转基因骨髓基质细胞对人脐血CD34+细胞体外扩增作用的研究

为探讨转染FL基因的人原代骨髓基质细胞对脐血CD34+ 细胞的体外扩增效应 ,建立了转基因骨髓基质细胞与人脐血CD34+ 细胞共培养体系。采用免疫磁珠法分离人脐血CD34+ 细胞 ,在不同的体外培养体系中进行培养并取样检测细胞总数 ,CFC和CD34+ 细胞百分率。结果表明 ,在不同组合的培养体系中 ,转基因骨髓基质细胞培养体系较骨髓基质细胞培养体系和无滋养层培养体系对有核细胞总数 ,CFC含量和CD34+ 细胞含量均具有明显的扩增作用 ,分别扩增了 12 2 .5± 4 .3,39.6± 2 .7和 11.8± 0 5 2倍 (P <0 .0 5 )。结论 :转染FL基因的人骨髓基质细胞协同其它细胞因子可增强对人脐血CD34+ 细胞的体外扩增作用。     

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.     

Stimulation of mouse bone marrow cells with kit ligand, FLT3 ligand, and thrombopoietin leads to efficient retrovirus-mediated gene transfer to stem cells, whereas interleukin 3 and interleukin 11 reduce transduction of short- and long-term repopulating cells

The effects of cytokine stimulation during retroviral transduction on in vivo reconstitution of mouse hematopoietic stem cells was tested in a murine competitive repopulation assay with alpha-thalassemia as a marker to distinguish donor and recipient red blood cells (RBCs) and the enhanced green fluorescent protein (EGFP) as a marker for gene transfer. After transplantation, EGFP was detected in up to 90% of circulating RBCs, platelets, and leukocytes, and in primitive progenitors in bone marrow (BM), spleen, and thymus of individual transplanted mice for observation periods of more than 6 months. Large quantitative differences in reconstitution were observed after transplantation with graded numbers (1000-30, 000) of EGFP(+) cells preconditioned with various combinations of Kit ligand (KL), FLT-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), and IL-11. Relative to nonmanipulated BM cells, repopulation of EGFP(+) cells was maintained by KL/FL/TPO stimulation, but approximately 30-fold reduced after KL/FL/TPO/IL-3, or KL/FL/IL-3/IL-11. These differences were not caused by changes in the ability of immature hematopoietic cells to home to the BM, which was only moderately reduced. In conclusion, these quantitative transplantation studies of mice demonstrate the importance of optimal ex vivo cytokine stimulation for gene transfer to stem cells with retention of their in vivo hematopoietic potential, and also emphasize that overall in vitro transduction frequency does not predict gene transfer to repopulating stem cells.     

人胎盘源贴壁细胞支持脐血CD34+细胞体外扩增

从胎盘中分离培养人胎盘源贴壁细胞 (humanplacentaderivedadherentcells,hPDAC) ,研究其对脐血CD34+细胞体外扩增作用。以酶消化法自人胎盘组织中分离培养hPDAC ,并以流式细胞术对其进行鉴定。进一步 ,采用免疫磁珠法分离人脐血CD34+细胞 ,建立以hPDAC为滋养层的CD34+细胞体外扩增培养体系 ,并与无滋养层的液体培养体系相比 ,观察不同培养体系对有核细胞总数、CFC及CD34+细胞百分率的影响。结果表明 ,人胎盘组织中可分离培养出hPDAC ,并证实其为混合性细胞群体 ,主要含有间充质细胞。以hPDAC为滋养层的共培养体系较无滋养层液体培养体系对有核细胞总数、CFC及CD34+细胞均具有明显的扩增效应 ,其中以SCF +IL 3+IL 6 +FL +hPDAC组扩增效果最强 ,分别扩增了 ( 12 6 .0± 6 .7)倍 ,( 4 9.8± 1.7)倍和 ( 8.3± 1.6 5 )倍。上述结果提示 ,hPDAC具有体外支持造血作用 ,并提供了一与脐血CD34+细胞体外扩增相适应的新滋养层。     

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。     

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   

Preferential ex vivo expansion of megakaryocytes from human cord blood CD34+-enriched cells in the presence of thrombopoietin and limiting amounts of stem cell factor and Flt-3 ligand

