Kojabad Amir Asri, Ghaleh Hadi Esmaeili Gouvarchin, Shahriary Alireza, Farzanehpour Mahdieh
Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Chemical Injuries Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Indian J Hematol Blood Transfus. 2023 Apr;39(2):173-182. doi: 10.1007/s12288-022-01576-4. Epub 2022 Nov 1.
HSC transplantation (HSCT) has emerged as a promising treatment option for hematological and immunological disorders. Unfortunately, many viral vectors are inefficient at transduction, limiting the number of cells available for gene therapy in cord blood HSC transplantation. Combining ex vivo expansion and genetic manipulation of cord blood cells is a potential gene therapy approach. We present a 3D co-culture method using a demineralized bone matrix scaffold to optimize lentiviral vector-mediated gene transduction. pLenti-III-miR-GFP-has-miR-124 was transduced into cord blood HSCs. Transduced CD34 + cells co-cultured on the stromal layer for 72 h under cytokine-free conditions. We performed flow cytometry, colony assays, real-time polymerase chain reaction, and SEM morphological analysis. Seventy-two hours after transduction, when pLentiIII-miR-GFP-has-miR-124 and control vector-transduced expanded cord blood HSCs were compared to non-transduced expanded cord blood HSCs, the findings revealed 15 ± 3.04 and 55 ± 3.05-fold increases in miR-124 mRNA expression, respectively. Compared to a control culture on the same day, the expansion of CD34+, CD38-HSCs in 3D culture increased 544 ± 31.09 fold. This result demonstrated that the 3D-culture system could emerge as a novel approach to overcoming the current limitations of cord blood HSC transduction. In the future, this research could be applied in a therapeutic setting.
造血干细胞移植（HSCT）已成为治疗血液学和免疫学疾病的一种有前景的治疗选择。不幸的是，许多病毒载体的转导效率低下，限制了脐带血造血干细胞移植中可用于基因治疗的细胞数量。将脐带血细胞的体外扩增与基因操作相结合是一种潜在的基因治疗方法。我们提出了一种使用脱矿骨基质支架的三维共培养方法，以优化慢病毒载体介导的基因转导。将pLenti-III-miR-GFP-has-miR-124转导到脐带血造血干细胞中。转导后的CD34 +细胞在无细胞因子条件下在基质层上共培养72小时。我们进行了流式细胞术、集落测定、实时聚合酶链反应和扫描电子显微镜形态分析。转导72小时后，将pLentiIII-miR-GFP-has-miR-124和对照载体转导的扩增脐带血造血干细胞与未转导的扩增脐带血造血干细胞进行比较，结果显示miR-124 mRNA表达分别增加了15±3.04倍和55±3.05倍。与同一天的对照培养相比，三维培养中CD34 +、CD38-HSCs的扩增增加了544±31.09倍。这一结果表明，三维培养系统可能成为克服当前脐带血造血干细胞转导局限性的一种新方法。未来，这项研究可应用于治疗环境。
