的PCNA-ASODN作用于体外扩增的CD34+细胞,应用流式细胞仪技术检测其扩增后各种细胞的数量,细胞内PCNA的表达及细胞周期的变化情况.结果与对照组相比,加入了低浓度PCNA-ASODN的实验组,其PCNA的表达水平明显较低,表达率为(27.2±3.6)%,且G0/G1期细胞占(55.9±4.4)%,扩增的细胞中CD34+细胞比例增高,达(33.4±3.2)%,而且CD34+CD38-细胞数量达到(57.8±9.9)倍.结论低浓度的PCNA-ASODN抑制CD34+细胞在增殖过程中PCNA表达,减缓扩增过程中的分化作用.     

高三尖杉酯碱对Ph+树突状细胞成熟与功能的影响

目的：观察不同浓度的高三尖杉酯碱(HHT)对慢性粒细胞白血病(CML)Ph^ 树突状细胞(DC)成熟与功能的影响。方法：采用Ficoll密度离心法分离制备单个核细胞(MNC)，在体外诱导扩增DC，在培养体系中添加不同浓度的HHT，通过细胞化学染色、流式细胞仪检测细胞表面分子表达水平、^3H—TdR掺入法检测细胞功能等方法，对所得细胞进行鉴定。结果：CML DC存在表型及功能障碍。在培养体系中添加HHT，能获得较多成熟CML DC，提高其细胞表面共刺激分子CD80以及黏附分子LFA-2的表达水平，并可改善其刺激淋巴细胞增殖的能力。结论：适合浓度的HHT可影响Ph^ DC的分化和成熟，并可改变DC细胞表面免疫分子的表达，增强其功能，     

粒细胞集落刺激因子动员对CD34^＋细胞黏附分子表达的影响

目的探讨粒细胞集落刺激因子（G—CSF）动员对CD34^＋细胞黏附分子表达的影响。方法应用流式细胞仪检测健康供者稳态及G—CSF动员过程中骨髓、外周血CD34^＋细胞的黏附分子表达变化，并应用结晶紫染色测定CD34^＋细胞的黏附功能。结果G-CSF动员后CD34^＋CD49d^＋细胞比例无显著下降，外周血CD34^＋CD621．^＋、CD34^＋CD54^＋和CD34^＋CDlla^＋细胞比例增高。动员后CD34^＋细胞表面CD49d的平均荧光强度显著减弱，但CD49e、CD62L、CDlla、CD54的平均荧光强度虽呈减弱趋势，却无统计学差异。动员后CD34^＋细胞黏附纤连蛋白能力下降。结论G—CSF通过降低造血干细胞与骨髓基质的黏附而产生动员效应。     

脐血造血干细胞向巨核细胞诱导分化的研究

目的:建立脐血CD34+造血干细胞向巨核细胞诱导分化的体系,探讨最佳的扩增方法。方法:免疫磁珠法分离获得CD34+细胞培养在无血清无基质培养液中,采用TPO加SCF加IL-3加IL-6、TPO加SCF加IL-3、TPO加SCF3种不同因子组合对其诱导分化及扩增。收集3、7、10、14d的扩增产物,运用荧光显微镜检测巨核细胞的表面标志;流式细胞术(FCM)检测巨核细胞的凋亡;并对巨核细胞形成单位(CFU-MK)及DNA含量进行检测。结果:分离获得的CD34+细胞在体外可以有效扩增,随培养时间的延长CD34+/CD41+细胞数第7天达最高值,之后逐渐下降;而CD41+、CD42b+、CD61+细胞随培养时间的延长表达量逐渐增高。加入IL-3和IL-6后,Annexin Ⅴ阳性细胞由(8.26±2.49)%降至(3.51±1.24)%。CFU-MK的数量在第10天时最高,且8倍体及8倍体以上的巨核细胞所占的的百分比增加,即成熟产板型巨核细胞增加。结论:脐血CD34+造血干细胞在体外可向巨核细胞诱导分化及有效扩增。3种因子组合中TPO加SCF加IL-3加IL-6组扩增效率最高。     

