Abstract
Background Human mesenchymal stromal/stem cells (hMSCs) hold great therapeutic potential due to their immunomodulatory and tissue regenerative properties. Enhancement of biological features of hMSCs by transfection has become a focus of investigation for cell- and gene-based therapies. However, many of the current transient transfection methods result in either low transfection efficiency or high cytotoxicity. Methods In order to find a transfection method that would address the current issues of low transfection efficiency and high cytotoxicity, 6 commercially available cationic lipid and polymer reagents were tested on human bone marrow-derived MSCs (hBM-MSCs) using GFP as a reporter gene. One transfection method using TransIT-2020 was selected and tested with an emphasis on cell quality (viability, identity, and yield), as well as efficacy with a human placental growth factor (PlGF) plasmid. Results TransIT-2020 yielded the highest fluorescence signal per cell out of the methods that did not decrease cell recovery. Transfecting GFP to 5 hBM-MSC donors using TransIT-2020 yielded 24–36% GFP-expressing cells with a viability of 85–96%. hBM-MSC identity was unaffected as CD90, CD105, and CD73 markers were retained (>95%+) after transfection. When this method was applied to PlGF expression, there was up to a 220-fold increase in secretion. Both growth and secretion of PlGF in overexpressing hBM-MSC were sustained over 7 days, confirming the sustainability and applicability of the TransIT-2020 transfection system. Discussion We report a simple and efficient method for transient transfection that has not been reported for hBM-MSCs, encompassing high levels of plasmid expression without significant changes to fundamental hBM-MSC characteristics.
Highlights
Multipotent human mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of stromal cells capable of supporting hematopoiesis, mediating tissue repair and immunomodulation [1]
Since the TransIT-2020 transfection system showed the highest GFP expression without decreasing cell recovery, it was selected for further validation of its performance
HBM-MSCs continued to grow up to 7 days, achieving over 400% growth with empty plasmid transfection and over 600% growth with placental growth factor (PlGF) transfection (Figure 3(i)). These results show that a functional plasmid, PlGF can be overexpressed in hBM-MSCs without lowering viability and growth
Summary
Multipotent human mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of stromal cells capable of supporting hematopoiesis, mediating tissue repair and immunomodulation [1] Based on these essential biological functions, their proliferative capacity and their immunoprivileged trait, MSCs have become a major focus of investigation for many potential therapeutic applications. In order to find a transfection method that would address the current issues of low transfection efficiency and high cytotoxicity, 6 commercially available cationic lipid and polymer reagents were tested on human bone marrow-derived MSCs (hBM-MSCs) using GFP as a reporter gene. HBM-MSC identity was unaffected as CD90, CD105, and CD73 markers were retained (>95%+) after transfection When this method was applied to PlGF expression, there was up to a 220-fold increase in secretion. We report a simple and efficient method for transient transfection that has not been reported for hBM-MSCs, encompassing high levels of plasmid expression without significant changes to fundamental hBM-MSC characteristics
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