Analysis of maternal and neonatal factors that influence the nucleated and CD34+ cell yield for cord blood banking

BACKGROUND: It would be beneficial to be able to predict the cord blood (CB) cell yield from volunteer donors before cell processing. STUDY DESIGN AND METHODS: The maternal and neonatal factors that influence the total nucleated cell (TNC), CD34+ cell, and CFU-GM yields in CB collected for the Chugoku-Shikoku Cord Blood Bank were evaluated. RESULTS: In a univariate analysis, the volume of CB collected was significantly correlated with the TNC, CD34+ cell, and CFU-GM yields (p < 0.001). A longer cord (p < 0.001), larger placenta (p < 0.001), and bigger baby (p < 0.001) were associated with a greater volume of CB. A female baby (p < 0.05) and longer gestational age (p < 0.005) were associated with a higher TNC concentration. A younger maternal age (p < 0.05), larger birth weight (p < 0.001), shorter gestational age (p < 0.001), and shorter time from collection to processing (p < 0.05) were associated with a higher CD34+ cell concentration. A multivariate linear regression analysis was performed to predict the yield and determine first-level selection criteria to start processing when the volume of CB units was on the borderline. However, this formula might not be suitable for actual use. CONCLUSION: Maternal and neonatal factors appeared to affect CB cell yields. These findings might be useful for efficiently collecting more qualified CB units.     

MARCH-I expression in cord blood CD34+KDR+ cells

Hematopoietic stem cells transplantation has been successfully used in the treatment of patients with hematological malignances. A better knowledge of the mechanisms beyond their ability to completely repopulate the entire hematopoietic system would help in the treatment of hematological diseases. For this reason we focused our studies on a cell population that has been demonstrated to have some peculiar characteristics among the stem cells: CD34+KDR+ cells. These cells, an extremely rare population among the CD34 (0.1%-0.5%) cells, have been demonstrated from different groups to have the potential to give rise to the hematopoietic and endothelial lineage. By a subtraction library approach we found different sequences more expressed in CD34+KDR+ than their CD34+KDR- counterpart. In particular, we found an open reading frame correspondent to a newly characterized E3 ligase, MARCH-I. This gene is part of a recently described family involved in immune response modulation through the proteosomal mediated degradation. MARCH-I expression in stem cells could be important for their intrinsic immune properties.     

Prospective flow cytometric evaluation of nucleated red blood cells in cord blood units and relationship with nucleated and CD34(+) cell quantification

BACKGROUND: Cord blood (CB) represents an alternate source of stem cells in transplantation. Nucleated red blood cells (NRBCs) are a physiological subset of CB population. Although it is important to have an accurate estimate of CD34(+) cell number, NRBCs could compromise white blood cell count and interfere with CD34(+) cell quantification. STUDY DESIGN AND METHODS: A total of 826 CB units were analyzed for total nucleated cells (TNCs), NRBCs, and CD34(+) cells by flow cytometry. NRBCs were also counted conventionally by manual microscopy. Percentages of CD34(+) cells corrected by NRBC count (CD34+c) were determined as follows: %CD34+c = CD34(+)/CD45(+) (x10(6))/(TNCs (x10(8)) - NRBCs (x10(8))). RESULTS: The mean percentages of CD34+ cells and NRBCs were 0.27 percent (range, 0.01%-1.25%) and 7.64 percent (range, 0.13%-84%), respectively. Comparison between flow cytometric and microscopic NRBC count showed a regression of y = 0.685 + 0.719x and a coefficient of determination of r(2) = 0.721. When corrected with NRBC count, the mean percentage of CD34(+) c cells was 0.295 percent (p = 0.0008 compared with CD34(+)%) and mean TNCc count was 14.8 x 10(8) (p < 10(-4) compared to TNC count). CONCLUSION: The determination of NRBCs with a flow cytometric method might represent a new strategy for providing satisfactory quality assurance controls of CB products.     

