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。     

体外扩增脐血CD34+细胞的实验研究

目的 探讨体外扩增脐血干／祖细胞用于成人脐血移植的可能性。方法 从１０份新鲜的脐血标本中纯化的ＣＤ３４＾＋细胞接种于含体积分数为２０％的胎牛血清（ＦＢＳ）的ＩＭＤＭ培养基的悬 培养体系中,分别加入由ＳＣＦ、Ｆｌｔ－３Ｌｉｇａｎｄ（ＦＬ）与ＩＬ－１β、ＩＬ－３、ＩＬ－６、Ｇ－ＣＳＦ、Ｅｐｏ（合称１３６ＧＥ）组成的３组细胞因子（Ａ组：１３６ＧＥ＋ＦＬ;Ｂ组：ＳＣＦ＋１３６ＧＥ;Ｃ组：ＦＬ＋ＳＣＦ＋１３     

Negative influence of IL3 on the expansion of human cord blood in vivo long-term repopulating stem cells

Identification of culture conditions that support expansion or even long-term maintenance of in vivo repopulating human hematopoietic stem cells is still a major challenge. Using a combination of FLT3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO) and Interleukin 6 (IL6), we cultured cord blood (CB) CD34+ cells for up to 12 weeks and transplanted their progeny into sublethally irradiated NOD/SCID mice. Bone marrow engraftment was considered successful when recipients contained measurable numbers of human CD45+, CD71+ and Glycophorin A+(GpA) cells 8 weeks after transplantation. Twelve-week expanded cells with FL+SCF+TPO+IL6 successfully engrafted all of the recipients and human CD45(+)+CD71(+)+GpA(+) cells represented 4.3 to 22.4% of bone marrow. Substitution of IL6 with IL3 led to an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture; however, LTC-IC output increased up to week 6 and then decreased and disappeared. By contrast, with FL+SCF+TPO+IL6, LTC-IC kept increasing up to week 12. Four-week cultured cells with FL+SCF+TPO+IL3 less efficiently engrafted NOD/SCID mice, both as measured by frequency of positive recipients (4 out of 10) and percentage of engrafted human cells (< or =2%). Six-week expanded cells failed to engraft. This study provides evidence that many, but not all, of the so-called "early acting" cytokines, can sustain long-term maintenance and even expansion of human primitive in vivo repopulating stem cells. In particular, in the culture conditions used in this study, the presence of IL3 greatly reduces the repopulating potential of expanded CD34+ CB cells.     

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     

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。     

Preclinical ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of culture; the media, serum supplements, and growth factors used; and engraftment in NOD/SCID mice

BACKGROUND: Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This study's purpose was to establish a clinically applicable culture system by investigating the use of cytokines, serum-free media, and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. STUDY DESIGN AND METHODS: Enriched CB CD34+ cells were cultured in four media (Iscove's modified Dulbecco's medium with FCS, Gibco; X-Vivo-10, BioWhittaker; QBSF-60, Quality Biological; and StemSpan SFEM, Stem Cell Technologies) with four cytokine combinations (thrombopoietin [TPO], SCF, Flt-3 ligand [FL] with and without G-CSF, and/or IL-6). The effect of autologous CB plasma was also investigated. The read-out measures were evaluated on Days 8 and 12. After expansion at the optimized condition, cultured cells were transplanted into sublethally irradiated NOD/SCID mice. The engraftment of human CD45+ cells and subsets in the bone marrow, spleen, and peripheral blood was determined. RESULTS: QBSF-60 or StemSpan SFEM supported high yields of early progenitors (CD34+ cells, 相似文献   