血管紧张素Ⅱ对定向造血祖细胞BFU-E体外扩增的影响

目的：研究血管紧张素Ⅱ(AngⅡ)参与造血调控的机制及其对造血干／祖细胞BFU-E定向优势分化的作用。方法：将脐血来源的CD34^ 细胞在不同的细胞因子组合中悬浮培养，使它们向红系造血祖细胞优势分化，分别在不同时间对培养细胞进行3种处理：一部分细胞提取：RNA，用RT-PCR方法检测AngⅡ受体：mR-NA的转录；一部分添加AngⅡ和其他细胞因子继续培养3天后转移到半固体培养基中进行集落计数；其余细胞在原来的条件下继续悬浮培养。结果：新分离的CD34^ 细胞表面无AngⅡ受体mRNA的表达，此时添加AngⅡ对造血祖细胞生长无明显作用；在CD34^ 向红系优势分化体系中悬浮培养7天后，细胞中能检测到AngⅡ受体mRNA的表达，此时添加AngⅡ可刺激BFU-E的扩增；在培养第14天，细胞中己无AngⅡ受体表达，此时添加AngⅡ对红系祖细胞的扩增已无明显作用。结论：红系祖细胞在分化过程中有AngⅡ受体mRNA的表达，AngⅡ可刺激早期红系造血细胞的增生，并可提高造血细胞向红系定向优势分化的效率。     

Circulating CD34+ cells in cord blood and mobilized blood have a different profile of adhesion molecules than bone marrow CD34+ cells

Abstract: The expression of adhesion molecules was studied on CD34+ hematopoietic precursors in cord blood, bone marrow and mobilized blood. The samples were labeled in a double immunofluorescence procedure with a CD34 monoclonal antibody and with antibodies against maturation and differentiation antigens and adhesion molecules. Myeloid precursors formed the majority of the CD34+ cells in all samples. In bone marrow a separate cluster of B-cell precursors with low forward scatter was present. Nearly all CD34+ cells in normal bone marrow expressed VLA-4 and VLA-5, PECAM-1, LFA-3 and HCAM. The majority of the CD34+ cells also had LFA –1 and L-selectin on the surface membrane. A small subset was VLA-2, VLA-3, ICAM-1 or Mac-1 positive. CD34+ cells expressing the vitronectin receptor or the CD11c antigen were rare. Cord blood and mobilized blood CD34+ cells had a lower expression of VLA-2, VLA-3 and VLA-5 and a higher expression of LFA-1, ICAM-1 and L-selectin than bone marrow CD34+ cells. Except for LFA-1, this was not due to the presence of more myeloid precursors in these samples. Low β₁ integrin expression may lead to less adhesion to the extracellular matrix. High expression of L-selectin may facilitate interaction with endothelial cells. Therefore, this phenotype may favour mobilization.     

Expression of adhesion molecules on CD34 cells does not correlate with property to adhere to normal marrow-derived stroma

The differences of engraftment kinetics after umbilical cord blood (CB) and mobilized peripheral blood (MPB) transplantation are not yet fully understood. Since homing into bone marrow microenvironment would certainly play crucial role during engraftment, we have investigated adhesion capacities of CB and MPB CD34⁺ cells to bone marrow stromal cells (SC) and expression of several molecules known to be important during homing process. Cells, at day 0 and after 48 hour culture with SCF+ IL-3, are plated for 2 hours on BM confluent stromal layer. The non adherent (na) and adherent (a) fractions are then recovered. Cells are counted and evaluated for the coexpression of CD34 and VLA-4 (CD49d), VLA-5 (CD49e), L-selectin (CD62L) or CXCR4. The adhesion molecule expression is expressed in term of antigen density (Mean Equivalent Soluble Fluorescence (MESF)x 10³).Our results show that a) cell adhesion capacity is significantly increased after culture in CB (78.6±4.0% vs 58.2±7.4%) as well in MPB (72.5±5.9% vs 51.5±5.4%) and is not different between the two sources of progenitors; b) VLA-4, VLA-5, CD62L and CXCR4 expression increased significantly after culture, in CB and in MPB; c) no specific expression on adherent cells could be observed except for L-selectin after 48h culture; d) the only significant difference between CB and MPB concerns the CXCR4 expression,lower on PB cells but independent of cell adhesion capacities. In conclusions, it seems that neither adhesion capacities nor expression of evaluated adhesion molecules could explain differences between CB and MPB engraftment.     

