Hematopoietic stem cells expanded by fibroblast growth factor-1 are excellent targets for retrovirus-mediated gene delivery
Hematopoietic stem cells expanded by fibroblast growth factor-1 are excellent targets for retrovirus-mediated gene delivery
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DOI：
10.1016/j.exphem.2005.09.001
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发表时间：
2005-12-01
影响因子：
2.6
通讯作者：
Chiba, S
中科院分区：
文献类型：
--
作者：
Crcareva, A;Saito, T;Chiba, S
Objective.为了研究目的基因在造血干细胞（HSC）中的功能和成功的基因治疗，开发有效的基因转移到HSC中的方法是基础。在小鼠实验中，已经努力通过修饰载体，向供体小鼠施用5-氟尿嘧啶（5-FU），选择细胞因子混合物以更好地维持干细胞的长期再增殖潜力，本研究的目的是检查是否使用成纤维细胞生长因子-1（FGF-1）-扩增的骨髓细胞为向HSC递送逆转录病毒基因提供了改进的来源。将来自一只小鼠的未分级骨髓细胞在含有FGF-1的无血清培养基中培养。将漂浮和附着的细胞转移到retronectin预包被的培养皿中，并用来自用pMY/GFP逆转录病毒稳定转导的MP34细胞的病毒上清液感染。感染3天后，分选出绿色荧光蛋白阳性部分，并将细胞移植到致死剂量照射的小鼠体内。实验表明，在用FGF-1培养3周后，骨髓来源的竞争性再增殖单位（CRU）的数量从每只小鼠600增加到9300。在扩增细胞的逆转录病毒转导后，分选的逆转录病毒转导的CRU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，我们在100%的受者中实现了辐射保护和长期骨髓重建，平均骨髓和淋巴嵌合体分别为70%和50%。总之，FGF-1扩增的骨髓细胞构成了一个很好的干细胞来源，可用于一系列基因递送方案。(c)2005年国际实验血液学学会。爱思唯尔公司出版
Objective. 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.Materials and Methods. 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.Results. 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.Conclusion. 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. (c) 2005 International Society for Experimental Hematology. Published by Elsevier Inc.