Large-scale expansion and transplantation of CD34(+) hematopoietic cells: in vitro and in vivo confirmation of neutropenia abrogation related to the expansion process without impairment of the long-term engraftment capacity

BACKGROUND: Herein are reported the results obtained in all multiple myeloma patients transplanted with peripheral blood hematopoietic progenitor cells submitted to ex vivo expansion. STUDY DESIGN AND METHODS: Patients had blood progenitor cell mobilization with cyclophosphamide and filgrastim. CD34+ cells were expanded for 10 days in a medium containing granulocyte-colony-stimulating factor (G-CSF), stem cell factor, and megakaryocyte growth and development factor (MGDF). Twenty-seven patients underwent transplantation with expanded and nonexpanded cells and 7 patients underwent transplantation with expanded cells only. RESULTS: The median fold cell expansion was 29.1. The number of colony-forming unit-granulocyte-macrophage (CFU-GM) and CD34+ cells, and the long-term culture-initiating cell (LTC-IC) activity increased with median fold values of 14.7, 2.75, and 2.25, respectively. Postmyeloablative neutropenia was abrogated in 24 of 27 patients transplanted with expanded cells plus nonexpanded cells. The median duration of severe neutropenia was 0 days and correlated with the number of cells and CFU-GM infused. Survival was similar to that of a historical control group. Our LTC-IC and NOD-SCID mice studies showed that the expanded cells are able of sustaining long-term hematopoiesis. Seven other patients received transplantation with expanded cells alone. Absolute neutropenia was abrogated in 6 patients. The median duration of neutropenia was 0 days. Two patients who received the lower number of total cells or CFU-GM had brief secondary neutropenia, which resolved after G-CSF injections. CONCLUSION: CD34+ cells expanded ex vivo can abrogate absolute and severe neutropenia after high-dose therapy. The results of the amplification process are strongly related to the delay of hematopoietic recovery.     

Expansion of mobilized peripheral blood progenitor cells under defined culture conditions rsing CD34+CD71-CD45- cells as a starting population

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions supporting the expansion of transplantable hematopoietic stem cells. We assessed the expansion potential of CD34+CD71-CD45- cells derived from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood under recently defined serum-free culture conditions. The CD34+CD71-CD45- cells in mobilized peripheral blood were found to contain the majority (92%+/-5.6) of primitive long-term culture initiating cells (LTCIC) and 53.5%+/-16.7 of the more committed colony-forming cells (CFC). Furthermore, this population represents 23.3%+/-4.1 of the total CD34+ cells and allows reduction of the cell density important for maintenance/expansion of primitive progenitor cells. CD34+ CD71- CD45- cells were cultured in defined serum-free media supplemented with 300 ng each of Flt-3 ligand and stem cell factor (SCF), 60 ng of interleukin (IL)-3, and 20 ng each of IL-6 and G-CSF. Mononuclear cells (MNC) and CFC were expanded 50-fold and 200-fold, respectively; primitive progenitor cells (LTC-IC) were maintained at input values after a total of 10 days of expansion. The addition of IL-15 to our cytokine cocktail expanded LTC-IC 2- to 3-fold and CFC to >500-fold. The data presented should allow clinical manipulation (purging) and expansion procedures with mobilized PBPC harvests without the loss of primitive progenitor cells and could be made applicable for large-scale clinical expansion.     