The high proliferative potential of cord blood (CB) stem cells and the identification of the key factor of megakaryopoiesis, thrombopoietin (TPO), permit the ex vivo expansion of megakaryocytes (MKs) for possible use in early post-transplant support of patients and the production of functional platelets for transfusion. However, culture conditions for the generation of adequate MKs for this purpose are not yet optimized. Therefore, we sought to define the mixture of early-acting cytokines and TPO that would promote the expansion of MK progenitors over other lineages and result in overall better MK expansion and platelet yields. CB CD34(+)-enriched cells were cultured in serum-free medium for 17 days in presence of TPO alone or in various combinations with early-acting cytokines used at different concentrations and addition times. MK expansion and polyploidy and platelet production were monitored by flow cytometry analysis using specific surface markers (CD41 and CD42b) and propidium iodide labeling. Our results showed that the use of high concentrations of stem cell factor (SCF) and Flt-3 ligand (FL) in early CB TPO-supplemented cultures was more favorable to monocytic and granulocytic cell expansion. However, we observed that their presence in limiting amounts was required for the preferential expansion of MK progenitors. The addition of SCF, FL, TPO, and interleukin-6 (IL-6) at high concentrations in secondary cultures of these expanded MKs resulted in optimal MK proportion (approximately 25% of MKs) and expansion (>300 MK per seeded cell), highest proportions of polyploid MKs (22% of mature MKs > or = 8N), and best platelet yields. Our results indicate that TPO-induced MK progenitors are more sensitive to early-acting cytokines than non-MK cells. We propose that MKs generated in the optimized conditions, in combination with immature stem/progenitor cells, could prove useful for the short-term platelet recovery following CB transplantation.     

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.     

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.     

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.     

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.     

人骨髓基质细胞促进脐血CD34+细胞的体外扩增和对NOD/SCID小鼠的植入

目的 研究人骨髓基质细胞(MSC)促进脐血CD34+细胞体外扩增及植入能力的作用.方法 分离、培养正常人的MSC作为滋养层细胞.在TPO、SCF、FL和G-CSF刺激下,比较有、无MSC滋养层细胞对扩增脐血CD34+细胞后,CD34+细胞和CFU数的增加倍数,以及植入非肥胖性糖尿病/重症联合免疫缺陷(NOD/SCID)小鼠的能力.结果 以骨髓MSC为滋养层细胞的培养体系可以更加有效地扩增脐血CD34+细胞.体外扩增1周总细胞数(TNC)、CD34+细胞和CFU的均数分别增加111.6、19.3和58.0倍;体外扩增2周后TNC、CD34+细胞和CFU的均数分别增加532.8、41.3和563.5倍.脐血细胞输入NOD/SCID小鼠6周后,移植未扩增脐血细胞的对照组小鼠骨髓细胞中人CD45+细胞比例仅为1.2%～3.7%;移植单纯细胞因子刺激扩增组小鼠骨髓中,人CD45+细胞比例为7.6%～12.1%;以MSC为滋养层扩增的脐血细胞移植组,人CD45+细胞比例达到45.3%～59.1%.结论 以人骨髓MSC为作为饲养层细胞,不但可以更加有效扩增脐血CD34+细胞,而且可以促进脐血细胞植入NOD/SCID小鼠的能力,有潜在的临床应用价值.     

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。     

免疫磁性分选系统大量分选CD34+细胞的实验研究

CD3 4 细胞的分离纯化在自体外周血干细胞、异基因骨髓移植 /外周血干细胞移植及干细胞研究中具有重要的应用价值。为了摸索大量分选CD3 4 细胞的方法 ,本研究应用Isolex3 0 0i磁性分选系统富集CD3 4 细胞 ,采用流式细胞术监测分选前后细胞表面标志 ,经CD3 4 细胞体外增殖实验及克隆形成实验验证分选获得的CD3 4 细胞生物学活性。结果显示 ,所完成的 5例次自体外周血富集CD3 4 细胞时 ,先收获单个核细胞约 ( 3 .5 -6.0 )× 10 10 ,CD3 4 细胞占单个核细胞百分率为 ( 0 .5 5 - 1.2 ) % ;收获的CD3 4 阳性细胞总数为 ( 2 - 3 )× 10 8,纯度为 ( 75 - 85 ) % ,回收率为 ( 4 0 - 65 ) %。体外实验表明 ,在SCF IL 3 FL TPO EPO存在下 ,经过 3 - 4天培养 ,可扩增 2 - 3倍 ;经CFU GM、BFU E集落形成实验显示具有形成集落的能力 ,证实分选后细胞具有造血祖细胞生物活性。结论 :应用Isolex3 0 0iCD3 4 细胞分选仪可以高效大量富集CD3 4 细胞 ,适于临床应用。     

