GM-CSF对脐血CD34+巨核祖细胞体外扩增及分化的影响

本实验旨在研究GM-CSF对脐血CD34^＋细胞诱导分化为巨核细胞的影响.采用免疫磁珠法分选CD34^＋细胞,在含有TPO＋IL-3＋SCF并添加了不同浓度（5、20、100ng/ml）的GM-CSF的无血清培养基中进行培养.培养6、10、14天后计数单个核细胞（MNC）,检测CD41^＋细胞比例和CFU-MK.结果表明,培养14天后3种不同浓度GM-CSF对MNC均有明显的扩增作用,其中以20和100ng/ml GM-CSF的扩增效果较好.3种不同浓度的GM-CSF均使CD41^＋细胞比例增加,20和100ng/ml与5 ng/ml GM-CSF相比更能提高CD41^＋细胞的比例.5和20 ng/ml的GM-CSF能促进CFU-MK的形成,但100ng/ml的GM-CSF却抑制CFU-MK的形成.结论：在TPO＋IL-3＋SCF细胞因子组合中添加GM-CSF有利于促进脐血CD34^＋细胞诱导分化为巨核细胞.     

脐血CD34+细胞及红系祖细胞扩增的实验研究

脐血是造血祖细胞的丰富来源之一,选择合适的培养条件,体外诱导其定向扩增为红系祖细胞,输入体内产生成熟红细胞。本实验旨在探讨脐血单个核细胞(MNC)体外红系定向扩增的理想因子组合(Flt3配基FL联合TPO、SCF、EPO及FL、SCF、TPO)对CD34 细胞扩增的影响。将单个核细胞接种至stemspan无血清培养液中,共分3组:A组为对照组,B组为TPO SCF FL EPO IGF1组,C组为TPO SCF FL组,C组在第6天及以后换液加入EPO和IGF1。于培养0、6、10、14天进行细胞计数,细胞集落测定,流式细胞术测定细胞的CD34、CD34CD71、CD71GPA细胞的比例。结果表明:经10天培养后,B组总细胞数扩增6.89倍,而C组3.06倍;B组CD34 细胞增加4.83,而C组2.47倍;B组集落形成细胞数增加4.3倍,而C组增加2.5倍;B组红系祖细胞BFUE和CFUE数增加5.4倍,而C组3.1倍;B组CD34 CD71 细胞数增加8.72倍,而C组3.37倍;B组CD71 GPA 细胞数增加53.4倍,而C组30.29倍。结论:脐血MNC在无血清培养液中加入FL SCF TPO实现了CD34 细胞及集落形成细胞的扩增。脐血MNC在无血清培养液中加入FL SCF TPO EPO IGF1短期液体培养获得红系祖细胞的扩增,在第0天比6天加入EPO获得更多红祖细胞(P<0.05)。由于TPO SCF FL EPO IGF1组的集落形成细胞数、CFUE和BFUE数于第10天最多,故培养后收获时     

不同细胞因子组合对人脐血单个核细胞体外扩增及CD49d和CXCR4表达的影响

为了探讨不同的细胞因子组合对脐血单个核细胞体外的扩增作用及扩增后CD49d和CXCR4的变化，将新鲜脐血标本分离的单个核细胞接种于含有不同细胞因子组合的无血清无基质培养体系中培养7天，在0天，7天检测有核细胞数，CD34^＋细胞数及CD34^＋CXCR4^＋，CD34^＋CD49d^＋的细胞数和集落形成单位（CFU）数．根据不同细胞因子组合实验分组为：对照组；SF组（SCF＋FL）；SFT组（SCF＋FL＋TPO）和SFT6组（SCF＋FL＋TP0＋IL-6）。结果表明，和对照组相比，SF组合仅能低水平支持脐血造血细胞扩增，加入TPO后即SCF／FL／TPO组合能有效的扩增脐血细胞，但SFT和SFT6两组之间差异却无明显发生（P〉0．05）；SF，SFT和SFT63组的细胞因子组合均可提高脐血CD34^＋细胞CD49d，CXCR4的表达，但3组之间差异无显著性（P〉0．05）。结论：SF组合可协同扩增人造血细胞，但协同作用较弱；TPO在脐血造血干／祖细胞体外扩增中起重要调节作用，而IL-6作用不显著；SCF／FL／TPO 3种因子组合不仅可促进脐血造血祖细胞的扩增，而且可上调脐血造血细胞CD49d，CXCR4表达。     

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.     