系统性红斑狼疮骨髓受累机制初步研究

目的 初步探讨系统性红斑狼疮(SLE)骨髓受累的可能机制.方法 对21例初治SEE患者骨髓标本21份,10例正常对照骨髓标本分别进行骨髓单个核细胞(BMMNC)Coombs试验并采用甲基纤维素法检测CFU-E、CFU-GM、BFU-E产率.结果 10份正常对照骨髓标本BMMNC-Coombs试验均为阴性,21例SLE患者,有12例(57.1%)阳性,其中17例合并血细胞减少者中有10例(58.2%)阳性;4例血细胞正常者中有2例(50.0%)阳性,SLE患者BMMNC-Coombs试验阳性率显著高于正常对照组(P＜0.05).而SLE合并血细胞减少组与血细胞正常组相比,阳性率差异无统计学意义(P＞0.05).BMMNC抗体类型与血细胞水平无显著相关性.SLE合并血细胞减少组的CFU-E、CFU-GM产率与正常对照组相比,差异无统计学意义(P＞0.05).SLE血细胞正常组CFU-E、CFU-GM产率显著高于正常对照组(P＜0.05).SLE患者中,合并血细胞减少组CFU-E、CFU-GM产率显著低于血细胞正常组(P＜0.05).SEE患者无论是否合并血细胞减少其BFU-E产率均显著低于正常对照组(P值均＜0.05).而SLE合并血细胞减少组与血细胞正常组患者相比,其BFU-E产率差异无统计学意义(P＞0.05).结论 SLE患者BMMNC膜表面被覆自身抗体;造血祖细胞增殖、分化功能无明显异常,某些SLE患者可能通过造血祖细胞的代偿增殖维持正常的血细胞水平.     

Clinical scale expansion of cryopreserved small volume whole bone marrow aspirates produces sufficient cells for clinical use.

Ex vivo expansion of bone marrow (BM) mononuclear cells (MNC) in a perfused culture system produces stem-progenitor cell type(s) in sufficient number(s) for hematopoietic reconstitution. The limitations in using fresh BM MNC for ex vivo expansion include additional cell processing and inflexibility in patient treatment. Cryopreservation of whole bone marrow (WBM) eliminates processing costs of MNC or CD34+ cell selection and allows for flexibility in patient treatment. We developed a convenient system to cryopreserve and thaw small volume WBM aspirations (n = 13) and then compared the expandability of unprocessed normal cryopreserved/thawed (C/T) WBM to that of fresh BM MNC cultured in the presence of erythropoietin, PIXY 321, and Flt3-ligand. Ex vivo expansion potential was retained in WBM aspirates after C/T. When initiated with 225 million viable nucleated cells, clinical scale expansion cultures (n = 6) yielded 9.7+/-2.8 x 10(8) total cells, which contained 10.4+/-5.8 x 10(6) colony-forming units-granulocyte-macrophage (CFU-GM), 1.3+/-1.4 x 10(4) LTCIC, and 2.2 x 10(6) CD34+Lin- cells, sufficient cell numbers for clinical use. These studies demonstrate that ex vivo perfusion culture expansion of unfractionated C/T WBM (< or =30 ml) provides doses of stem-progenitor cells similar in composition to expanded fresh BM MNC, previously demonstrated to achieve hematopoietic reconstitution.     

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.     

Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex vivo expansion protocols

BACKGROUND: Cord blood is a useful source of HPCs for allogeneic transplantation. HPC ex vivo expansion of a cord blood graft has been proposed as a way to increase the speed of engraftment and thus to reduce the occurrence of transplantation-related complications. OBJECTIVE: The purpose of this study was to optimize a method for CD34+ cell selection of thawed cord blood grafts under clinical grade conditions, intended for application in a static, serum-free expansion culture. MATERIAL AND METHODS: Twelve samples were thawed and washed with dextran, albumin, and rHu-deoxyribo-nuclease I (RHu-DNase) to avoid clumping. CD34+ cells were selected by using a sensitized immunomagnetic bead and 9C5 MoAb complex. A buffer containing rHu-DNase, citrate, albumin, and immunoglobulin in PBS was used during the procedure. CD34+ cells were eluted and detached by using an immunomagnetic cell selection device. Cells from the enriched fraction were cultured for 6 days in serum-free medium supplemented with rHu-SCF, rHu-IL-3, rHu fetal liver tyrosine kinase 3 ligand, and rHu thrombopoietin (50 ng/mL each). Cells were expanded in well plates and in two semipermeable bags. RESULTS: A mean of 1.94 x 10(6) (+/- 1.55) CD34+ cells was obtained, yielding a CD34+ cell recovery of 52 +/- 12 percent. Nonspecific loss of CD34+ cells was 32 +/- 10 percent. CFU-GM and BFU-E/CFU-Mixed recoveries were 33 +/- 15 percent and 27 +/- 12 percent, respectively. CD34+ cells obtained were functionally comparable with fresh CD34+ cells selected for clonogenic potential. The capacity for expansion was not significantly different in the two types of bags studied. HPCs in wells were expanded 33 +/- 14-fold for CD34+ cells and 42 +/- 19-fold for overall colonies. The expansion rates observed in wells were significantly superior to those obtained in bags. CONCLUSION: The feasibility of a clinical-scale cord blood selection procedure based on a direct immunomagnetic method after thawing, followed by an ex vivo expansion culture using semipermeable bags, is shown. After 6 days of expansion, it was possible to generate a 9-fold increase in CD34+ cells, a 6-fold increase in CFU-GM and a 13-fold increase in BFU-E/CFU-Mixed colonies.     