体外扩增的脐血单个核细胞植入NOD/SCID小鼠的研究

为了探讨在无血清、无基质培养条件下SCF、FL和TPO 3种因子组合体外扩增的脐血单个核细胞(MNC)的最佳移植时机及植入潜能,将SCF,FL和TPO 3种因子组合体外扩增的脐血单个核细胞培养14天,在0、7、10和14天检测有核细胞数(TNC),CD34 细胞数,CD34 CXCR4 细胞数,CD34 CD49d 的细胞数及集落形成单位(CFU)数,并将SCF FL TPO 3种因子组合的无血清无基质条件下扩增培养7天前后的脐血单个核细胞移植给经亚致死量照射的NOD/SCID小鼠,6周后用流式细胞术,PCR法检测存活小鼠体内的人源性细胞.结果表明,经过14天的培养,脐血细胞得到了有效的扩增,TNC数,CD34 细胞数,CD34 CD49d 的细胞数于7天达高峰,其后开始下降,而CFU数,CD34 CXCR4 细胞数于第10天达高峰.在移植6周后,扩增脐血移植组的NOD/SCID小鼠的存活率和人源性CD45 细胞的检出率分别为56.25%和(1.39±0.63)%,高于新鲜脐血移植组31.25%和(0.73±0.16)%,亦高于生理盐水移植组(0和0),差异有显著性(p＜0.05),扩增脐血移植组有6只NOD/SCID小鼠骨髓细胞中可检测到人特异ALU序列的表达.结论体外培养7-10天可能是收获细胞的最佳时机;SCF FL TPO 3种因子组合扩增7天的脐血单个核细胞能够植入NOD/SCID小鼠,其植入水平优于未扩增的脐血;上调脐血造血细胞上CXCR4,CD49d的表达可能会增加脐血造血细胞的植入能力.     

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.     

Thrombopoietin is synergistic with other cytokines for expansion of cord blood progenitor cells

We investigated the effects of recombinant human thrombopoietin (TPO) in combination with various cytokines including erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor (SCF) on megakaryopoiesis, and the expansion of CD34+CD41a+ cells from human cord blood CD34+ cells with these cytokines under serum-free conditions. Human cord blood CD34+ cells were cultured in Megacult (Stem Cell Technologies Inc. Vancouver, Canada) in the presence of recombinant growth factors. Colony-forming unit-megakaryocyte (CFU-M) colonies were counted on day 14. CD34+CD41a+ and CD34-CD41a+ cell expansion was analyzed using a serum-free liquid culture system for 7 days with recombinant growth factors. TPO alone had a concentration-dependent effect on megakaryocyte colony growth. At concentrations above 1 ng/ml, TPO supported significant CFU-Meg colony formation in a concentration-dependent manner. The combination of TPO plus other cytokines, including EPO, IL-3, and SCF, resulted in a synergistic enhancement of the number of CFU-Meg colonies, but IL-6 failed to enhance the effect of TPO. The number of CD41a+ cells increased after 7 days in liquid culture of human cord blood CD34+ cells with various cytokines (EPO, IL-3, IL-6, SCF) combined with TPO, but SCF plus TPO only resulted in a significant synergistic increment of CD34+CD41a+ cells compared with TPO alone. The results of the present study indicate that EPO, IL-3, and SCF can be synergistic with TPO to stimulate proliferation of CFU-Meg and suggest that SCF plus TPO can expand CD34+CD41a+ cells to effect the rapid recovery of platelets in patients following stem cell transplantation.     

无血清脐血巨核系祖细胞体外扩增的研究

脐血造血干细胞移植后血小板恢复延迟是一大难题,目前认为这主要与脐血中巨核系祖细胞数量不足及脐血巨核细胞分化成熟延迟有关,而将部分脐血进行巨核系祖细胞体外扩增后输注受者体内是有望解决这一难题的重要途径。但适用于临床应用的扩增条件至今仍未确立。本课题采用人脐血单个核细胞(MNC)在无血清培养体系中使用TPO,IL-3,SCF,IL-6等细胞因子进行不同的组合,在培养的0,6,10,14天进行MNC、CD41^ 细胞及CFU-MK数的检测。以寻找最佳的细胞因子组合及最佳的收获时机。结果表明：无血清条件下TPO与IL-3,SCF,IL-6等细胞因子联用可实现脐血巨核系祖细胞有效的体外扩增,各因子组中以TPO IL-3 SCF IL-6组扩增效果为最佳。其CFU-MK数于第10天最多,扩增达6．8倍,CD41^ 细胞扩增达8．8倍。结论：在人脐血MNC无血清培养条件下。TPO IL-3 SCF IL-6组为巨核系祖细胞体外扩增较佳的因子组合。由于TPO IL-3 SCF IL-6组的CFU-MK数于第10天最多,CD41^ 细胞数亦为同期最高,故培养后收获时间宜控制在其体外培养的第10天。     