Megakaryocyte ex vivo expansion potential of three hematopoietic sources in serum and serum-free medium.

Megakaryocytes (MK) were expanded from purified human CD34+ cells obtained from three sources, bone marrow (BM), mobilized peripheral blood progenitor cells (PB), and umbilical cord (UC) blood. CD34+-selected cells were cultured for 12 days with 10 ng/ml thrombopoietin (TPO), 10 ng/ml IL-3, 10 ng/ml TPO + 10 ng/ml IL-3, or 200 ng/ml promegapoietin (PMP), a chimeric dual agonist of the c-Mpl and human IL-3 receptors. MK production was compared in serum-free versus human serum-supplemented liquid media. PMP and the combination of TPO and IL-3 (TPO + IL-3) increased MK production similarly. Culturing CD34+ cells with PMP in serum-free medium resulted in a twofold increase in MK yield compared with serum-supplemented medium. CD34+ cells from UC proliferated more than those from either BM or PB in liquid culture, resulting in much greater MK production under all conditions. Phenotypic analysis of the uncultured CD34+ cells showed that BM had a higher frequency of CD34+/CD41+ cells than PB or UC. TPO + IL-3 or PMP produced larger and greater numbers of BFU-MK and CFU-MK per seeded CD34+/CD41+ cell from UC than from either BM or PB. Thus, although uncultured CD34+-selected BM cells contained a higher frequency of committed mature MK progenitors, UC CD34+ cells had a greater proliferative capacity and, therefore, were more productive. PMP induced megakaryocytopoietic activity comparable to that achieved with TPO + IL-3 and may be useful for ex vivo expansion of MK for clinical trials.     

不同组合细胞因子与人参总皂苷对体外培养CD34^＋细胞分化的诱导效应

背景:造血生长因子可分别作用于不同的造血细胞和细胞的不同分化阶段。红细胞生成素可促进晚期红系祖细胞的增殖分化,白细胞介素3为多潜能集落刺激因子,对多种造血细胞的分化成熟具有促进作用。目的:观察细胞因子的不同组合及人参总皂苷对CD34 细胞体外定向诱导分化为红系血细胞的影响。设计:观察对比实验。单位:重庆医科大学。材料:实验于2002-07/2004-01在重庆医科大学组织胚胎学教研室、基础医学研究所、重庆市生物化学与分子药理学重点实验室完成。正常人骨髓细胞来自第三军医大学大坪医院胸外科无血液系统疾病患者手术摘除的肋骨,所有患者知情同意。人参总皂苷和试剂由重庆中药研究所惠赠。胎牛血清、FITCCD34 抗体、FITC标记同型对照小鼠IgG1为BectonDickinson公司产品,CD71 抗体为SantaCruz公司产品,血小板生成素、Flt-3配基、干细胞因子、白细胞介素-3为SantaCruz公司产品,红细胞生成素为Amgen公司。方法:应用阴性分选策略,以StemsepTM系统从正常人骨髓细胞中分离CD34 造血干/祖细胞。经造血细胞因子不同组合(白细胞介素3 红细胞生成素、干细胞因子 白细胞介素3 红细胞生成素、Flt-3配基 血小板生成素 干细胞因子 白细胞介素3 红细胞生成素)进行液体培养,并以干细胞因子 白细胞介素3 红细胞生成素为对照组,在其中加入终浓度分别为10,20,50,70,100mg/L人参总皂苷为加药组,检测细胞总数、CD71 细胞比例;在CD34 细胞培养体系中加入不同剂量的人参总皂苷(剂量同前)为药物组,以不加人参总皂苷为对照组,通过甲基纤维素半固体培养法检测人参总皂苷诱导CD34 造血干/祖细胞向红系祖细胞(早期红系祖细胞、晚期红系祖细胞)增殖与分化能力。主要观察指标:①不同的细胞因子组CD34 细胞体外增殖情况。②人参总皂苷对CD34 细胞定向诱导分化为红系血细胞的影响。③人参总皂苷对CD34 细胞形成红系祖细胞能力的影响。结果:①各因子组合中以Flt-3配基 血小板生成素 干细胞因子 白细胞介素3 红细胞生成素细胞因子组合诱导CD34 细胞分化为红系血细胞的能力最强,2周时CD71 细胞比例为(61.20±5.31)%。②人参总皂苷20mg/L是液体培养诱导CD34 细胞向红系分化的最佳浓度,与干细胞因子 白细胞介素3 红细胞生成素协同作用CD34 细胞2周后,CD71 细胞比例从(48.39±5.07)%增加到(56.20±1.40)%。③人参总皂苷10～50mg/L均能提高CD34 细胞形成早期红系祖细胞、晚期红系祖细胞的集落产率。结论:含有Flt-3配基 血小板生成素 干细胞因子 白细胞介素3 红细胞生成素的细胞因子组合,可诱导CD34 细胞定向生成大量的红系细胞;人参总皂苷能促进CD34 细胞定向诱导分化为红系血细胞。     