Maternal and neonatal factors associated with the high yield of mononuclear low-density/CD34+ cells from placental/umbilical cord blood

Placental/umbilical cord blood (CB) contains nucleated cells and hematopoietic stem/progenitor cells (CD34(+) cells). However it is difficult to predict the number of nucleated/CD34(+) cells in each CB before cell processing. Despite many previous studies from institutes affiliated with CB banks in metropolitan areas, little information is available regarding the characteristics of CB units from other medical facilities. The purpose of the present study was to analyze the maternal/neonatal factors on the yield of cells in CB units. A total of 176 CB units were obtained from single-birth and normal vaginal deliveries. Mononuclear low-density (LD) cells were separated using Ficoll-Paque within 24 hrs after CB collection and then processed for the purification of CD34(+) cells. A multiple linear regression analysis was performed to assess the correlations between the yield of cells and maternal/neonatal factors including maternal age, gravid status, duration of labor, gestational age, neonatal height and weight, cord length, and meconium in the amniotic fluid. The total LD cells per CB unit had a weak positive correlation with the maternal age of primigravidae. The total LD cells per CB unit from the primigravidae aged > or = 25 were significantly higher than those from the primigravidae aged < or = 24. The total CD34(+) cells per CB unit from the 1-gravidae were significantly higher than those from the 2-gravidae and 3-gravidae, respectively among all donors. These results indicate that the CB units from the primigravidae aged > or = 25 are more likely to contain higher yield of LD/CD34(+) cells.     

CD34+CD38- is a good predictive marker of cloning ability and expansion potential of CD34+ cord blood cells

BACKGROUND : Ex vivo expansion of HPCs is an attractive approach to overcoming the current limitations of human cord blood transplantation. It is important not only to define the optimal culture conditions but also to know the number of progenitor cells that can be obtained. CD34+ cells have a great variability in their cloning capacity and in their ability to expand HPCs. This study was carried out to assess whether this variability could be due to intrinsic or extrinsic factors.
STUDY DESIGN AND METHODS : CD34+ cells were analyzed for the expression of CD38, CD133, and CD117 and cultured in serum-free culture medium with four cytokine combinations: SCF plus thrombopoietin plus flt3 ligand (STF), STF plus IL-3, STF plus IL-6, and STF plus IL-6 plus IL-3. After a 1-week culture, the numbers of CD34+ cells and CFUs were determined.
RESULTS : The variability observed both in the cloning ability of CD34+ isolated cells and in their expansion capacity was inversely related to the frequency of the more immature CD34+CD38– cells. When more mature CD34+CD38+ cells were present within CD34+-isolated cells, a higher cloning ability, measured as CFUs, and a higher expansion capacity were observed.
CONCLUSION : Enumeration of CD34+CD38– cells is correlated with the number of committed progenitors and the capacity of generating CD34+ cells, an important parameter if expansion protocols must be used in clinical transplantation.     

脐血CD34^＋细胞和外周血单核细胞两种来源的树突状细胞特性？…

目的 体外大量扩增和纯化具有典型表型、形态和功能的树突状细胞（ＤＣ）、以进行相关基础研究和临床应用。方法 采用免疫磁珠江分离脐血ＣＤ３４＾＋细胞及外周血去Ｂ、去Ｔ淋巴细胞的单个核细胞（单核细胞）,然后以ＧＭ－ＣＳＦ、ＩＬ－４、ＴＮＦα、Ｆｌｔ３配基（ＦＬ）、ＳＣＦ等不同的细胞因子配伍分别诱生ＤＣ,通过流式细胞仪、电镜、光镜分析其特性,同时检测其刺激同种Ｔ细胞增殖的能力。结果 脐民外周血诱生ＤＣ的方     

In vitro clinical-grade generation of red blood cells from human umbilical cord blood CD34+ cells