Different expression of adhesion molecules on myeloid and B-lymphoid CD34+ progenitors in normal bone marrow

Abstract: The expression of adhesion molecules was studied on B lymphoid and myeloid CD34⁺ precursors in normal bone marrow. Bone marrow aspirates were labelled in a double fluorescence procedure with the CD34 monoclonal antibody 43A1 and with antibodies directed against maturation and differentiation antigens and adhesion molecules. Three clusters of CD34⁺ cells could be distinguished by their light scatter characteristics in flow cytometry. The population with the lowest forward scatter contained B-lymphoid precursors while the two others showed phenotypic characteristics of, respectively, early and late myeloid precursors. Nearly all CD34⁺ cells in the 3 subpopulations expressed VLA-4, VLA-5, LFA-3 and H-CAM. B-lymphoid progenitors showed a higher density of VLA-4 and VLA-5 than the myeloid progenitors. Myeloid precursors, and particularly the late subset, expressed more HCAM than the B-lymphoid progenitors. The majority of the CD34⁺ cells also expressed LFA-1 and L-selectin. Higher numbers of positive cells were found in the myeloid subset. The early myeloid subset showed the highest positivity for L-selectin. We conclude that B lymphoid and early and late myeloid CD34⁺ precursors in normal bone marrow show a different profile of adhesion molecules. These profiles could reflect a higher tendency of the myeloid CD34⁺ precursors to circulate.     

Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation

Adhesion molecules play a role in the migration of hematopoietic progenitor cells and regulation of hematopoiesis. To study whether the mobilization process is associated with changes in expression of adhesion molecules, the expression of CD31, CD44, L-selectin, sialyl Lewisx, beta 1 integrins very late antigen 4 (VLA-4) and VLA-5, and beta 2 integrins lymphocyte function-associated 1 and Mac-1 was measured on either bone marrow (BM) CD34+ cells or on peripheral blood CD34+ cells mobilized with a combination of granulocyte colony- stimulating factor (G-CSF) and chemotherapy. beta 1 integrin VLA-4 was expressed at a significantly lower concentration on peripheral blood progenitor cells than on BM CD34+ cells, procured either during steady- state hematopoiesis or at the time of leukocytapheresis. No differences in the level of expression were found for the other adhesion molecules. To obtain insight in which adhesion molecules may participate in the homing of peripheral blood stem cells (PBSCs), the number of CD34+ cells expressing these adhesion molecules present in leukocytapheresis material was quantified and correlated with hematopoietic recovery after intensive chemotherapy in 27 patients. The number of CD34+ cells in the subset defined by L-selectin expression correlated significantly better with time to platelet recovery after PBSC transplantation (r = - .86) than did the total number of CD34+ cells (r = -.55). Statistical analysis of the relationship between the number of CD34+L-selectin+ cells and platelet recovery resulted in a threshold value for rapid platelet recovery of 2.1 x 10(6) CD34+ L-selectin+ cells/kg. A rapid platelet recovery (< or = 14 days) was observed in 13 of 15 patients who received > or = 2.1 x 10(6) CD34+ L-selectin+ cells/kg (median, 11 days; range, 7 to 16 days), whereas 10 of 12 patients who received less double positive cells had a relative slow platelet recovery (median, 20 days; range, 13 to 37 days). The L-selectin+ subpopulation of CD34+ cells also correlated better with time to neutrophil recovery (r = - .70) than did the total number of reinfused CD34+ cells (r = -.51). However, this latter difference failed to reach statistical significance. This study suggests that L-selectin is involved in the homing of CD34+ cells after PBSC transplantation.     

Different expression of adhesion molecules on CD34+ cells in AML and B-lineage ALL and their normal bone marrow counterparts.