脐血来源树突状细胞的体外诱导及扩增

本研究的目的是分析脐血的细胞组成 ,研究加入细胞因子培养前后脐血树突状细胞的变化 ,探索体外诱导、扩增树突状细胞的方法并进行表型鉴定。选择正常成人外周血 9份 ,脐血 12份 ,分离单个核细胞。在脐血单个核细胞中加入细胞因子GM CSF、IL 3、SCF和EPO ,培养 4周。应用流式细胞仪和CD4、CD8、CD19、CD34、CD38、CD1a、CD11c及CDw12 3单克隆抗体测定正常成人外周血、培养前后 1,2 ,3,4周脐血细胞表面抗原及树突状细胞情况。结果表明 :正常成人外周血CD34 细胞 0 .0 2× 10 5 ml,CD1a 细胞 0 .0 1× 10 5 ml,CD11c 细胞 4 .32×10 5 ml,CD83 细胞 1.31× 10 5 ml,CDw12 3 细胞 1.4 1× 10 5 ml。新鲜脐血中CD34 细胞 0 .2 2× 10 5 ml,CD1a 细胞 0 .2 7× 10 5 ml,CD11c 细胞 5 .87× 10 5 ml,CD83 细胞 1.94× 10 5 ml,CDw12 3 细胞 2 .73× 10 5 ml。加入细胞因子GM CSF ,IL 3,SCF ,EPO后培养 1- 4周的脐血单个核细胞分化为CD1a ,CD11c ,CD83 ,CDw12 3 树突状细胞 ,在培养的 2 - 4周 ,脐血树突状细胞数量明显增多 ,此后逐渐减少。通过培养 ,树突状细胞数量增加 ,CD1a 细胞达 11.0 2× 10 5 ml,CD11c 细胞 2 8.2 4× 10 5 ml,CD83 细胞 10 .5 7× 10 5 ml,CDw12 3 细胞 18.7× 10 5     

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。     

人脐血巨核系祖细胞体外扩增的实验研究

本研究旨在探讨各种细胞因子对人脐血CD34 细胞体外扩增生成巨核细胞的作用,以建立人脐血巨核系祖细胞体外扩增的最佳体系。采用Ficoll分离液分离脐血单个核细胞,免疫磁珠法分离纯化CD34 细胞,进行体外半固体集落培养和液体培养,观察各种细胞因子组合对CD41 细胞和巨核细胞集落形成单位(CFU-MK)的影响。结果显示:TPO IL-6 IL3 FLT-3L4因子组合体外培养14天效果最好,在第7、14天CD41 细胞分别扩增了154.67±32.21倍、193.23±25.24倍。结论:本实验建立的巨核系祖细胞体外扩增体系,为促进脐血移植后血小板恢复奠定了实验基础。     