sIL-6R，IL-6和SCF对人脐血CD34^＋细胞的扩增作用

1995年,我们发现由于细胞因子(SCF)和白细胞介素6(IL-6)/可溶性白细胞介素6受体(sIL-6R)复合物同时激活c-kit和gp130信号系统,有利于CD34~ 细胞的扩增。在此基础上,我们进一步研究了同时激活这两个信号系统对更原始的脐血长期培养启动细胞(long-term culture-initiating cell,LTC-IC)和混合造血细胞集落形成单位(CFU-Mix)的扩增作用。     

小鼠骨髓细胞体外扩增及其造血重建的研究

目的：观察干细胞因子（ＳＣＦ）与白细胞介素（ＩＬ）－１或ＩＬ－３联合对经５氟尿嘧啶（５ＦＵ）处理的骨髓细胞的体外扩增作用和体外扩增细胞移植的小鼠造血恢复能力。方法：在含有细胞因子的液体培养体系中培养５ＦＵ处理的骨髓细胞，观察粒－巨噬细胞集落形成单位（ＣＦＵ－ＧＭ）和高增殖潜能集落形成细胞（ＨＰＰ－ＣＦＣ）数量的增加以及移植扩增细胞的小鼠外周血细胞恢复。结果：单用ＳＣＦ无扩增作用，但ＳＣＦ与ＩＬ－１或ＩＬ－３联合时，与对照组相比较ＣＦＵ－ＧＭ分别增加３３．７±１８．１倍和１８．１±６．３倍。ＨＰＰ－ＣＦＣ分别扩增１７．８±１０．５倍和１２．７±９．１倍。移植体外扩增细胞组与对照组比较，外周血细胞恢复时间缩短１～３天，并且生存率也显著提高。结论：在液体培养体系中ＳＣＦ与ＩＬ－１或ＩＬ－３联合对造血细胞扩增有协同作用，并且体外扩增后的骨髓细胞能加速移植受体的早期造血重建。     

免疫相关全血细胞减少患者骨髓造血祖细胞增殖功能及辅助性T淋巴细胞功能观察

目的　了解与免疫相关全血细胞减少患者骨髓造血祖细胞增殖分化功能以及辅助性T(Th)淋巴细胞在此类患者发病中的作用。方法　采用甲基纤维素法培养 2 5例初诊、15例治疗后血常规恢复的免疫相关全血细胞减少患者和 10名正常人的CFU GM、CFU E、BFU E ;采用RT PCR方法 ,检测 2 5例初诊、15例治疗后血常规恢复的免疫相关全血细胞减少患者、19例其他全血细胞减少症患者和 10名正常人未经体外刺激的骨髓单个核细胞 (BMMNC)中Th1细胞的代表因子IFN γ、IL 2和Th2细胞的代表因子IL 4、IL 10的基因表达。结果　初诊组、治疗后血常规恢复正常的免疫相关全血细胞减少患者组、正常对照组BMMNC培养CFU E、CFU GM、BFU E产率 ,三组差异均无显著性 (P均 >0 .0 5 ) ,Th2型细胞因子IL 4、IL 10mRNA的表达在初诊组明显增高 ,与正常对照组比较 ,P均 <0 .0 1;经治疗血常规恢复正常后趋于正常 ,与正常对照组比较 ,P均 >0 .0 5。Th1型细胞因子IFN γ、IL 2mRNA的表达在所有免疫相关全血细胞减少患者均无增高 ,与正常对照组比较 ,P均 >0 .0 5。结论　引起免疫相关全血细胞减少患者全血细胞减少的原因可能并非骨髓造血祖细胞本身质的异常 ,而是造血祖细胞以外的因素 ;Th细胞亚群功能失衡、Th2细胞功能亢进可能是此类患者B淋巴细     