Comparison of the hematopoietic activity of flt-3 ligand and granulocyte-macrophage colony-stimulating factor acting alone or in combination

The hematopoietic sequelae of intramuscular administration of flt-3 ligand (FL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) alone, or in combination, were compared in BALB/c mice. Changes in hematopoiesis were measured in the marrow, spleen and blood using an in vitro colony-forming unit (CFU) assay and flow cytometrically (expression of CD34 and stem cell antigen (Sca)-1). FL administration was associated with a significant increase in the absolute number of CFU and CD34+ cells in the marrow and CFU, CD34+, Sca-1+, and CD34+ Sca-1+ cells in the spleen and blood. These data demonstrate that FL expands and mobilizes a range of hematopoietic progenitors. By comparison, GM-CSF administration was associated with a significant increase in the number of CFU in the spleen and a significant reduction in marrow CD34+, Sca-1+, and CD34+Sca-1+ cells. These data suggest that GM-CSF-driven expansion of CFU may be at the expense of more primitive cells. The pattern of progenitor cell expansion associated with FL + GM-CSF administration was similar to that of FL alone with the following exceptions. The numbers of spleen and blood CFU were significantly greater and the number of marrow CD34+Sca-1+ cells were significantly less, than with FL alone. These data suggest that co-administration of these cytokines may combine the expansion of the more primitive cell populations (associated with FL) with the expansion of the more mature CFU population (associated with GM-CSF) to yield a greater overall CFU expansion and elevation of CFU in the blood. However, increasing the expansion and mobilization of the relatively mature, rather than the more primitive, hematopoietic progenitors, may be of limited value as a mobilization strategy, if the goal is the expansion and isolation of increased numbers of "high-quality," primitive cells for transplantation.     

Analysis of interleukin 6 receptor and gp130 expressions and proliferative capability of human CD34+ cells

We recently demonstrated that stimulation of gp130 by a combination of soluble interleukin 6 receptor (sIL-6R) and IL-6 but not IL-6 alone significantly stimulates the ex vivo expansion of primitive hematopoietic progenitors and the generation of erythroid cells from human CD34+ cells in the presence of stem cell factor (SCF). Here, we show that gp130 is found low positively on most CD34+ cells, whereas IL- 6R is expressed on only 30-50% of these cells. Although most of the colonies generated from FACS-sorted CD34+IL-6R+ cells were granulocyte/macrophage (GM) colonies, CD34+IL-6R- cells gave rise to various types of colonies, including erythroid bursts, GM, megakaryocytes, and mixed colonies in methylcellulose culture with a combination of IL-6, sIL-6R, and SCF. Similar results were obtained in culture supplemented with a combination of IL-3, IL-6, SCF, granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. A limiting dilution analysis of long-term culture-initiating cells (LTC- IC) showed that the CD34+IL-6R- cells contained a larger number of LTC- IC than did the CD34+IL-6R+ cells. In a serum-free suspension of CD34+IL-6R- cells, the addition of sIL-6R to the combination of IL-6 and SCF dramatically increased the total and multipotential progenitors, whereas CD34+IL-6R+ cells failed to do so under the same conditions. These results indicate that most of the erythroid, megakaryocytic, and primitive human hematopoietic progenitors are included in the IL-6R- populations, and the activation of gp130 on these progenitors can be achieved by a complex of IL-6-sIL-6R, but not by IL-6 alone. The present culture system using IL-6, sIL-6R, and SCF may provide a novel approach for ex vivo expansion of human primitive hematopoietic progenitors.     