提高多药耐药基因导入人CD34^＋细胞转导效率的探讨

目的 探讨逆转录病毒介导的多药耐药基因导入人ＣＤ３４＾＋细胞的影响因素,以提高基因转导效率。方法 用流式细胞术（ＦＣＭ）检测不同组合细胞因子及人骨髓基质细胞＋细胞因子支持的基因转导效率;用造血祖细胞集落培养观察耐药性;用ＦＣＭ检测不同浓度柴杉醇素对基因转导细胞的作用。     

人骨髓间充质干细胞与脐血CD34+细胞体外扩增

为了研究骨髓间充质干细胞(MSC)及细胞因子对脐血CD34 造血祖细胞体外扩增的作用,及其扩增作用 对细胞黏附分子的影响,用免疫磁珠富集脐血CD34 细胞,然后接种到含有或不含有MSC和细胞因子的24孔培 养板,体外培养1周,观察不同指标并进行组间比较。结果表明:①SDF-1α SCF TPO FL因子组合与SCF TPO FL因子组合对脐血CD34 细胞的扩增作用无显著性差异(无论有无MSC细胞层存在)(P>0.05);②MSC 与上述细胞因子共存的培养体系优于相应的单纯细胞因子培养体系(P<0.05);③扩增前与扩增后脐血造血祖 细胞黏附分子CD44的表达没有明显变化。结论:趋化因子SDF-1α对SCF TPO FL因子组合的扩增作用无显 著影响;MSC增加细胞因子的脐血细胞体外扩增的作用;体外扩增不影响跻血细胞黏附分子CD44的表达。     

动员人外周血巨核祖细胞体外扩增的研究

本研究探讨多种细胞因子(TPO、SCF、FL、IL-1、IL-3、IL-6)组合的几种培养体系对人外周血CD34+细胞体外诱导扩增生成巨核细胞的作用,研究人外周血来源的巨核细胞体外扩增的最佳细胞因子组合及培养时间。用Ficoll-Hapaque分离法分离动员的外周血(MPB)单个核细胞,免疫磁珠法分离纯化CD34+细胞,并将其在含胎牛血清的液体培养体系中、各组细胞因子诱导下培养15天。在不同时间点采用血细胞计数板进行细胞计数,采用流式细胞术检测培养体系中CD41+细胞的含量;同时采用甲基纤维素半固体培养法进行巨核细胞集落培养,测定巨核细胞集落形成单位(CFU-MK)的数量。结果表明经过15天的培养,在MPB来源的CD34+细胞体外诱导并扩增巨核祖细胞体系中,以TPO/FL/IL-6/IL-3组合的扩增效果最好,明显高于其它3组,CD41+细胞第5天、10天分别扩增了93.97±17.27倍、131.23±18.26倍。第15天CD41+细胞含量及CD41+细胞数迅速下降。CFU-MK产率(/1×103个细胞)第5天、10天分别为83.33±10.02个、120.67±13.01个,明显高于其余3组。结论以TPO/FL/IL-6/IL-3因子组合为体外诱导扩增巨核祖细胞的最佳组合,动员外周血的巨核祖细胞体外诱导扩增以培养第10天为宜。本实验建立了动员人外周血来源的巨核祖细胞体外扩增体系。     