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。     

Establishment of mouse embryonic fibroblast cell lines that promote ex vivo expansion of human cord blood CD34+ hematopoietic progenitors

The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic progenitor cells (HPC) is vital to stem cell transplantation. The use of a monolayer of stromal cells on which to grow HPC in direct contact allows high efficiency ex vivo expansion of HPC. Here, we report an establishment of three murine embryonic fibroblast stromal cell lines from adherent cells of day-12 mouse embryos. Among them, HYMEQ-5 was most efficient in supporting long-term maintenance of human umbilical cord blood (CB) CD34(+) cells. Human CB CD34(+) cells cultured on HYMEQ-5 in the presence of stem cell factor (SCF), thrombopoietin, and flk-ligand (FL) showed high expansion of CD34(+)CD38(-) cells and highly proliferative potential-colony forming cells (HPP-CFC). Direct cell-to-cell contact between CD34(+) cells and HYMEQ-5 was important for this expansion. RT-PCR analysis showed that HYMEQ-5 produced FL, SCF, interleukin-6, and macrophage colony-stimulating factor (M-CSF). Expanded CB CD34(+) cells efficiently reconstituted hematopoiesis in nonobese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. These findings suggest that HYMEQ-5 provides a milieu that supports long-term human hematopoiesis as well as ex vivo expansion of human CB CD34(+) HPC. This cell line may facilitate elucidation of the mechanism of cellular interactions between HPC and stromal 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.     

FL与SCF、IL-3、GM-CSF及EPO协同调控脐血造血干/祖细胞的体外扩增

目的　探讨造血细胞因子的不同组合对脐血造血干 /祖细胞的扩增作用。方法　应用免疫磁珠法分离纯化脐血CD3 4+ 造血干 /祖细胞 ,在体外液体培养体系中经各种不同细胞因子组合扩增 1周 ,用流式细胞仪检测CD3 4+ 造血干 /祖细胞并进行甲基纤维素法半固体培养 2周 ,在倒置显微镜下计数集落产率。结果　在FL、SCF、IL 3、GM CSF、EPO造血细胞因子的不同组合下脐血造血干 /祖细胞得以扩增 ,以IL 3+SCF +FL +EPO组的扩增效率最高 ,其有核细胞数、CD3 4+ 细胞及CFU GM、BFU E集落分别扩增 46 2± 175、3.47± 1.6 4、2 6 4± 10 5和 12 8± 6 7倍。结论　FL对扩增CD+ 3 4细胞具有较强的协同作用 ,合理的细胞因子组合扩增的脐血造血干 /祖细胞可成为异基因造血干细胞移植的主要来源。     

Human accessory cells have a humoral bystander effect on CAFC growing on murine feeder

Human early hematopoietic progenitors from bone marrow (BM) and leukapheresis products (LP) are highly proliferative in presence of accessory cells in standard culture on the murine FBMD-1 cell feeder with weekly addition of human interleukin-3 (HuIL-3) and granulocyte-colony stimulating factor (HuG-CSF). If however purified CD34+ cells are cultured under otherwise identical conditions, cobblestone areas (CAFC) formed by the same number of target cells are diminished by more than 1 log, as we showed previously. This suggests that mature cells are involved in growth of early progenitors. To determine whether this bystander effect is mediated by soluble growth factors, or by direct cell-to-cell contact with early progenitors, we stimulated mature plastic adherent cells separately and tested the resulting conditioned supernatant (ACS) on CAFC and colony-forming unit-granulocyte-macrophage (CFU-GM) production. In ACS-complemented standard cultures of purified CD34+ cells, the yield of CAFC was up to 1 log higher if compared to parallel cultures without ACS. Likewise, the CFU-GM production was enhanced in presence of ACS, especially in the adherent fraction of the culture. When CD34+ cell cultures were performed with ACS but without added interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF), CAFC production was in the same range as if these growth factors were added alone. Addition of anti-G-CSF antibody (Ab) to ACS decreased CAFC recruitment significantly, whereas anti-IL-3 Ab had no significant effect. These findings suggest that ACS complemented with IL-3 and G-CSF replaces the accessory cells largely; this is not only due to presence of G-CSF, because ACS in combination with recombinant growth factors mounts CAFC yield higher than saturating amounts of growth factors alone do. There must be further synergizing soluble factors in the supernatant.     

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.