Hanley M E, Nolta J A, Parkman R, Kohn D B
Children's Hospital Los Angeles, Department of Pediatrics, University of Southern California School of Medicine 90027, USA.
Blood Cells. 1994;20(2-3):539-43; discussion 544-6.
Human umbilical cord blood (UCB) can be a source of hematopoietic stem cells for gene therapy, as an alternative to allogeneic bone marrow transplantation, for the treatment of a number of genetic diseases. To determine conditions that yield maximal gene transfer into UCB progenitor cells, we examined a number of variables. We used cell-free retroviral vector supernatants that convey neomycin (G418) resistance and measured the percentage of G418-resistant progenitor-derived colonies. Adding retroviral supernatant to the UCB cells in basal medium once a day for 3 days produced a threefold increase of G418-resistant colonies (9.8%) compared to a single exposure to supernatant (3.1%). To establish whether recombinant human growth factors are beneficial during transduction, the presence of interleukin-3 (IL-3), IL-6, and mast cell growth factor (MGF, a c-kit ligand) were compared in different combinations. Inclusion of the three factors together caused a threefold increase of gene transfer (30.4%) compared to transduction in basal medium. When the UCB cells were precultured in medium containing IL-3, IL-6, and MGF for 3 days before addition of the retroviral supernatant on days 4, 5, and 6, the average extent of gene transfer was 21.8%, compared with an average of 34.4% when UCB cells were transduced on days 1, 2, and 3. The presence of marrow stroma during the transduction of the UCB cells did not further increase gene transfer. We conclude that UCB progenitor cells can be efficiently transduced with the use of recombinant human growth factors IL-3, IL-6, and MGF and may be a suitable source for gene therapy.
人脐带血（UCB）可作为造血干细胞的来源用于基因治疗，作为异基因骨髓移植的替代方法，用于治疗多种遗传疾病。为了确定能使基因最大程度转移至UCB祖细胞的条件，我们研究了多个变量。我们使用携带新霉素（G418）抗性的无细胞逆转录病毒载体上清液，并测量G418抗性祖细胞衍生集落的百分比。与单次暴露于上清液（3.1%）相比，在基础培养基中每天向UCB细胞添加一次逆转录病毒上清液，持续3天，可使G418抗性集落增加三倍（9.8%）。为了确定重组人生长因子在转导过程中是否有益，我们比较了不同组合下白细胞介素-3（IL-3）、IL-6和肥大细胞生长因子（MGF，一种c-kit配体）的存在情况。与在基础培养基中转导相比，同时包含这三种因子可使基因转移增加三倍（30.4%）。当在第4、5和6天添加逆转录病毒上清液之前，将UCB细胞在含有IL-3、IL-6和MGF的培养基中预培养3天时，基因转移的平均程度为21.8%，而当UCB细胞在第1、2和3天进行转导时，平均基因转移程度为34.4%。在UCB细胞转导过程中存在骨髓基质并不会进一步增加基因转移。我们得出结论，使用重组人生长因子IL-3、IL-6和MGF可有效转导UCB祖细胞，并且其可能是基因治疗的合适来源。