血管紧张素Ⅱ对脐血CD34+细胞体外分化为巨核细胞的影响

为了探讨血管紧张素Ⅱ对脐血CD34^＋细胞诱导分化为巨核细胞的影响，采用免疫磁珠法（MACS）分选8例健康产妇足月顺产胎儿脐血中的CD34^＋细胞，在舍血小板生成素（TPO50ng／ml）、白介素-3（IL-310ng／ml）、干细胞刺激因子（SCF50ng／l）的无血清培养液中添加浓度分别为50、100、1000μg／ml的血管紧张素Ⅱ作为实验组；同时以未添加血管紧张素Ⅱ的基础培养液作为对照组，培养14天后观察结果。细胞计数仪计数单个核细胞数（MNC）；流式细胞仪计数培养体系中的CD41^＋细胞数、血小板数，及分析细胞周期；利用CD41单克隆抗体免疫荧光染色观察培养体系中的细胞情况。结果表明：与对照组比较，实验组单个核细胞数无明显改变（P〉0．05）；而CD41^＋细胞和血小板数量有明显的增加（P〈0.05）；细胞周期分析显示，实验组的4倍体细胞增加，并存在明显的凋亡（P〈0．05）；荧光显微镜下观察对照组和实验组均可见大小不一的CD41^＋细胞。结论：血管紧张素Ⅱ可以促进脐血中CD34^＋细胞诱导分化为巨核细胞，并能促进巨核细胞产生血小板。     

体外扩增对脐带血CD34~+细胞归巢相关分子影响的研究

目的体外探讨脐带血CD34+细胞经细胞因子刺激后的增殖、分化以及表面归巢相关分子的表达。方法磁珠分选出脐血CD34+细胞,无血清培养基培养14d。实验分组:A组:培养基对照组;B组:SCF+TPO+Flt3;C组:SCF+TPO+Flt3+IL-3;应用流式细胞仪检测扩增前后CD34+细胞CD33、CD41、CD71、CD62L、CD162、CLA、CD44、CD11a、CD18、CD184、CD26表达;用半固体培养基培养集落形成单位(CFU)。结果 B、C两组有核细胞、CD34+细胞和CFU都得到有效扩增,C组的有核细胞、CFU扩增倍数明显高于B组(P<0.01),但CD34+细胞扩增倍数低于B组(P<0.01);扩增后的CD34+细胞表达CD33、CD41、CD71的比例增加,其中C组的增加尤为显著;扩增后CD62L、CD162、CD44的表达减低,其中C组CD62L、CD44减低更明显;而CLA、CD11a、CD18、CD184、CD26表达均上调,C组的CLA、CD11a、CD18、CD184上调更为明显。结论脐血CD34+细胞经过细胞因子的扩增,虽然有核细胞及祖细胞的量增加了,但细胞发生分化,分化因子的作用更为明显。扩增的CD34+细胞表面部分与归巢相关的分子表达上调,有促进细胞归巢的作用。     

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.     

人脐血造血干/祖细胞体外定向诱导分化过程中端粒酶的表达

探讨脐血造血干/祖细胞体外诱导分化过程中端粒酶的表达,为造血干细胞产品的临床应用提供一个监测细胞增殖潜能和安全性的指标。用源自人脐血的CD34~+细胞及不同细胞因子组合(SCF+IL-3+IL-6+G-CSF,SCF+IL-3+IL-6+EPO)在体外进行诱导分化;用TRAP聚丙烯酰胺凝胶电泳法、TRAP-ELISA法检测CD34~+细胞及诱导分化细胞的端粒酶活性;用Western印迹法在蛋白水平检测端粒酶催化亚基hTERT的表达,用RT-PCR在细胞转录水平检测端粒酶催化亚基hTERT mRNA的表达。结果显示,在体外诱导分化14-21天为细胞生长峰值,细胞总数可增加(1006.4±103.2)倍,随着培养时间的延长,细胞数量不再增加。CD34~+细胞低度表达端粒酶活性和hTERT基因,在细胞诱导分化过程中端粒酶活性及hTERT表达逐渐升高,细胞诱导14天后端粒酶活性及hTERT表达下降,28天端粒酶活性及hTERT基因检测不出。结论:利用CD34~+细胞在体外定向诱导分化出的大量细胞,不具有无限增殖的特性,可安全地应用于临床,且利用端粒酶的检测可为临床应用诱导分化细胞的时机提供依据。     