The expression of adhesion molecules on CD34+ cells in acute myeloid leukemia (AML) and B-lineage acute lymphoblastic leukemia (B-lineage ALL) was compared with that on the myeloid and B-lymphoid CD34+ cells in normal bone marrow. Bone marrow aspirates of 10 patients with AML, 8 patients with B-lineage ALL and of 6 healthy volunteers were examined. The phenotype of the CD34+ cells was determined with a double immunofluorescence method and flow cytometry. CD34+ cells in AML and B-lineage ALL showed a lower expression of VLA-2 and VLA-3 and a higher expression of ICAM-1 and LFA-3 than their normal bone marrow counterparts. AML CD34+ cells had less L-selectin but more VLA-5 on their surface membrane than normal myeloid CD34+ cells. B-lineage ALL CD34+ cells showed an overexpression of LFA-3. In individual patients deficiencies or over-expression of the beta1 integrin chain, VLA-4, PECAM-1 or HCAM also occurred. An abnormal adhesive capacity of the leukemic cells may influence their proliferation, their localisation and apoptosis. An aberrant expression of adhesion molecules may be used for the detection of minimal residual leukemia in these patients.     

A systematic strategy to optimize ex vivo expansion medium for human hematopoietic stem cells derived from umbilical cord blood mononuclear cells

OBJECTIVE: In this study, a serum-free, stroma-free, and chemically defined medium for hematopoietic stem cell (HSC) expansion was systematically developed and optimized using factorial design and the steepest ascent method. MATERIALS AND METHODS: Mononuclear cells (MNCs) were isolated from umbilical cord blood (UCB). HSCs were stimulated to proliferate ex vivo in the MNC culture system with variable serum substitutes, cytokines, and basal media according to experimental design. The expanded cells were assessed for cellular characteristics by surface antigen analysis, colony-forming cell assay (CFC assay), and long-term culture-initiating cell assay (LTC-IC assay). RESULTS: The optimal compositions of serum substitutes and the cytokine cocktail for HSC expansion in the MNC culture system were BIT (4 g/L BSA, 0.71 microg/mL insulin, and 27.81 microg/mL transferrin), and CC-9 (5.53 ng/mL TPO, 2.03 ng/mL IL-3, 16 ng/mL SCF, 4.43 ng/mL FL, 2.36 ng/mL IL-6, 1.91 ng/mL G-CSF, 1.56 ng/mL GM-CSF, 2.64 ng/mL SCGF, and 0.69 ng/mL IL-11) in the Iscove's modified Dulbecco's medium. After 6-day culture, the absolute fold expansions for white blood cells, CD34+ cells, CD34+CD38- cells, CFC, and LTC-IC were 1.4-, 30.4-, 63.9-, 10.7-, 2.8-fold, respectively. CONCLUSION: Using the statistic methodology to develop HSC medium, our formula had lower cytokine concentrations comparing to other literatures and commercial media, but had superior or comparable expansion ability on HSC growth.     

Ex vivo expansion of umbilical cord blood (UCB) CD34+ cells alters the expression and function of α4β1 and α5β1 integrins

We have investigated the influence of ex vivo expansion of human CD34(+) cord blood cells on the expression and function of adhesion molecules involved in the homing and engraftment of haematopoietic progenitors. Ex vivo expansion of umbilical cord blood CD34(+) cells for 6 d in the presence of interleukin 3 (IL-3), IL-6 and stem cell factor (SCF) or IL-11, SCF and Flt-3L resulted in increased expression of alpha 4, alpha 5, beta 1, alpha M and beta 2 integrins. However, a significant decrease in the adhesion of progenitor cells to fibronectin was observed after the ex vivo culture (adhesion of granulocyte-macrophage colony-forming units (CFU-GM) was 22 +/- 4% in fresh cells versus 5 +/- 2% and 2 +/- 2% in each combination of cytokines). Incubation with the beta 1 integrin-activating antibody TS2/16 restored adhesion to fibronectin. Transplantation of ex vivo expanded umbilical cord blood CD34(+) cells was associated with an early delayed engraftment in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Incubation of cells with the monoclonal antibody TS2/16 before transplantation almost completely abrogated NOD/SCID repopulating ability of both fresh and expanded CD34(+) cells. The seeding efficiency of fresh and expanded CD34(+) cells was similar, but markedly reduced after incubation with the TS2/16 monoclonal antibody. Our results show that functional activation of beta 1 integrins could overcome the decreased very late antigen (VLA)-4- and VLA-5-mediated adhesion observed after ex vivo expansion of haematopoietic progenitors. However, in vivo, these effects induced an almost complete abrogation of the homing and repopulating ability of CD34(+) UCB cells.     