BACKGROUND: There is no appropriate alternative source of red blood cells (RBCs) to relieve the worsening shortage of blood available for transfusion. Therefore, in vitro generation of clinically available RBCs from hematopoietic stem cells could be a promising new source to supplement the blood supply. However, there have been few studies about the generation of clinical‐grade RBCs by coculture on human mesenchymal stem cells (MSCs) and various cytokine supplements, even though the production of pure RBCs requires coculture on stromal cells and proper cytokine supplements. STUDY DESIGN AND METHODS: Umbilical cord blood (CB) CD34+ cells were cultured in serum‐free medium supplemented with two cytokine sets of stem cell factor (SCF) plus interleukin‐3 (IL‐3) plus erythropoietin (EPO) and SCF plus IL‐3 plus EPO plus thrombopoietin (TPO) plus Flt‐3 for 1 week, followed by coculture upon MSCs derived from bone marrow (BM) or CB for 2 weeks. RESULTS: Almost pure clinical‐grade RBCs could be generated by coculturing with CB‐MSCs but not BM‐MSCs. Expansion fold and enucleation rate were significantly higher in coculture with CB‐MSCs than BM‐MSCs. Despite a 2.5‐fold expansion of erythroblasts in the presence of TPO and Flt‐3 for 8 days, the final RBC count was higher without TPO and Flt‐3. CONCLUSIONS: This study is the first report on generating clinical‐grade RBCs by in vitro culture with human MSCs and compared effectiveness of several cytokines for RBC production. This provides a useful basis for future production of clinically available RBCs and a model of erythropoiesis that is analogous to the in vivo system.     

GFP在人脐血CD34~+细胞中的表达及意义

目的探讨慢病毒载体在CD34+脐血细胞(CBCs)中的基因转导效率,为基因治疗的临床应用提供关键材料。方法应用1型人类免疫缺陷病毒(HIV-1)改造而成的第三代自身失活(self-inactivating,SIN)慢病毒载体(lentiviralvector)系统,通过流式细胞术检测基因导入细胞百分比,评价该载体系统在人CBCs中的基因转导效率。结果 HIV载体的转导效率在95%以上。结论基于慢病毒载体基因转导的高效性,该载体系统可作为CD34+CBCs基因转导的极好工具。     

血小板第4因子对脐血CD34+细胞黏附功能的影响

目的研究血小板第4因子(PF4)对新鲜脐血CD34+细胞及扩增后脐血CD34+细胞黏附功能的影响;PF4对脐血CD34+细胞上的黏附分子CD49d及基质细胞趋化因子(SDF-1)受体CXCR4的作用.方法采用免疫磁珠法(MACS)分选CD34+细胞,结晶紫染色测定细胞总黏附性,免疫荧光标记流式细胞仪测定CD49d及CXCR4的表达.结果①PF4 可使新鲜脐血CD34+细胞总黏附性提高,且与剂量相关.②SDF-1 100 ng/ml可使脐血CD34+细胞总黏附性提高.③脐血CD34+细胞扩增10 d后未加PF4刺激的自发以及经SDF-1诱导的黏附功能开始下降,在扩增脐血CD34+细胞不同时间段加入100ng/ml PF4,脐血CD34+细胞对基质层的黏附能力始终保持较高水平,以0天时脐血CD34+细胞黏附性为100%,扩增14 d时脐血CD34+细胞黏附性PF4组为(262.04±64.81)%,同期对照组为(64.35±8.29)%,经SDF-1诱导下扩增14 d的CD34+细胞的总黏附性PF4组为(138.31±32.39)%,同期对照组为(67.66±12.44)%.④PF4 100 ng/ml作用于CD34+细胞时,CD49d表达增长13.02%,CXCR4表达增长17.33%.结论 PF4可使新鲜及扩增后的脐血CD34+细胞黏附功能增强,并促进CD49d及CXCR4的表达,提示PF4可能有助于脐血干细胞的归巢.     