间充质干细胞对脐血CD34^＋细胞扩增及其细胞特性变化的影响

本研究探讨脐带间充质干细胞（MSC）对CD34^＋细胞（HSPC）体外扩增的支持作用及对CD34^＋细胞表面标志、归巢黏附分子、集落形成能力等干细胞特征变化的影响。用免疫磁珠法从新鲜分离的脐血单个核细胞分离CD34^＋造血干祖细胞（HSPC）；用MSC饲养层（feeder）制备经^137Cs照射的间充质干细胞饲养细胞（MSC feeder cells）。将CD34^＋细胞接种在不同的培养体系中，实验分为3组：HSPC＋CK组为培养液中加入细胞因子组合（SCF、FL和TPO），HSPC＋MSC组为CD34^＋细胞接种在MSC feeder上，HSPC＋MSC＋CK组同时加入细胞因子组合及MSC饲养细胞。培养后4、7、10、14天计数有核细胞总数（MNC），计算细胞扩增情况；用流式细胞术检测不同处理组间CD34^＋细胞及亚群免疫表型、归巢黏附分子和集落形成能力。结果表明：在2周的培养时间里，3组MNC和CD34^＋细胞均明显增加，MNC扩增数依次HSPC＋MSC＋CK组〉HSPC＋CK组〉HSPC＋MSC组。体外扩增10天内HSPC＋MSC＋CK组MNC得到大量的扩增，同时CD34^＋细胞的扩增亦较高。培养4天3组细胞CD34^＋比例较0天有明显下降（P〈0．01）；扩增后CD34^＋细胞比例：HSPC＋MSC组〉HSPC＋MSC＋CK组〉HSPC＋CK组（P〈0．01）；各组CD34^＋细胞亚型细胞比例有所不同，HSPC＋CK组4天时CD34^＋CD38^-细胞有一过性升高（62．71％），之后迅速降低，7天时为0．05％；HSPC＋MSC组7天时CD34^＋CD38^-细胞比例为18．92％，与HSPC＋CK组比较差异有统计学意义（P〈0．05）。从集落形成分析结果看出：MSC、细胞因子混合组扩增后细胞集落形成能力在不同时间点均维持在较高水平。结论：脐血CD34^＋细胞在体外短期培养（〈7天）下，MSC和细胞因子联合应用能同时使CD34^＋细胞得到明显的扩增并维持造血干祖细胞的生物学特征。     

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。     

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.     

Thrombopoietin to replace megakaryocyte‐derived growth factor: impact on stem and progenitor cells during ex vivo expansion of CD34+ cells mobilized in peripheral blood

BACKGROUND: The first protocol of ex vivo expansion that enabled almost total abrogation of postmyeloablative chemotherapy neutropenia was based on a three‐cytokine cocktail (stem cell factor [SCF], granulocyte–colony‐stimulating factor [G‐CSF], pegylated‐megakaryocyte growth and development factor [PEG‐MGDF]) in a serum‐free medium. Since the clinical‐grade molecule MGDF is no longer available on the market, we evaluated its substitution by thrombopoietin (TPO). STUDY DESIGN AND METHODS: CD34+ cells of myeloma patients were expanded for 10 days in serum‐free cultures with SCF, G‐CSF, or MGDF (100 ng/mL) or with TPO (2.5, 10, 20, 50, and 100 ng/mL) instead of MGDF. Day 10 amplifications of total nucleated cells, CD34+ cells, committed progenitors (CFCs), the capacity of engraftment of NOD/SCID mice (SCID repopulating cells [SRCs]), and the immunophenotype of cells in expansion product (CD13, CD14, CD33, CD41, CD61) were analyzed. RESULTS: TPO in doses of 2.5 and 10 ng/mL exhibits an effect comparable to that of MGDF (100 ng/mL) on total, CD34+, and CFCs amplification. Compared to MGDF, TPO (starting at 10 ng/mL) enhances two‐ to threefold the percentage of megakaryocyte lineage cells (CD41+ and CD61+). Finally, TPO maintains or even enhances (depending on dose) SRC activity. CONCLUSIONS: The use of TPO instead of MGDF in our protocol is feasible without any negative effect on progenitor cell expansion. Furthermore, applied in dose of 10 or 100 ng/mL it could enhance both the stem cell activity and the percentage of megakaryocyte lineage cells in expansion product.     