Ex vivo expansion of apheresis-derived peripheral blood hematopoietic progenitors

Because the administration of hematopoietic growth factors and the use of stem cell support often fails to alleviate the neutropenic phase induced by cytotoxic drugs, several investigators have attempted to expand ex vivo hematopoietic progenitors for clinical use. These attempts have clearly shown that the cultured cells are functional and can be safely administered to patients, but that the in vivo performance is disappointing and the concept as a whole is not yet clinically useful. The major reasons for these unsuccessful attempts are thought to be cumbersome cell fractionation techniques, contamination, prolonged incubation, and the use of less than ideal cytokine combinations. In response, we have developed a simple procedure for ex vivo expansion of myeloid progenitor cells. In this assay, unfractionated mononuclear cells from apheresis donors are incubated in nonpyrogenic plastic bags for 7 days in the presence of culture medium either containing fetal calf serum or human plasma, granulocyte colony-stimulating factor, and stem cell factor. We have demonstrated that under these conditions the number of colony-forming units (CFU) granulocyte-macrophage (CFU-GM) and of CFU-granulocyte-macrophage-erythroid-megakaryocyte (CFU-GEMM) increased 7- and 9-fold, respectively, by day 7 and the number of burst-forming units-erythroid (BFU-E) increased 2.7-fold by day 5 of culture. Significant increases in the numbers of cells expressing CD34+, CD34+/CD38+, CD34+/CD33+, CD34+/CD15+, and CD34+/CD90+ and significant declines in the numbers of cells expressing CD34+/CD38- and CD19 surface antigens were also observed. The relative numbers of cells expressing T-cell markers and CD56 surface antigen did not change. By using different concentrations of various hematopoietic growth factor combinations, we can increase the number of mature and immature cells of different hematopoietic lineages.     

Ex vivo expansion of early and late megakaryocyte progenitors

Our goal is to produce ex vivo-expanded human megakaryocytes (MK) cells from peripheral blood progenitor cell (PBPC) harvests for use in supplementing conventional autografts. In this paper we show the megakaryocytopoietic productivity of small-scale in vitro serum-free cultures of human CD34+ cells containing MK growth and development factor (MGDF) and stem cell factor (Kit ligand; SCF) +/- granulocyte colony-stimulating factor (G-CSF). Cultures were characterized after 3, 6, 9, and 13 days by flow cytometry and clonogenic assays. CD34+ cells expanded 5.2- and 3.4-fold, and produced 2.2 and 2.4 CD34+/41(+) cells per seeded CD34+ cell after 6 and 9 days in culture, respectively. None were detected at day 13. CD41+ cells expanded exponentially over 13 days. Colony-forming unit-megakaryocyte (CFU-MK) also expanded exponentially, but the proportion of the most primitive CFU-MK dropped from 45% to 1.5% and to <1% after 6 and 9 days, respectively. G-CSF increased total cell expansion, but decreased CD41+ frequency, yielding no gain in MK production. We also found that PB CD34+ cells cultured for 3-6 days are richer in primitive MK progenitors, while those cultured for 9-13 days have greater numbers of more differentiated MKs. Overall, the combination of MGDF+SCF proved sufficient for expanding CD34+/CD41+ cells. As the stage of ex vivo MK differentiation most conducive to optimal platelet production in vivo is not known, we are planning a clinical trial to determine the efficacy of ex vivo-expanded MKs on platelet recovery in relation to MK maturity.     