Characterization of multiple CD34+ cell populations in cord blood

Unlike granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood, which show a single homogeneous population of CD34(+) cells, umbilical cord blood (CB) CD34(+) cells are present as multiple populations, CD34(regular) and CD34(bright) (the latter comprising 7.0-58.2% of the total CD34(+) cells), using the ProCOUNT trade mark procedure or with anti-CD34 labeling of immunoselected cells. The CD34(regular) population contains cells with high forward scatter (CD34(regular)FSC(high)) and with low forward scatter (CD34(regular) FSC(low)). Immunomagnetically selected CD34(+) cells, sorted into CD34(regular), CD34(regular) FSC(high), CD34(regular)FSC(low), and CD34(bright) cell populations, were used in in vitro assays: only the CD34(regular)FSC(high) population transmigrated and showed growth of colony-forming unit (CFU) and long-term culture initiating cells (LTC-IC) colonies. The absolute number of CD34(+) cells in CB samples was determined by ProCOUNT trade mark and Stem Kit trade mark enumeration protocols. In liquid stored CB units, ProCOUNT trade mark and Stem Kit trade mark count differences are accounted for by the enumeration of CD34(bright) cells. Differences between ProCOUNT trade mark and Stem Kit trade mark counts using cryopreserved/thawed samples are accounted for by increased CD34(regular) FSC(low) cell numbers (2.0 +/- 1.4% in liquid stored and 27.8 +/- 14.6% in cryopreserved/thawed samples). The ProCOUNT trade mark assay includes the nonfunctional CD34(bright) and CD34(regular)FSC(low) cells as part of the CD34(+) cell count, thereby elevating the absolute number of CD34(+) cells. Using the Stem Kit trade mark assay method of gating, CD34(bright) and CD34(regular)FSC(low) cells are not counted. Our data indicate that the CD34(regular)FSC(high) cell population has functional characteristics based on the in vitro assays and a more accurate count of these cells can be achieved using the Stem Kit trade mark assay.     

脐血CD34+细胞与单个核细胞的体外扩增特性研究

目的　探讨CD34+ 富集细胞和单个核细胞 (MNC)的体外扩增特性。方法　利用Min iMACS系统富集CD34+ 细胞 ,在相同条件下与同批MNC进行对照培养 ;观察了再次富选和MNC培养上清 (MNC SN)对CD34+ 富集细胞扩增的影响 ;并尝试了MNCCD34- 细胞的培养。结果　虽然CD34+ 富集细胞具有很高的扩增潜力 ,但在培养过程中 ,其集落密度和CD34 + 细胞含量却始终呈下降趋势 ,而MNC在培养中却出现了一个上升的趋势 ,集落密度和CD34+ 细胞含量分别由第 0天的 (4 12± 16 7) 10 5细胞和 (1.12± 0 .4 2 ) %增至第 7天的 (116 2± 5 6 6 ) 10 5细胞和 (4 .17± 1.4 4 ) % ;再次富选可以使培养过的CD34+ 富集细胞的总细胞和CD34+ 细胞扩增能力大大提高 ;MNCCD34- 细胞具有集落形成和转化为CD34+ 细胞的能力 ;MNC SN对CD34+ 富集细胞的集落形成有促进作用 ,而同时又对CD34+ 细胞有促分化作用。结论　CD34+ 富集细胞在体外大量扩增的同时存在大量分化 ,其在培养过程中产生的CD34-细胞对CD34+ 细胞的扩增有抑制作用 ;脐血MNC中大量的CD34- 细胞含有造血干 祖细胞 ,其分泌的细胞因子有促进CD34+ 细胞向较为成熟的集落形成祖细胞分化的作用。     

脐血CD34+细胞体外扩增诱导成熟树突状细胞实验研究

目的建立体外诱导和扩增人脐血树突状细胞（DC）的方法,并进行生物学鉴定。方法脐血细胞经免疫磁珠法分离纯化为CD34^＋细胞,加入细胞因子（GM-CSF和TNF-α培养约2周,光镜观察培养的DC形态学特征;通过与同种T细胞混合培养,采用MTT比色分析法测定不同浓度的DC激发同种T细胞增殖的能力。结果培养的DC胞浆突起大而长,呈树突状,具有DC的典型形态,并且具有强烈的激发同种异体T细胞增殖的能力。结论从脐血细胞分离纯化CD34^＋细胞,加入细胞因子（GM-CSF和TNF-α培养,能获得大量、较高纯度的DC。DC具有强烈的激发同种异体T细胞增殖的能力。     

