Non-viral gene delivery to human mesenchymal stem cells: a practical guide towards cell engineering

Abstract

In recent decades, human mesenchymal stem cells (hMSCs) have gained momentum in the field of cell therapy for treating cartilage and bone injuries. Despite the tri-lineage multipotency, proliferative properties, and potent immunomodulatory effects of hMSCs, their clinical potential is hindered by donor variations, limiting their use in medical settings. To address this challenge, gene delivery technologies have emerged as a promising approach to modulate the phenotype and commitment of hMSCs towards specific cell lineages, thereby enhancing osteochondral repair strategies. This review provides a comprehensive overview of current non-viral gene delivery approaches used to engineer MSCs, highlighting key factors such as the choice of nucleic acid or delivery vector, transfection strategies, and experimental parameters. Additionally, it outlines various protocols and methods for qualitative and quantitative evaluation of their therapeutic potential as a delivery system in osteochondral regenerative applications. In summary, this technical review offers a practical guide for optimizing non-viral systems in osteochondral regenerative approaches. hMSCs constitute a key target population for gene therapy techniques. Nevertheless, there is a long way to go for their translation into clinical treatments. In this review, we remind the most relevant transfection conditions to be optimized, such as the type of nucleic acid or delivery vector, the transfection strategy, and the experimental parameters to accurately evaluate a delivery system. This survey provides a practical guide to optimizing non-viral systems for osteochondral regenerative approaches.

Keywords: Mesenchymal stem cells; Non-viral gene delivery vectors; Osteochondral repair; Regenerative medicine; Tissue engineering.

Fig. 1

Schematic of the variables and factors affecting non-viral gene delivery to MSCs

Fig. 2

Schematic representation of the correlation between the dose of NAs per 10⁵ cells, ranging from low (from 0 to 1 µg/10⁵ cells), medium (from 1 to 5 µg/10⁵ cells), and high dose (from 5 to 20 µg/10⁵ cells), and the cell density, ranging from low (from 0 to 1 × 10⁴ cells/cm²), medium (from 1 to 5 × 10⁴ cells/cm²), and high cell density (from 5 × 10⁴ to 2.5 × 10⁵ cells/cm²). The trend line (R² = 0.79) represents the tendency of the data taken from articles (references [, , , , , , , , , , –, , , , , –203]), where the cell density (cells/cm²) and the dose (µg) are specified

Fig. 3

Transfection efficiency, expressed as luciferase activity normalized to the total protein content (RLU/mg of protein), following transfection with bPEI/pluc complexes prepared at N/P 10 on hMSCs isolated from two patients (i.e., patient #1 and patient #2, right and left panels, respectively) as a function of the pDNA dose (0.1, 0.5, and 1 µg/cm²) and the cell density (5 × 10³, 1.5 × 10⁴, 5 × 10⁴, and 1 × 10⁵ cells/cm²). Luciferase activity was evaluated 24, 48, and 72 h post-transfection. Results are expressed as mean ± SD (n = 3, * p < 0.05)

Fig. 4

Common colorimetric indicators used to evaluate the cytotoxicity of non-viral gene delivery carriers

Fig. 5

Schematic classification of the most popular methods and technologies used to gauge the transfection efficiency of non-viral gene delivery carriers

Fig. 6

Most used therapeutic genes and markers to induce and evaluate osteochondral differentiation. Abbreviations: BMP-2, -6, -7, -9: bone morphogenic protein 2, 6, 7, 9; siRNA: small interfering RNA; miRNA: micro-RNA; TGF-β1 and -β3: transforming growth factor-β1 and -β3; SOX9: sex-determining region Y-type high mobility group box 9; RUNX2: core binding factor alpha 2; OSX: osterix; ALP: alkaline phosphatase; COL-I, -II, -X: type-I, -II, and -X: collagen; OCN: osteocalcin; ACAN: aggrecan