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

我们利用干细胞因子（SCF）、Flk2/Flt3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-6R）的复合物，证明了能够在NOD/SCID小鼠中再生的脐带血造血干细胞的体外扩增。将1 ~ 2x104个脐带血CD34+细胞和SCF+FL、SCF+FL+TPO和SCF+FL+TPO+IL-6/sIL-6R培养的细胞移植到NOD/SCID小鼠体内，移植后10 ~ 12周，6/25（24%）、7/16（44%）、7/14（50%）和13/16（81%）的受体成功移植，移植后的受体骨髓细胞中分别含有1.4%、2.3%、6.7%和11.5%的人CD45+细胞。移植SCF+FL+TPO+IL-6/sIL-6R培养的细胞后，受者体内的CD45+细胞具有多种细胞系和大量的CD34+细胞，在体外克隆培养中形成人造血集落。与12个样本的新鲜CD34+细胞与SCF+FL+TPO+IL-6/sIL-6R培养的后代相比，后者在受体骨髓中CD45+细胞的比例高出10倍，表明长期再生的造血干细胞显著扩增。用极限稀释法估计膨胀率为4.2倍。在细胞因子组合中加入IL-3会破坏扩增细胞的再生能力。本研究为人类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.