Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute-NHLBI/National Institute of Diabetes and Digestive and Kidney Diseases-NIDDK, National Institutes of Health-NIH, Bethesda, MD 20892, USA.
Gene Ther. 2011 Nov;18(11):1078-86. doi: 10.1038/gt.2011.63. Epub 2011 May 5.
Cytokines are required for γ-retroviral transduction of human CD34+ cells. However, cytokines may reduce engraftment of CD34+ cells and may not be necessary for their lentiviral transduction. We sought to optimize transduction and engraftment of human CD34+ cells using lentiviral vectors. Single 24 h transduction of human CD34+ cells with human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors in media containing stem cell factor (SCF), FMS-like tyrosine kinase 3 (FLT3) ligand, thrombopoietin (each 100 ng ml⁻¹) and 10% fetal bovine serum was compared with various cytokine conditions during ex vivo culture and assayed using humanized xenograft mice for 6 months after transplantation. Serum-free media improved transduction efficiency of human CD34+ cells. Interleukin-3 (20 ng ml⁻¹) had little effect on transduction efficiency or engraftment. Threefold higher cytokine mixture (each 300 ng ml⁻¹) reduced engraftment of CD34+ cells. SCF alone (100 ng ml⁻¹) proved insufficient for maintaining engraftment ability and reduced transduction efficiency. Short-term prestimulation had little effect on transduction efficiency or engraftment, yet 24 h prestimulation showed higher transduction efficiency, higher gene expression levels and lower engraftment. In summary, 24 h prestimulation followed by single 24-h lentiviral transduction in serum-free media with SCF, FLT3 ligand and thrombopoietin yields high transduction efficiency to engrafting human CD34+ cells, and is applicable in human clinical gene therapy trials.
细胞因子对于 γ-逆转录病毒转导人 CD34+细胞是必需的。然而，细胞因子可能会降低 CD34+细胞的植入率，并且对于慢病毒转导可能不是必需的。我们试图通过慢病毒载体优化人 CD34+细胞的转导和植入。在含有干细胞因子 (SCF)、FMS 样酪氨酸激酶 3 (FLT3) 配体、血小板生成素（每种 100ng/ml）和 10%胎牛血清的培养基中，对人 CD34+细胞进行单次 24 小时的 HIV1 基于慢病毒载体的转导，与 ex vivo 培养期间的各种细胞因子条件进行比较，并在移植后 6 个月用人源化异种移植小鼠进行检测。无血清培养基可提高人 CD34+细胞的转导效率。白细胞介素-3（20ng/ml）对转导效率或植入率影响不大。三倍高的细胞因子混合物（每种 300ng/ml）降低了 CD34+细胞的植入率。单独的 SCF（100ng/ml）不足以维持植入能力并降低转导效率。短期预处理对转导效率或植入率影响不大，但 24 小时预处理显示出更高的转导效率、更高的基因表达水平和更低的植入率。总之，24 小时预处理后，在无血清培养基中进行单次 24 小时的慢病毒转导，用 SCF、FLT3 配体和血小板生成素进行转导，可获得高转导效率以植入人 CD34+细胞，并且适用于人类临床基因治疗试验。
