Kim H J, Okamoto Y, Ito M, Takaue Y, Kawano Y, Watanabe T, Yamaue T, Tohda Y, Ogose T, Shimada T, Shimosaka A, Kuroda Y
Department of Pediatrics, University of Tokushima, Japan.
Stem Cells. 1997;15(5):347-52. doi: 10.1002/stem.150347.
We examined cell culture conditions with various combinations of cytokines including thrombopoietin (TPO) to obtain the most efficient transduction of recombinant retrovirus vectors into G-CSF-mobilized blood CD34+ cells which were obtained from children and purified with an Isolex 50 system (Baxter; Deerfield, IL). Three different 4-day culture conditions for the stimulation of CD34+ cells were compared in terms of a cell-cycle analysis by fluorometry and gene transduction efficiency as determined by resistance to G418 and NeoR polymerase chain reaction (PCR) for individual colony-forming unit-granulocyte/macrophage (CFU-GM) grown in a methylcellulose culture system. The cytokines tested were: A) interleukin (IL)-6 + stem cell factor (SCF); B) IL-3 + IL-6 + SCF, and C) IL-3 + IL-6 + SCF + TPO. Without a cell culture, the percentage of CD34+ cells in the cell cycle (the percentage of cells in phases S and G2/M) was 4.6%. After a four-day culture (n = 5), this value increased with the addition of IL-3 (22%) or IL-3 + TPO (27%, p < 0.05) as compared to that with the baseline cocktail of IL-6 + SCF (15%). The cell number uniformly increased approximately 10-fold in each culture condition. The average efficiency of gene transfer into incubated CD34+ cells with the corresponding combinations of cytokines was, respectively, 57%, 47%, and 30% for G418-screened CFU-GM and 72%, 68%, and 51% for polymerase chain reaction-positive CFU-GM. A statistically significant difference (p < 0.01) was found for G418/CFU-GM with IL-3 + IL-6 + SCF (57%) versus IL-3 + IL-6 + SCF + TPO (30%). Hence, it is likely that the increased cell proliferation produced by the addition of TPO was not necessarily translated into an increased rate of retroviral-mediated gene transduction, possibly because TPO preferentially induced the differentiation of stem cells into mature progenitors in these culture systems.
我们研究了包括血小板生成素（TPO）在内的多种细胞因子组合的细胞培养条件，以实现重组逆转录病毒载体对从儿童获取并经Isolex 50系统（百特公司；伊利诺伊州迪尔菲尔德）纯化的G-CSF动员的血液CD34+细胞进行最有效的转导。通过荧光法进行细胞周期分析以及通过对甲基纤维素培养系统中生长的单个集落形成单位-粒细胞/巨噬细胞（CFU-GM）的G418抗性和NeoR聚合酶链反应（PCR）来确定基因转导效率，比较了三种不同的用于刺激CD34+细胞的4天培养条件。所测试的细胞因子为：A）白细胞介素（IL）-6 +干细胞因子（SCF）；B）IL-3 + IL-6 + SCF；C）IL-3 + IL-6 + SCF + TPO。未经细胞培养时，处于细胞周期中的CD34+细胞百分比（处于S期和G2/M期的细胞百分比）为4.6%。经过4天培养（n = 5）后，与IL-6 + SCF的基线组合（15%）相比，添加IL-3（22%）或IL-3 + TPO（27%，p < 0.05）时该值增加。在每种培养条件下细胞数量均均匀增加约10倍。对于经G418筛选的CFU-GM，用相应细胞因子组合对培养的CD34+细胞进行基因转移的平均效率分别为57%、47%和30%，对于聚合酶链反应阳性的CFU-GM则分别为72%、68%和51%。发现IL-3 + IL-6 + SCF（57%）与IL-3 + IL-6 + SCF + TPO（30%）的G418/CFU-GM存在统计学显著差异（p < 0.01）。因此，添加TPO所产生的细胞增殖增加不一定转化为逆转录病毒介导的基因转导速率增加，这可能是因为在这些培养系统中TPO优先诱导干细胞分化为成熟祖细胞。
