van Hennik P B, Verstegen M M, Bierhuizen M F, Limón A, Wognum A W, Cancelas J A, Barquinero J, Ploemacher R E, Wagemaker G
Institute of Hematology, Erasmus University Rotterdam, The Netherlands; and the Department of Cryobiology and Cell Therapy, Institut de Recerca Oncologica, Barcelona, Spain.
Blood. 1998 Dec 1;92(11):4013-22.
Purified CD34(+) and CD34(+)CD38(-) human umbilical cord blood (UCB) cells were transduced with the recombinant variant of Moloney murine leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope proteins. Using a 2-day growth factor prestimulation, followed by a 2-day, fibronectin fragment CH-296-supported transduction, CD34(+) and CD34(+)CD38(-) UCB subsets were efficiently transduced using either vector. The use of the SF-EGFP/PG13 retroviral packaging cell combination consistently resulted in twofold higher levels of EGFP-expressing cells than the MFG-EGFP/Am12 combination. Transplantation of 10(5) input equivalent transduced CD34(+) or 5 x 10(3) input equivalent CD34(+)CD38(-) UCB cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages of 8% and 5%, respectively, which showed that the in vivo repopulating ability of the cells had been retained. In addition, mice engrafted after transplantation of transduced CD34(+) cells using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP with median values of 2% and 23% of human CD45(+) cells, respectively, which showed that the NOD/SCID repopulating cells were successfully transduced. EGFP+ cells were found in all human hematopoietic lineages produced in NOD/SCID mice including human progenitors with in vitro clonogenic ability. EGFP-expressing cells were also detected in the human cobblestone area-forming cell (CAFC) assay at 2 to 6 weeks of culture on the murine stromal cell line FBMD-1. During the transduction procedure the absolute numbers of CAFC week 6 increased 5- to 10-fold. The transduction efficiency of this progenitor cell subset was similar to the fraction of EGFP+ human cells in the bone marrow of the NOD/SCID mice transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced CD34(+) cells, ie, 6% and 27%, respectively. The study thus shows that purified CD34(+) and highly purified CD34(+)CD38(-) UCB cells can be transduced efficiently with preservation of repopulating ability. The SF-EGFP/PG13 vector/packaging cell combination was much more effective in transducing repopulating cells than the MFG-EGFP/Am12 combination.
使用莫洛尼鼠白血病病毒（MoMLV）的重组变体MFG - EGFP或SF - EGFP转导纯化的CD34(+)和CD34(+)CD38(-)人脐带血（UCB）细胞，其中EGFP的表达由脾集落形成病毒（SFFV）和小鼠胚胎干细胞病毒（MESV）的杂交启动子驱动。感染性MFG - EGFP病毒由嗜性病毒生产细胞系（GP + envAm12）产生。SF - EGFP在为长臂猿白血病病毒（GaLV）包膜蛋白假型化的PG13细胞系中产生。使用2天的生长因子预刺激，随后进行2天的纤连蛋白片段CH - 296支持的转导，使用任一载体均可有效转导CD34(+)和CD34(+)CD38(-)UCB亚群。使用SF - EGFP/PG13逆转录病毒包装细胞组合产生的表达EGFP的细胞水平始终比MFG - EGFP/Am12组合高两倍。将10⁵个输入当量的转导CD34(+)或5×10³个输入当量的CD34(+)CD38(-)UCB细胞移植到非肥胖糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠中，中位植入率分别为8%和5%，这表明细胞的体内再增殖能力得以保留。此外，使用MFG - EGFP/Am12或SF - EGFP/PG13组合转导CD34(+)细胞后移植的小鼠，分别有2%和23%的人CD45(+)细胞表达EGFP，这表明NOD/SCID再增殖细胞被成功转导。在NOD/SCID小鼠产生的所有人类造血谱系中都发现了EGFP⁺细胞，包括具有体外克隆形成能力的人类祖细胞。在小鼠基质细胞系FBMD - 1上培养2至6周的人鹅卵石区域形成细胞（CAFC）试验中也检测到了表达EGFP的细胞。在转导过程中，第6周CAFC的绝对数量增加了5至10倍。该祖细胞亚群的转导效率与移植了MFG - EGFP/Am12或SF - EGFP/PG13转导的CD34(+)细胞的NOD/SCID小鼠骨髓中EGFP⁺人细胞的比例相似，分别为6%和27%。因此，该研究表明纯化的CD34(+)和高度纯化的CD34(+)CD38(-)UCB细胞可以在保留再增殖能力的情况下被有效转导。SF - EGFP/PG13载体/包装细胞组合在转导再增殖细胞方面比MFG - EGFP/Am12组合有效得多。
