Piacibello Wanda, Bruno Stefania, Sanavio Fiorella, Droetto Sara, Gunetti Monica, Ailles Laurie, Santoni de Sio Francesca, Viale Andrea, Gammaitoni Loretta, Lombardo Angelo, Naldini Luigi, Aglietta Massimo
Department of Oncological Sciences, University of Torino Medical School, Torino, Italy.
Blood. 2002 Dec 15;100(13):4391-400. doi: 10.1182/blood.V100.13.4391.
The ability of advanced-generation lentiviral vectors to transfer the green fluorescent protein (GFP) gene into human hematopoietic stem cells (HSCs) was studied in culture conditions that allowed expansion of transplantable human HSCs. Following 96 hours' exposure to flt3/flk2 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), and interleukin-6 (IL-6) and overnight incubation with vector particles, cord blood (CB) CD34(+) cells were further cultured for up to 4 weeks. CD34(+) cell expansion was similar for both transduced and control cells. Transduction efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) was assessed by transplants into NOD/SCID mice. Mice that received transplants of transduced week 1 and week 4 expanded cells showed higher levels of human engraftment than mice receiving transplants of transduced nonexpanded cells (with transplants of 1 x 10(5) CD34(+) cells, the percentages of CD45(+) cells were 20.5 +/- 4.5 [week 1, expanded] and 27.2 +/- 8.2 [week 4, expanded] vs 11.7 +/- 2.5 [nonexpanded]; n = 5). The GFP(+)/CD45(+) cell fraction was similar in all cases (12.5% +/- 2.9% and 12.2% +/- 2.7% vs 12.7% +/- 2.1%). Engraftment was multilineage, with GFP(+)/lineage(+) cells. Clonality analysis performed on the bone marrow of mice receiving transduced and week 4 expanded cells suggested that more than one integrant likely contributed to the engraftment of GFP-expressing cells. Serial transplantations were performed with transduced week 4 expanded CB cells. Secondary engraftment levels were 10.7% +/- 4.3% (n = 12); 19.7% +/- 6.2% of human cells were GFP(+). In tertiary transplants the percentage of CD45(+) cells was lower (4.3% +/- 1.7%; n = 10); 14.8% +/- 5.9% of human cells were GFP(+), and human engraftment was multilineage. These results show that lentiviral vectors efficiently transduce HSCs, which can undergo expansion and maintain proliferation and self-renewal ability.
在允许可移植人类造血干细胞（HSCs）扩增的培养条件下，研究了新一代慢病毒载体将绿色荧光蛋白（GFP）基因导入人类造血干细胞的能力。脐血（CB）CD34(+)细胞在暴露于fms样酪氨酸激酶3/胎儿肝激酶2配体（FL）、血小板生成素（TPO）、干细胞因子（SCF）和白细胞介素-6（IL-6）96小时并与载体颗粒过夜孵育后，进一步培养长达4周。转导细胞和对照细胞的CD34(+)细胞扩增相似。通过移植到非肥胖糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠中来评估NOD/SCID重建造血干细胞（SRCs）的转导效率。接受第1周和第4周扩增的转导细胞移植的小鼠比接受未扩增的转导细胞移植的小鼠表现出更高水平的人类造血植入（移植1×10(5)个CD34(+)细胞时，CD45(+)细胞的百分比分别为20.5±4.5 [第1周，扩增]和27.2±8.2 [第4周，扩增]，而未扩增组为11.7±2.5；n = 5）。所有情况下GFP(+)/CD45(+)细胞比例相似（分别为12.5%±2.9%、12.2%±2.7%和12.7%±2.1%）。造血植入是多谱系的，存在GFP(+)/谱系(+)细胞。对接受第4周扩增的转导细胞移植的小鼠骨髓进行的克隆性分析表明，可能不止一种整合体促成了表达GFP细胞的造血植入。用第4周扩增的转导CB细胞进行了连续移植。二次造血植入水平为10.7%±4.3%（n = 12）；19.7%±6.2%的人类细胞为GFP(+)。在三次移植中，CD45(+)细胞的百分比更低（4.3%±1.7%；n = 10）；14.8%±5.9%的人类细胞为GFP(+)，且人类造血植入是多谱系的。这些结果表明，慢病毒载体能有效地转导造血干细胞，这些造血干细胞能够扩增并维持增殖和自我更新能力。