Ex vivo expansion of CD34+ umbilical cord blood cells in a defined serum-free medium (QBSF-60) with early effect cytokines

To investigate the clinically applicable conditions that support substantial expansion of both primitive and more mature hematopoietic cells of umbilical cord blood (UCB) for transplantation in adults, enriched CD34+ cells from 8 fresh UCB samples and 4 expanded UCB products were cultured in defined serum-free medium (QBSF-60) in the presence of a cytokine combination of SCF, Flt-3-ligand (FL), thrombopoietin (TPO), IL-3 for up to 2 weeks. Fresh medium with cytokines was supplemented or exchanged at day 4, day 7, and day 10. The proliferative response was assessed at day 7, day 10, and day 14 by evaluating the following parameters: nucleated cell (NC), clonogenic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM], burst-forming unit-erythrocyte [BFU-E], CFU-GEMM, and high-proliferative potential colony-forming cell [HPP-CFC]), immunophenotypes (CD34+ cells and CD34+ subpopulations), and LTCIC. Simultaneously numerical expansion of various stem/progenitor cells, including primitive CD34+CD38-HLA-DR- subpopulation and LTCIC, CD34+ cells, and clonogenic progenitors to mature nucleated cells, were continuously observed during the culture. An average 103.32 +/- 71.37 x 10(6) CD34+ cells (range 10.12 x 10(6)-317.9 x 10(6)) could be obtained from initial 1.72 +/- 1.13 x 10(6) UCB CD34+ cells after 10-14 days cultured under the described conditions. Sufficient CD34+ cells (>50.0 x 10(6)) for transplantation in adults would be available in all but one UCB collections after 10-14 days expansion. The expanded CD34+ cells sustained most of the in vitro characteristics of initial unmanipulated CD34+ cells, including clonogenic efficiency (of both primitive and committed progenitors), the proportion of CD34+CD38-HLA-DR- subpopulation, and the expansion potential. Initial addition of IL-3 to the cocktail of SCF + FL + TPO had positive effects on the expansion of both primitive and, especially, the more mature hematopoietic cells. It accelerated the expansion speed and shortened the optimal culture time from 14 days to 10 days. These results indicated that our proposed short-term culture system, consisting of QBSF-60 serum-free medium with a simple early acting cytokine combination of SCF + FL + TPO, could substantially support simultaneous expansion of various stem/progenitor cell populations involved in the different phases of engraftment. It would be a clinically applicable protocol for ex vivo expansion of CD34+ UCB cells.     

基质细胞对体外扩增造血细胞造血重建活性的促进作用

本研究探讨骨髓单个核细胞(13MMNC)在不同的培养条件下扩增后是否具有造血重建的活性。在小鼠BMMNC液体培养体系中，分别在含有几种细胞因子组合和(或)基质细胞层存在的条件下进行体外扩增，然后将不同条件下扩增的细胞经尾静脉回输至经致死量照射的小鼠体内，动态观察小鼠的骨髓有核细胞数及各种祖细胞集落数，以观察其造血重建的效果。结果表明：单纯细胞因子介导BMMNC体外扩增后并不能增进这些细胞的造血重建功能，但含有骨髓基质细胞底层的体外扩增，无论是否加入细胞因子，均有明显的促进移植受体造血功能重建的作用。结论：骨髓基质细胞支持下体外扩增的造血细胞具有促进移植受者造血重建的功能。证实了在维持体外扩增造血细胞的移植活性中，基质细胞具有非常重要的作用。     

