Wang Lan, Guan Xin, Wang Huihui, Shen Bin, Zhang Yu, Ren Zhihua, Ma Yupo, Ding Xinxin, Jiang Yongping
Biopharmaceutical R&D Center, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, China.
Biopharmagen Corp, Suzhou, China.
Stem Cell Res Ther. 2017 Jul 18;8(1):169. doi: 10.1186/s13287-017-0625-z.
Accumulated evidence supports the potent stimulating effects of multiple small molecules on the expansion of hematopoietic stem cells (HSCs) which are important for the therapy of various hematological disorders. Here, we report a novel, optimized formula, named the SC cocktail, which contains a combination of three such small molecules and four cytokines.
Small-molecule candidates were individually screened and then combined at their optimal concentration with the presence of cytokines to achieve maximum capacity for stimulating the human CD34 cell expansion ex vivo. The extent of cell expansion and the immunophenotype of expanded cells were assessed through flow cytometry. The functional preservation of HSC stemness was confirmed by additional cell and molecular assays in vitro. Subsequently, the expanded cells were transplanted into sublethally irradiated NOD/SCID mice for the assessment of human cell viability and engraftment potential in vivo. Furthermore, the expression of several genes in the cell proliferation and differentiation pathways was analyzed through quantitative polymerase chain reaction (qPCR) during the process of CD34 cell expansion.
The SC cocktail supported the retention of the immunophenotype of hematopoietic stem/progenitor cells remarkably well, by yielding purities of 86.6 ± 11.2% for CD34 cells and 76.2 ± 10.5% for CD34CD38 cells, respectively, for a 7-day culture. On day 7, the enhancement of expansion of CD34 cells and CD34CD38 cells reached a maxima of 28.0 ± 5.5-fold and 27.9 ± 4.3-fold, respectively. The SC cocktail-expanded CD34 cells preserved the characteristics of HSCs by effectively inhibiting their differentiation in vitro and retained the multilineage differentiation potential in primary and secondary in vivo murine xenotransplantation trials. Further gene expression analysis suggested that the small-molecule combination strengthened the ability of the cytokines to enhance the Notch pathway for the preservation of HSC stemness, and inhibited the ability of the cytokines to activate the Wnt pathway for HSC differentiation.
We developed an optimal small-molecule/cytokine combination for the enhancement of HSC expansion via inhibition of differentiation. This approach indicates promising application for preparation of both the HSCs and the mature, functional hematopoietic cells for clinical transplantation.
越来越多的证据支持多种小分子对造血干细胞（HSC）扩增具有强大的刺激作用，这对各种血液系统疾病的治疗至关重要。在此，我们报告一种新型的优化配方，名为SC鸡尾酒，它包含三种此类小分子和四种细胞因子的组合。
对小分子候选物进行单独筛选，然后在细胞因子存在的情况下以最佳浓度进行组合，以实现体外刺激人CD34细胞扩增的最大能力。通过流式细胞术评估细胞扩增程度和扩增细胞的免疫表型。通过体外额外的细胞和分子试验确认HSC干性的功能保存。随后，将扩增的细胞移植到经亚致死剂量照射的NOD/SCID小鼠体内，以评估人细胞在体内的活力和植入潜力。此外，在CD34细胞扩增过程中，通过定量聚合酶链反应（qPCR）分析细胞增殖和分化途径中几个基因的表达。
SC鸡尾酒能非常好地支持造血干/祖细胞免疫表型的保留，在7天培养中，CD34细胞纯度分别为86.6±11.2%，CD34CD38细胞纯度为76.2±10.5%。在第7天，CD34细胞和CD34CD38细胞扩增的增强分别达到最大值28.0±5.5倍和27.9±4.3倍。SC鸡尾酒扩增的CD34细胞通过在体外有效抑制其分化而保留了HSC的特征，并在原发性和继发性体内小鼠异种移植试验中保留了多谱系分化潜力。进一步的基因表达分析表明，小分子组合增强了细胞因子增强Notch途径以保存HSC干性的能力，并抑制了细胞因子激活Wnt途径促进HSC分化的能力。
我们开发了一种最佳的小分子/细胞因子组合，通过抑制分化来增强HSC扩增。这种方法为临床移植制备HSC和成熟的功能性造血细胞显示出有前景的应用。
