Analysis of the molecular mechanism regulating the development of hematopoietic stem cells in cord blood and their application to ex vivo expansion

我们证明了使用干细胞因子（SCF）、Flk 2/Flt 3配体（FL）、血小板生成素（TPO）和白细胞介素（IL）-6和可溶性IL-6受体（IL-6/sIL-6 R）的复合物的组合能够在NOD/SCID小鼠中重新增殖的脐带血造血干细胞的显著离体扩增。将1 ~ 2 × 104脐血CD_（34）~+细胞和SCF+FL、SCF+FL+TPO、SCF+ FL +TPO+IL-6/sIL-6 R培养7天的细胞移植到NOD/SCID小鼠体内，移植成功率分别为6/25（24%）、7/16（44%）、7/14（50%）和13/16（81%）。移植后10 ~ 12周，移植受者骨髓细胞中人CD 45+细胞的含量分别为1.4%、2.3%、6.7%和11.5%。SCF+FL+TPO+IL-6/sIL-6 R培养的细胞移植受体的CD 45+细胞由多种谱系组成，并含有大量CD 34+细胞，体外克隆培养形成人造血集落。12例新鲜CD 34+细胞与SCF+FL+TPO+IL-6/sIL-6 R培养的子代细胞重建情况比较，后者受者骨髓中CD 45+细胞比例高10倍，表明长期重建的造血干细胞明显扩增。通过有限稀释法估计扩增率为4.2倍。向细胞因子组合中加入IL-3废除了扩增细胞的再增殖能力。本研究为临床应用的人可移植造血干细胞的扩增提供了一种新的培养方法。

We demonstrated a significant ex vivo expansion of cord blood 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 interleukin (IL)-6 and soluble IL-6 receptor (IL-6/sIL-6R). When 1 to 2x104 cord blood CD34+ cells and the cells cultured with SCF+FL, SCF+FL+TPO and SCF+FL+TPO+IL-6/sIL-6R for 7 days were transplanted into NOD/SCID mice, 6/25 (24%), 7/16 (44%), 7/14 (50%) and 13/16 (81%) recipients revealed successful engraftment, and the bone marrow cells of the engrafted recipients contained 1.4%, 2.3%, 6.7% and 11.5% of human CD45+ cells, respectively, 10 to 12 weeks after the transplantation. The CD45+ cells in the recipients engrafted with the cells cultured with SCF+FL+TPO+IL-6/sIL-6R consisted of various lineages and a large number of CD34+ cells, which formed human hematopoietic colonies in in vitro clonal culture. In comparion of the reconstitution between fresh CD34+ cells from 12 samples and their progenies cultured with SCF+FL+TPO+IL-6/sIL-6R, the proportion of CD45+ cells in recipient marrow was 10 times higher in the latter, indicating the significant expansion of the long-term repopulating hematopoietic stem cells. The expansion rate was estimated at 4.2-fold by a limiting dilution method. The 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 aimed for clinical applications.