Polybrene Inhibits Human Mesenchymal Stem Cell Proliferation during Lentiviral Transduction

Abstract

Human mesenchymal stem cells (hMSCs) can be engineered to express specific genes, either for their use in cell-based therapies or to track them in vivo over long periods of time. To obtain long-term expression of these genes, a lentivirus- or retrovirus-mediated cell transduction is often used. However, given that the efficiency with these viruses is typically low in primary cells, additives such as polybrene are always used for efficient viral transduction. Unfortunately, as presented here, exposure to polybrene alone at commonly used concentratons (1–8 µg/mL) negatively impacts hMSC proliferation in a dose-dependent manner as measured by CyQUANT, EdU incorporation, and cell cycle analysis. This inhibition of proliferation was observable in culture even 3 weeks after exposure. Culturing the cells in the presence of FGF-2, a potent mitogen, did not abrogate this negative effect of polybrene. In fact, the normally sharp increase in hMSC proliferation that occurs during the first days of exposure to FGF-2 was absent at 4 µg/mL or higher concentrations of polybrene. Similarly, the effect of stimulating cell proliferation under simulated hypoxic conditions was also decreased when cells were exposed to polybrene, though overall proliferation rates were higher. The negative influence of polybrene was, however, reduced when the cells were exposed to polybrene for a shorter period of time (6 hr vs 24 hr). Thus, careful evaluation should be done when using polybrene to aid in lentiviral transduction of human MSCs or other primary cells, especially when cell number is critical.

Lentiviral Transduction of CD34+ Cells Induces Genome-Wide Epigenetic Modifications

“…Groups 1 and 3 were very comparable since only 24 genes were found to differ by 20% of methylation level when polybrene was added in addition to cytokines and compared to the unstimulated control group. This is in line with the recent observation showing that polybrene used during lentiviral transduction inhibits the cell cycle [25]. By contrast, LV transduction significantly stimulated methylation changes in comparison to any other group of cells, with a marked tendency for augmenting CpG methylation (Fig.…”

Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

“…As polycations improve adenovirusmediated gene transfer, less virus would have to be used, which would improve the therapeutic index by reducing unwanted responses associated with high doses of virus. On the other hand, multiple reports indicate that polycations could exhibit nonspecific cytotoxicity in vivo as well as in vitro (73,74), with some studies demonstrating unacceptable cytotoxicity for DEAE-dextran (74,75) and Polybrene (76,77), at least under some experimental conditions. Therefore, while our study conceptually demonstrates the feasibility of the polycation-mediated improvement of VSV-based OV therapy in vitro, future studies are needed to compare Polybrene and DEAE-dextran to other polycations that could be used safely and effectively in vivo in combination with VSV and ruxolitinib.…”

A Nontoxic Transduction Enhancer Enables Highly Efficient Lentiviral Transduction of Primary Murine T Cells and Hematopoietic Stem Cells

“…However most of these enhancers have toxic effects that limit their use. For example, polybrene is a widely used polycationic enhancer for lentiviral transduction but disrupts the transmembrane potential in some sensitive cells 19 . PS is less toxic than polybrene but does not enhance the lentiviral vector-based transduction of primary murine T cells 13 .…”