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.     

Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent

BACKGROUND: The liquid culture of murine bone marrow cells at 1-percent oxygen maintains the balance between primative progenitor cell renewal and clonogenic progenitor expansion better than that at 20-percent oxygen. These results are of potential interest for the ex vivo expansion of human progenitor cells, as low O(2) tension could preserve the engraftment potential of cultured apheresis products. STUDY DESIGN AND METHODS: G-CSF-mobilized blood cells collected by apheresis, now the main source of progenitor cells for autologous transplantation, were cultured at 1-percent and 20-percent O(2) for 7 days in serum-free liquid cultures in the presence of IL-3 and SCF (5 ng/mL). The growth of the clonogenic progenitors (CFU-GM, BFU-E, CFU-Mix) and of the more primitive human HPCs that are capable of generating clongenic progenitors in secondary liquid culture, as well as the proliferation and differentiation of total and CD34+ cells, was analyzed. RESULTS: The expansion of CD34+ cells and of clonogenic progenitors was significantly lower in liquid cultures at 1-percent O(2) than at 20-percent O(2). On the contrary, the primitive human HPCs were better maintained and expanded at 1-percent O(2), although the number of CD34+ cells remaining quiescent was lower. After 7 days of liquid culture at 1-percent or 20-percent O(2) the percentage of CD34+ cells was similar. However, the CD34+ cells that divided more than four times (PKH2 staining) were more numerous in liquid cultures incubated at 1-percent O(2). CONCLUSION: When cultured at 1-percent O(2) for 7 days in presence of IL-3 and SCF, the CD34+ cells present in apheresis components underwent more cell divisions and better maintained their primitive progenitor cell potential. As suggested by previous results in mice, our data on human cells emphasize the potential interest of cultures at low O(2) tension (1%) for cell therapy protocols aimed at expanding primitive HPCs in autografts.     

Evaluation of serum-free culture conditions able to support the ex vivo expansion and engraftment of human hematopoietic stem cells in the human-to-sheep xenograft model

To develop culture conditions devoid of serum that would support the ex vivo expansion and maintenance of hematopoietic stem cells (HSC) with engraftment capability, we performed in vitro studies in which phenotypic and functional expansion of putative HSC populations were evaluated. We then used the human-sheep xenograft model to evaluate the engraftment potential of the ex vivo expanded cells. Adult human bone marrow CD34+-enriched cells were cultured in QBSF-60 for 14 days with or without fetal bovine serum (FBS) in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), and analyzed at days 0, 3, 7, and 14 for expansion, phenotype, clonogenic ability, and cell cycling status. Although there was a progressive expansion of numbers of cells in both groups, the group cultured with serum exhibited more than twice the expansion seen in the group without serum at all time points. The phenotypic analysis of the cultured cells showed an increase in the absolute numbers of CD34+ cells in both groups. However, when we evaluated the presence of CD34+CD38- cells, this population persisted in significantly higher numbers in the group cultured without serum, with maximal output of CD34+CD38- cells seen at 3 and 7 days. A higher total clonogenic potential was found in the serum-free cultures. To evaluate the in vivo engraftment potential of these cultured cells, 19 sheep fetuses were each injected i.p. with 9 x 10(5) cells either fresh or cultured in the conditions described above. Although all the transplanted fetal sheep showed the presence of human cells in their bone marrow (BM), the highest levels of long-term engraftment in primary recipients were obtained with the fraction of cells cultured for 3 days followed by 7 days in the absence of serum. In the secondary sheep recipients, the highest level of long-term engraftment was also achieved in sheep that received cells from primary recipients that had received cultured cells in serum-free conditions for 3 days.     

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.