Platelet Precursor Cells Can Be Generated from Cultured Human CD34+ Progenitor Cells But Display Recirculation into Hematopoietic Tissue upon Transfusion in Mice

BACKGROUND: Whereas ex vivo expanded megakaryocytic progenitor cells have been investigated for their ability to support platelet regeneration, the question whether more mature platelet-like particles expanded from hematopoietic progenitor cells may be useful for transfusion purposes remains largely elusive. METHODS: Human peripheral blood progenitor cells (PBPCs) were enriched using surface expression of CD34 by immunoselection. CD34+ enriched PBPCs were expanded ex vivo in serum-free medium supplemented with cytokines. As a proof-of-principle, distribution of expanded CD61+ particles was analyzed after transfusion into Non-Obese Diabetic/ Severe Combined Immunodeficiency (NOD/SCID) mice. RESULTS: Highest ex vivo expansion for CD41+/CD61 + cells was achieved when medium was supplemented with SCF, TPO and IL-3. During expansion culture, CD34 marker expression decreased from 85 to 2-8%, while megakaryocytic cells appeared and CD41 and CD61 expression increased from 3 to about 30%. After transfusion of the expanded cells in NOD/SCID mice, CD61 + cells located mainly to bone marrow and to a lesser degree to spleen, but also circulated in blood. CONCLUSIONS: Platelet-like particles using cytokine-substituted serumfree medium can be generated efficiently from CD34+ expansion cultures, but mainly home to hematopoietic tissue.     

红细胞生成素和重组细胞因子对动员后外周血造血祖细胞、红系爆式祖细胞和粒巨噬系祖细胞的集落形成和自我更新的作用

本研究探讨红细胞生成素（EPO）和重组细胞因子（G－CSF，SCF，IL－3和GM－CSF）对患者和健康供者动员后的外周造血祖细胞的红系爆式祖细胞（BFU－E）和粒巨噬系祖细胞（CFU－GM）的集落形成和自我更新的作用。为了更好的了解上述那一种细胞因子和如何联合对干细胞的扩增更为有效，采用甲基纤维素半固体培养法，观察在单独用EPO、G－CSF的基础上，比较联合SCF，IL－3和GM－CSF对BFU－E和CFU－GM的作用。结果显示：①在EPO＋IL－3和EPO＋SCF＋IL－3组，外周血BFU－E自我更新显著增加，而EPO＋SCF组则明显增加。②患者与正常供者之间BFU－E的自我更新无显著差别。③G－CSF联合SCR，IL－3和GM－CSF后CFU－GM集落形成显著增加。患者组仅G－CSF本身可使CFU－GM集落形成显著增加，而G－CSF＋SCF和GMix组CFU－GM集落形成明显增加。④当G－CSF联合SCF，IL－3和GM－CSF可能显著增加CFU－GM的自我更新（AUC）。GMix是CFU－GM的集落形成和祖细胞扩增的较佳组合。⑤正常供者比患者有较高的AUC即自我更新，特别是在G－CSF组比患者具有显著差异（P＝0．0067）。     

细胞因子组合对脐血干 /祖细胞的调控作用

INTRODUCTIONIntheclinicalapplication,suchashematopoeticstemcellorpro-genitortransplantation,genetictherapyandtumordefecation,whetherthehematopoieticstemcellorprogenitorcankeeptheam-plificationacquiredmuchattention犤1犦.MATERIALSANDMETHODSSamplecollection23samplesofumbilicalbloodwerefromdepartmentofobstetricsandgynecologyofHuashanHospital.Anti-coagulationwithheparinandmeancollectiveamountwas50～100ml.Collectmonocytes(MNC)regularlywithin24hoursaftercollectionandreservethemafterwash…     

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.