Adhesion molecules on CD 34+ hematopoietic cells in normal human bone marrow and leukemia

Summary Expression of selected adhesion molecules of the integrin and immunoglobulin family was investigated on CD 34+ leukemic cells in 19 AML and 11 ALL cases to evaluate phenotypic differences in adhesive properties of malignant hematopoietic precursor cells in comparison to normal bone marrow CD 34+ cells. Of the 2-integrin family, CD 11a was expressed on > 50% of CD 34+ cells in normal bone marrow and almost all leukemias, whereas CD 11 b and CD 11 c were not expressed on CD 34+ cells in normal bone marrow, but were found on CD 34+ blasts in some leukemias of a heterogeneous immunophenotype. Of the 1-family, CDw 49d (VLA-4) was strongly expressed on normal CD 34+ bone marrow cells and on the blasts of all 30 CD 34+ leukemic samples, whereas CDw 49 b (VLA-2) was absent on CD 34+ cells in normal bone marrow, but detected on CD 34+ cells in a few leukemias which did not constitute a clinical or phenotypic entity according to the FAB classification or immunocytological analysis. The lymphocyte-homing-associated adhesion molecule CD 44 (HCAM) and CD 58 (LFA-3) were expressed on CD 34+ cells in all investigated cases of normal and leukemic bone marrow. ICAM-1 (CD 54), the inducible receptor ligand for CD 11 a/CD 18, although present on CD 34+ cells in normal bone marrow, was lacking on blast cells of some ALL and AML cases. So far, the variable expression of 2-integrins as well as of VLA-2 and of ICAM-1 could indicate distinct differences in cell-cell or cell-matrix adhesion of leukemic cells in ALL and AML patients.     

Different behaviour of fresh and cultured CD34+ cells during immunomagnetic separation

In-vitro expansion of human cord blood (CB) cells could enhance peripheral blood recovery and ensure long-term engraftment of larger recipients in the clinical transplant setting. Enrichment of CD34+ cells using the MiniMACS column has been evaluated for the preparation of CB CD34+ cells before and after expansion culture. Repurification of CD34+ cells after culture would assist accurate phenotypic and functional analysis. When fresh CB mononuclear cells (MNC) were separated, the MACS positive (CD34+) fraction (90.1% pure) contained a mean (+/- SD, n = 5) of 93.0 +/- 8.0% of the eluted CD34+ cells, 99.6 +/- 0.7% of the CFU-GM and all of the eluted long-term culture-initiating cells (LTC-IC). Cord blood CD34+ cells were then cultured for 14 d with IL-3, IL-6, SCF, G-CSF and GM-CSF, each at 10 ng/ml. The total cell expansion was 2490 +/- 200-fold and the CD34+ cell expansion was 49 +/- 17-fold. The percentage of CD34+ cells present after expansion culture was 1.2 +/- 0.85%. When these cells were repurified on the MiniMACS column, the MACS positive fraction only contained 40.3 +/- 13.4% of the eluted CD34+ cells which was enriched for the mature CD34+ CD38+ subset, 24.4 +/- 8.8% of the eluted CFU-GM and 79.5 +/- 11.0% of the LTC-IC. The remaining cells were eluted in the MACS negative fraction. In conclusion, repurification of cultured CD34+ cells does not yield a representative population and many progenitors are lost in the MACS negative fraction. This can give misleading phenotypic and functional data. Cell losses may be important in the clinical setting if cultured cells were repurified for purging.