Crcareva Aleksandra, Saito Toshiki, Kunisato Atsushi, Kumano Keiki, Suzuki Takahiro, Sakata-Yanagimoto Mamiko, Kawazu Masahito, Stojanovic Aleksandar, Kurokawa Mineo, Ogawa Seishi, Hirai Hisamaru, Chiba Shigeru
Department of Hematology/Oncology, University of Tokyo, Japan.
Exp Hematol. 2005 Dec;33(12):1459-69. doi: 10.1016/j.exphem.2005.09.001.
For the study of the function of genes of interest in hematopoietic stem cells (HSCs) and for successful gene therapy, it is fundamental to develop a method of efficient gene transfer into HSCs. In mice experiments, efforts have been made to raise the transduction efficiency by modifying the vectors, administrating 5-fluorouracil (5-FU) to donor mice, selecting cytokine cocktails to better sustain the long-term repopulating potential of the stem cells, and so on. The objective of this study is to examine whether the use of fibroblast growth factor-1 (FGF-1)-expanded bone marrow cells provide an improved source for retroviral gene delivery to HSCs.
Unfractionated bone marrow cells from one mouse were cultured in serum-free medium containing FGF-1. Both floating and attached cells were transferred to retronectin precoated dishes and infected with virus supernatant from MP34 cells stably transduced with pMY/GFP retrovirus. After 3-day infection, the green fluorescence protein-positive fraction was sorted and the cells were transplanted to lethally irradiated mice.
The experiments illustrated that the number of bone marrow-derived competitive repopulation units (CRUs) was increased from 600 to 9300 per mouse after a 3-week culture period with FGF-1. Following retroviral transduction of the expanded cells, the absolute number of sorted retrovirus-transduced CRUs was 4200. Using these retrovirus-transduced cells in noncompetitive reconstitution assay, we achieved radiation protection and long-term bone marrow reconstitution in 100% of the recipients with average myeloid and lymphoid chimerisms of 70% and 50%, respectively, even if we transplanted 150 recipients with cells derived from a single donor mouse.
In conclusion, FGF-1-expanded bone marrow cells constitute an excellent source of stem cells that could be used in a range of gene delivery protocols.
为了研究造血干细胞（HSCs）中感兴趣基因的功能以及实现成功的基因治疗，开发一种将基因高效导入造血干细胞的方法至关重要。在小鼠实验中，人们已通过修饰载体、给供体小鼠施用5-氟尿嘧啶（5-FU）、选择细胞因子组合以更好地维持干细胞的长期重建潜力等方式来提高转导效率。本研究的目的是检验使用成纤维细胞生长因子-1（FGF-1）扩增的骨髓细胞是否能为逆转录病毒基因导入造血干细胞提供更好的来源。
将一只小鼠的未分离骨髓细胞在含有FGF-1的无血清培养基中培养。将悬浮细胞和贴壁细胞转移至经纤连蛋白预包被的培养皿中，并用来自稳定转导了pMY/GFP逆转录病毒的MP34细胞的病毒上清液进行感染。感染3天后，对绿色荧光蛋白阳性部分进行分选，并将细胞移植到经致死剂量照射的小鼠体内。
实验表明，在使用FGF-1培养3周后，每只小鼠骨髓来源的竞争性重建单位（CRUs）数量从600增加到了9300。对扩增后的细胞进行逆转录病毒转导后，分选得到的逆转录病毒转导CRUs的绝对数量为4200。在非竞争性重建试验中使用这些逆转录病毒转导的细胞，即使我们用来自一只供体小鼠的细胞移植了150只受体小鼠，仍有100%的受体实现了辐射防护和长期骨髓重建，平均髓系和淋巴系嵌合率分别为70%和50%。
总之，FGF-1扩增的骨髓细胞构成了一种优秀的干细胞来源，可用于一系列基因递送方案。
