Rice A, Barbot C, Lacombe F, Dubosc-Marchenay N, Marit G, Hau F, Boiron J M, Reiffers J
Laboratoire de Greffe de Moelle, URA CNRS 1456, Université de Bordeaux II, France.
Stem Cells. 1993 Jul;11(4):326-35. doi: 10.1002/stem.5530110411.
Peripheral blood stem cells (PBSC) contain a mixture of mature and immature hematopoietic progenitors. Resistance to 5-Fluorouracil (5-FU) has been used to identify and characterize primitive quiescent stem cells among bone marrow (BM) cells. To see if the same technique could be used to isolate a similar population of cells among PBSC, low-density peripheral blood mononuclear cells (PBMNC) were collected by cytapheresis in the regenerative phase after high-dose chemotherapy from patients with hematological malignancies. These PBMNC were incubated with increasing concentrations of 5-FU for 24 h. The viable 5-FU resistant cells were then cultured in semi-solid media in the presence of either single cytokines: TCM 5637, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), or a combination of cytokines: interleukin 1 (IL-1) IL-1 + IL-3 + 5637, IL-1 + IL-3 + Stem Cell Factor (SCF). Low concentrations (5-10 micrograms/ml 5-FU) eliminated mature day 7 Colony Forming Units-Granulocyte Macrophage (CFU-GM) and spared day 7 clusters while enriching for day 14 CFU-GM, irrespective of the growth factors used. Higher concentrations of 5-FU (15, 20, 25 micrograms/ml) selected for later forming clonogenic elements. A combination of synergistic growth factors was required for the development of morphologically identifiable clonogenic elements resistant to 25 micrograms/ml 5-FU at day 21 of culture. Further experimentation demonstrated that SCF could effectively replace TCM 5637 in the cytokine combination for the detection of primitive late forming clonogenic elements. The presence of SCF potentiated colony formation by 5-FU resistant PBMNC. It was confirmed that GM-CSF alone was unable to support colony formation by PBMNC resistant to 25 micrograms/ml. These observations demonstrate that PBSC contain a heterogenous mixture of hematopoietic progenitors and that incubation with 25 micrograms/ml 5-FU permits access to a quiescent primitive stem cell population that requires a combination of synergistic growth factors for the development of morphologically identifiable clonogenic elements at day 21. Taken together, these results suggest that PBSC have similar characteristics to BM derived stem cells.
外周血干细胞（PBSC）包含成熟和未成熟造血祖细胞的混合物。对5-氟尿嘧啶（5-FU）的抗性已被用于鉴定和表征骨髓（BM）细胞中的原始静止干细胞。为了确定相同技术是否可用于在PBSC中分离出类似的细胞群体，在血液系统恶性肿瘤患者接受大剂量化疗后的再生期，通过血细胞分离术收集低密度外周血单个核细胞（PBMNC）。将这些PBMNC与浓度递增的5-FU孵育24小时。然后将存活的5-FU抗性细胞在半固体培养基中培养，培养基中存在单一细胞因子：TCM 5637、粒细胞巨噬细胞集落刺激因子（GM-CSF），或细胞因子组合：白细胞介素1（IL-1）、IL-1 + IL-3 + 5637、IL-1 + IL-3 +干细胞因子（SCF）。低浓度（5 - 10微克/毫升5-FU）可消除第7天的成熟粒细胞巨噬细胞集落形成单位（CFU-GM），保留第7天的集落，同时富集第14天的CFU-GM，无论使用何种生长因子。较高浓度的5-FU（15、20、25微克/毫升）选择了后期形成克隆形成元件的细胞。在培养第21天时，需要协同生长因子组合才能形成对25微克/毫升5-FU具有抗性的形态可识别克隆形成元件。进一步的实验表明，在细胞因子组合中，SCF可有效替代TCM 5637来检测原始晚期形成的克隆形成元件。SCF的存在增强了5-FU抗性PBMNC的集落形成。已证实单独的GM-CSF无法支持对25微克/毫升5-FU具有抗性的PBMNC的集落形成。这些观察结果表明，PBSC包含造血祖细胞的异质混合物，并且与25微克/毫升5-FU孵育可获得静止的原始干细胞群体，该群体在第21天时需要协同生长因子组合才能形成形态可识别的克隆形成元件。综上所述，这些结果表明PBSC具有与骨髓来源干细胞相似的特征。