人脐血CD34+CD38-细胞免疫表型和染色体核型特征分析

目的 分离脐血干／祖细胞(CD34^ CD38)进行体外长期培养，观察分析其增殖、细胞表面分子标志和染色体核型的特征。方法 用流式细胞仪分选CD34-FITC和CD38-PE标记的CD34^ CD38脐血原始细胞，在含细胞生长因子IL-3、IL-6、GM-CSF、EPO、SCF和胰岛素样生长因子的干细胞培养基中培养6个月，用流式细胞术检测体外培养30d的干／祖细胞表面标记，并用G显带方法分析其染色体核型。结果 在一定培养条件下，经7～12d培养，脐血干／祖细胞(CD34^ CD38)开始增殖。培养6个月后，每孔接种1个细胞，细胞数增殖至250～350个；每孔接种10个细胞，细胞数可增殖至400～500个。每孔接种1个细胞其细胞增殖峰持续时间(8～9代)比接种10个细胞(6～7代)长：经体外长期培养增殖，细胞仍强烈显示十／祖细胞表面分子标记(CD34^ CD38^-)；细胞染色体数目、结构未见异常。结论 脐血干／祖细胞(CD34^ CD38^ )经体外特异性培养增殖，可为大量脐血干／祖细胞移植提供细胞来源。     

脐血浆神经营养活性蛋白成分对CD34+细胞增殖分化的影响

背景:迄今为止,人们对于脐血浆中是否存在活性成分及其对于神经发育及神经损伤修复的作用认识尚不足,有待深入研究.目的:检测脐血浆生物活性成分,观察其在神经发育及神经损伤修复中的作用.方法:采用抗体芯片技术对脐血浆和健康青年女性静脉血浆活性成分进行对比分析,采用免疫磁珠从脐血中分选出CD34+细胞,经体外培养计数观察CD34+细胞增殖能力,通过免疫组化法观察细胞分化情况.结果与结论:脐血浆中有31种蛋白分子的含量显著高于静脉血浆,其中具有促进神经再生和神经修复潜能的活性蛋白10种,包括FGF4、Frizzled-3、IL-3、RAGE、CRIM-1、Neuritin、Neuropilin-2、Neurturin、SFRP-3、Tomoregulin-1;在CD34+细胞培养液中加入脐血浆能显著促进细胞增殖,促进细胞向神经细胞分化.     

明胶酶A、B在脐血CD34+细胞及部分白血病细胞系中的表达

目的　研究骨髓及脐血CD34+ 细胞及白血病细胞系的明胶酶 ,即基质金属蛋白酶 9(MMP 9,明胶酶B)和基质金属蛋白酶 2 (MMP 2 ,明胶酶A)的产生 ,探讨它们在CD34+ 细胞迁移、归巢中的意义及在白血病发病中的作用。方法　通过MiniMACS磁珠分离技术分离脐血及骨髓的CD34+细胞 ,通过酶谱分析法测定脐血、骨髓CD34 + 细胞及白血病细胞系的无血清条件培养液中明胶酶的表达。结果　在脐血CD34+ 细胞的条件培养液中可测出相对分子质量为 92× 10 3的亮带 ,骨髓CD34+ 细胞的条件培养液中未测到亮带。部分白血病细胞系U937、KG 1a、HL 6 0可产生相对分子质量为 92×10 3和 72× 10 3的亮带 ,J6 1、J6 2细胞系可产生相对分子质量为 92× 10 3的亮带 ,而HEL、Namalva、CEM、K5 6 2、LCL H不产生亮带。结论　脐血CD34+ 细胞可产生MMP 9,而骨髓CD34+ 细胞不产生 ,脐血CD34+ 细胞具有较强的穿透基底膜的迁移能力并与MMP 9相关。在白血病细胞系中 ,部分髓系白血病细胞系产生MMP 9和MMP 2 ,而非髓系白血病细胞系不产生。