血管紧张素Ⅱ对脐血CD34+细胞体外扩增的作用

血管紧张素Ⅱ (AngⅡ )是肾素 血管紧张素系统的一种主要生物活性物质。为了研究它在造血系统中的影响 ,本实验通过体外细胞培养的方法 ,探讨AngⅡ与不同的细胞因子联合刺激脐血CD34+ 细胞生长和分化的作用。研究结果发现 ,悬浮培养体系中的AngⅡ可同时刺激BFU E和CFU GM的扩增 ,BFU E和CFU GM的数量在一定范围内随AngⅡ浓度 (0 0 1- 0 1μmol/L)的升高而增多 ;在半固体培养基中的AngⅡ则仅刺激CFU GM的形成 ,却不影响BFU E ;悬浮培养体系中AngⅡ与细胞因子SCF +G CSF +GM CSF +IL 3联合可使CFU GM的扩增倍数由 2 3± 0 8升高到 7 8± 1 9倍 ,与SCF +EPO +TPO +IL 3联合 ,BFU E的扩增倍数由 3 1± 1 8提高到 9 2± 2 3倍。结论 :AngⅡ与其他细胞因子联合可刺激脐血造血干 /祖细胞体外扩增。     

胚胎成纤维细胞与含LIF的培养体系支持鼠造血祖细胞体外扩增

为了探讨胚胎成纤维细胞与含白血病抑制因子 (LIF)的培养体系对鼠骨髓造血祖细胞体外扩增效应 ,并观察其对造血表面归巢相关黏附分子VLA 4 (CD4 9d)、VLA 5 (CD4 9e)、LFA 1(CD11a)、HCAM (CD4 4 )、L selectin(CD62L)的表达影响 ,将丝裂霉素C灭活的鼠胚胎成纤维细胞 (MEF)和含LIF的培养体系与鼠骨髓单个核细胞(BMMNC)体外共同培养 7天 ,检测BMMNC细胞总数、CFC、Sca 1 细胞百分率、细胞凋亡率和Sca 1 细胞表面黏附分子CD4 9d、CD4 9e、CD11a、CD4 4、CD62L的表达率。结果表明 :MEF和含LIF的培养体系明显扩增了BMMNC细胞总数、CFC和Sca 1 细胞 ,抑制细胞凋亡 (P <0 .0 5 ) ,而去掉MEF和LIF后的对照组培养体系BMMNC细胞总数明显下降 ,CFC和Sca 1 细胞完全死亡 ,细胞大部分凋亡 (P <0 .0 1)。扩增后Sca 1 细胞表面各黏附分子CD4 9d、CD4 4和CD61L表达与扩增前相当 (P <0 .0 5 ) ,而CD4 9e和CD11a表达明显高于扩增前 (P <0 .0 5 )。结论 :胚胎成纤维细胞和含LIF的培养体系体不仅可以体外显著扩增鼠骨髓造血祖细胞 ,而且扩增后的造血祖细胞(HPC)总体上保持其表面归巢相关黏附分子的表达 ,扩增后HPC归巢功能不受影响。     

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。     

再生障碍性贫血患者骨髓造血干/祖细胞体外增殖分化特征的研究

OBJECTIVE: To investigate the quality of bone marrow stem cells from aplastic anemia patients and their in vitro responses to hemopoietic growth factors(HGF). METHODS: CD34 positive bone marrow cells from 10 chronic aplastic anemia(CAA), 5 severe aplastic anemia(SAA) patients and 5 healthy subjects were detected with immunoflurescence assay, enriched by Panning way, and then cultured in vitro for CFU-GM, BFU-E and CFU-E in the presence of recombinant HGFs. RESULTS: CD34 positive rates of the bone marrow mononuclear cells(MNC) of CAA, SAA and control groups were (1.05 +/- 0.51)%, (0.70 +/- 0.37)% and (1.27 +/- 0.45)%, respectively, and there was no difference among them (P > 0.05). After being enriched, CD34 positive cells in MNC of CAA, SAA and control groups increased to a similar level(P > 0.05). In the presence of G-CSF or GM-CSF, the enriched CD34 positive MNC of CAA and SAA patients formed similar CFU-GM number to those of normal controls. The numbers of BFU-E and CFU-E formed from the enriched CD34 positive MNC of CAA and SAA patients were also the same as that of normal control groups under the stimulation of erythropoietin and interleukin-3. Stem cell factor could cooperate with G-CSF, GM-CSF, and erythropoietin significantly increasing the numbers of CFU-GM, BFU-E and CFU-E of CAA, SAA and normal control's enriched CD34 positive MNC. CONCLUSION: The bone marrow CD34 positive cells from aplastic anemia patients appears to be normal in percentages and in in vitro proliferation/differentiation capacities.     

