Kobari L, Giarratana M C, Poloni A, Firat H, Labopin M, Gorin N C, Douay L
Centre de Thérapie Cellulaire, CHU Saint-Antoine, INSERM U417, Hôpital Saint-Antoine, Paris, France.
Bone Marrow Transplant. 1998 Apr;21(8):759-67. doi: 10.1038/sj.bmt.1701199.
The aim of the study is to define the ability of Flt3 ligand, MGDF, Epo and G-CSF to modulate the expansion of different hematopoietic compartments in association with a basic cocktail of SCF + IL-3 + IL-6 (S36). CD34+ cells from normal bone marrow were cultured in stroma-free, serum-free medium for 10 days. Using various concentrations of cytokines, total cells could be expanded up to 5200-fold, CD34+ cells up to 78-fold, CFU-GM up to 143-fold, BFU-E up to 46-fold, CFU-MK up to six-fold and LTC-IC up to four-fold. The results were assessed by multiparametric analysis of variance. Three factors had a significant stimulatory effect on the late precursor compartment: Epo (P < 10(-5)), G-CSF (P=5 x 10(-3)) and FL (P=10(-5)). Two were critical for CD34+ cell expansion: FL (P=4 x 10(-5)) and Epo (P=6 x 10(-5)), while two were critical for BFU-E expansion: MGDF (P=8 x 10(-4)) and FL (P=0.017). FL strongly stimulated CFU-GM expansion (P < 10(-5)), whereas none of the growth factors studied had any effect on CFU-MK. FL (P=10(-4)) and MGDF (P=0.002) were essential to obtain high levels of expansion of LTC-IC as determined in limiting dilution assays. In the light of the above results showing a preferential effect on the expansion of precursor cells (3080-fold), CD34+ cells (53-fold), CFU-GM (134-fold), BFU-E (46-fold) and LTC-IC (five-fold), the combination SCF, IL-3, IL-6, FL, MGDF, Epo and G-CSF was chosen as a putative cytokine cocktail for further studies on long-term culture. Sustained production of precursor cells, progenitor cells, LTC-IC and E-LTC-IC for up to 100 days reflects the persistence of very primitive stem cells. This suggests that these populations are probably able to undergo self-renewal divisions. The above combination of cytokines meets the required criterion for potential clinical application, which may be defined as an effective capacity to expand all cell compartments, using as the starting material high concentrations of low purity CD34+ cells.
本研究的目的是确定Flt3配体、巨核细胞生长发育因子（MGDF）、促红细胞生成素（Epo）和粒细胞集落刺激因子（G-CSF）与干细胞因子（SCF）+白细胞介素-3（IL-3）+白细胞介素-6（IL-6）的基础组合（S36）联合调节不同造血细胞区室扩增的能力。将来自正常骨髓的CD34+细胞在无基质、无血清培养基中培养10天。使用不同浓度的细胞因子，总细胞可扩增至5200倍，CD34+细胞可扩增至78倍，粒-巨噬细胞集落形成单位（CFU-GM）可扩增至143倍，爆式红系集落形成单位（BFU-E）可扩增至46倍，巨核细胞集落形成单位（CFU-MK）可扩增至6倍，长期培养启动细胞（LTC-IC）可扩增至4倍。结果通过多参数方差分析进行评估。有三个因素对晚期前体细胞区室有显著刺激作用：Epo（P < 10^(-5)）、G-CSF（P = 5×10^(-3)）和Flt3配体（FL，P = 10^(-5)）。有两个因素对CD34+细胞扩增至关重要：FL（P = 4×10^(-5)）和Epo（P = 6×10^(-5)），而有两个因素对BFU-E扩增至关重要：MGDF（P = 8×10^(-4)）和FL（P = 0.017）。FL强烈刺激CFU-GM扩增（P < 10^(-5)），而所研究的生长因子均对CFU-MK无任何影响。根据有限稀释分析确定，FL（P = 10^(-4)）和MGDF（P = 0.002）对于获得高水平的LTC-IC扩增至关重要。鉴于上述结果显示对前体细胞（3080倍）、CD34+细胞（53倍）、CFU-GM（134倍）、BFU-E（46倍）和LTC-IC（5倍）的扩增有优先作用，选择SCF、IL-3、IL-6、FL、MGDF、Epo和G-CSF的组合作为一种假定的细胞因子组合用于长期培养的进一步研究。前体细胞、祖细胞、LTC-IC和延长长期培养启动细胞（E-LTC-IC）持续产生长达100天，这反映了非常原始的干细胞的持久性。这表明这些细胞群体可能能够进行自我更新分裂。上述细胞因子组合符合潜在临床应用的所需标准，这可定义为使用高浓度低纯度CD34+细胞作为起始材料有效扩增所有细胞区室的能力。
