Improving Lentiviral Transduction of Human Adipose-Derived Mesenchymal Stem Cells

Abstract

Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34⁺ hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications.

Keywords: gene therapy; lentiboost; lentivirus; mesenchymal stem cell; transduction enhancer.

Figure 1.

Experimental scheme. Human adipose-derived MSCs were expanded in culture to passage 3 and then plated at a density of 0.2 × 10⁶ cells/well in six-well plates, using two replicates per donor. MSCs were transduced with LV-GFP (MOI = 3) overnight in the presence or absence of TE(s). Cells were washed the following day and fresh media were added. Analysis included FACS for GFP-expressing cells, ddPCR to determine the vector copy number per cell, trypan blue live cell staining, and CFU-fibroblast assay (created with Biorender.com). CFU, colony-forming unit; ddPCR, droplet digital PCR; FACS, flow cytometry; MOI, multiplicity of infection; MSCs, mesenchymal stem cells; TE, transduction enhancer.

Figure 2.

Effects of TEs and TE combinations on lentiviral transduction of MSCs. Dose–response relationships were observed for transduction efficiency with the use of LB alone and in combination with PS (A, B). Strong dose–response relationships were observed for MFI with the use of LB in combination with PS, but not to the same extent with LB alone (C, D). A strong dose–response relationship was observed for VCN with the use of LB alone and in combination with PS (E). LB, LentiBOOST; MFI, median fluorescence intensity; VCN, vector copy number.

Figure 3.

LB and protamine sulfate do not affect viability or colony-forming potential of MSCs. No significant differences were noted between LB concentration (either alone or in combination with PS) and cell proliferation, as determined by cell counts at 7 and 14 days post-transduction (A, B). No significant differences were noted between LB concentration (either alone or in combination with PS) and differentiation potential, as determined by CFU assay, although the use of polybrene was associated with significantly impaired colony-forming potential (C).