抗TGF-β抗体对人脐血CD34+细胞体外扩增及黏附分子表达的影响

本研究的目的是证实抗TGF-β抗体在人脐血CD34 细胞体外扩增过程中可以增加CD34 细胞的扩增效率,并观察其对脐血CD34 细胞粘附分子表达的影响。从6份新鲜脐血标本中富集CD34 细胞,将其接种于含抗TGF-β抗体 SCF Flt-3L TPO IL-3因子组合的SFEM无血清培养体系中,培养6天后,细胞计数,并用FACS检测CD34、c-kit(CD117)、CD11a、CD49d、CD33表达情况,同时进行集落分析。结果表明:①实验组有核细胞数、CD34 细胞数、CD34 c-kit 细胞数及CFU-GEMM分别扩增了41.82±13.49,15.62±6.95,13.36±6.12,11.07±4.05倍,明显高于对照组(P分别=0.001,0.002,0.003,0.002),其中实验组中更为早期的CD34 c-kit-亚群的扩增倍数更多(69.10±41.06),多于有核细胞、CD34 细胞、CD34 c-kit 细胞的扩增倍数(P=0.024)。②TGF-β抗体对CD11a和CD49d的表达无明显影响(P值均大于0.05)。结论:抗TGF-β抗体可协同早期干细胞生长因子有效扩增脐血CD34 细胞,同时不降低CD34 细胞黏附分子的表达。     

Umbilical cord blood progeny cells that retain a CD34+ phenotype after ex vivo expansion have less engraftment potential than unexpanded CD34+ cells

BACKGROUND: Because of the limitation of cell numbers associated with cord blood harvests, there is a need to determine the efficacy of using ex vivo-expanded cord blood cells in a transplantation setting. In this study, limiting-dilution analysis was used in nonobese diabetic mice with severe combined immunodeficiency (NOD/SCID) to compare the engraftment potential of progeny cells expressing the CD34+ phenotype after expansion with that of uncultured CD34+ cells. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured in Iscove's modified Dulbecco medium supplemented with 10-percent fetal calf serum (FCS) and IL-6, SCF, megakaryocyte growth and development factor, and Flt3 ligand. The resulting ex vivo-expanded products were assessed for total numbers of nucleated cells, CD34+ cells, and CFUs and long-term culture-initiating cell activity. The engraftment potentials of cultured progeny CD34+ cells and uncultured CD34+ cells were determined by using NOD/SCID mice. RESULTS: After 14 days of culture, total nucleated cell counts increased over input values by 180 +/- 59-fold, CD34+ cell numbers by 44 +/- 13-fold, CFU activity by 23 +/- 5-fold, and long-term culture-initiating cell activity by 20 +/- 6-fold (mean +/- SD; n = 6). The frequency of SCID-repopulating cells (SRC) in mice transplanted with uncultured products was 1 per 20,000 CD34+ cells (95% CI, 1:10,000-1:38,000) and that in mice receiving ex vivo-expanded products was 1 per 418,000 progeny CD34+ cells (95% CI, 1:158,000-1:1,100,000). Taken together, these data indicated that, after 2 weeks of culture, there was a modest twofold increase in the total number of SRCs. However, the levels of human CD45 cell engraftment in NOD/SCID recipients of progeny CD34+ cells were significantly lower than those in mice receiving equivalent numbers of uncultured CD34+ cells (p<0.05). CONCLUSION: Umbilical cord blood progeny cells retaining a CD34+ phenotype after ex vivo expansion have less engraftment potential than do unexpanded CD34+ cells.