Human Hematopoietic Stem Cells Co-cultured in 3D with Stromal Support to Optimize Lentiviral Vector-mediated Gene Transduction.

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.