Chondrogenic Differentiation of Human Mesenchymal Stem Cells via SOX9 Delivery in Cationic Niosomes

Abstract

Gene transfer to mesenchymal stem cells constitutes a powerful approach to promote their differentiation into the appropriate cartilage phenotype. Although viral vectors represent gold standard vehicles, because of their high efficiency, their use is precluded by important concerns including an elevated immunogenicity and the possibility of insertional mutagenesis. Therefore, the development of new and efficient non-viral vectors is under active investigation. In the present study, we developed new non-viral carriers based on niosomes to promote the effective chondrogenesis of human MSCs. Two different niosome formulations were prepared by varying their composition on non-ionic surfactant, polysorbate 80 solely (P80), or combined with poloxamer 407 (P80PX). The best niosome formulation was proven to transfer a plasmid, encoding for the potent chondrogenic transcription factor SOX9 in hMSC aggregate cultures. Transfection of hMSC aggregates via nioplexes resulted in an increased chondrogenic differentiation with reduced hypertrophy. These results highlight the potential of niosome formulations for gene therapy approaches focused on cartilage repair.

Keywords: SOX9; chondrogenesis; human mesenchymal stem cells; nioplexes; niosomes.

Figure 1

(A) Particle size and zeta potential. (B) DNase protection ability and SDS-induced release visualized by agarose electrophoresis of polysorbate 80 (P80, mauve squares) and polysorbate 80 combined with poloxamer 407 (P80PX, dark purple circles); nioplexes formed at DOTMA/DNA mass ratios of 2.5/1, 5/1, 10/1, 15/1 and 20/1. 0: naked placZ; OC: open circular; SC: supercoiled * depicts p < 0.05 and ** p < 0.01 when comparing both nioplexe formulations at the same DOTMA/DNA ratio.

Figure 2

DNA complexation efficiency of P80 (mauve squares) and P80PX (dark purple circles) nioplexes formed at DOTMA/DNA mass ratios of 2.5/1, 5/1, 10/1, 15/1 and 20/1. ** depicts p < 0.01 and *** p < 0.001 when comparing both nioplexe formulations at the same DOTMA/DNA ratio.

Figure 3

(A) β-galactosidase activity (left), pGFP transfection efficiency (right) and (B) Cell viability of P80 (mauve) and P80PX (dark purple) nioplexes formed at DOTMA/DNA mass ratios of 2.5/1, 5/1, 10/1, 15/1 and 20/1. The commercial reagent Lipofectamine (LPF) was used as positive transfection control. * depicts p < 0.05, ** p < 0.01 and *** p < 0.001, when compared with denoted groups.

Figure 4

Immunohistochemical and histological analyses of hMSC aggregates cultured in chondrogenic medium (control; negative control) and transfected with psox9 via P80PX (P80PX/psox9) or LPF (LPF/psox9). Samples were kept in culture for 21 days and processed for (A) Immunodetection of SOX9, type-I, type-II and type-X collagen, and toluidine blue (all representative images; magnification 4X; scale bar 50 µm) or Hematoxylin/Eosin (H&E) stainings (all representative images; magnification 10x; scale bar 100 µm). (B) Histomorphometrical analyses (control: grey; P80PX/psox9: dark purple; LPF/psox9: white) realized as described in Materials and Methods (Section 2.13.2; Figure S5). * depicts p < 0.05, ** p < 0.01 and *** p < 0.001, when compared with denoted groups.

Figure 5

Real time RT-PCR analysis of hMSC aggregates cultured in chondrogenic medium (control; negative control: grey) and transfected with psox9 via P80PX (P80PX/psox9: dark purple) or LPF (LPF/psox9: white) complexes after 3 weeks in vitro. The genes analysed included aggrecan (ACAN), the transcription factor SOX9, type-II collagen (COL2A1), type-X collagen (COL10A1) and type I collagen (COL1A1) with GAPDH serving as a housekeeping gene and internal control (primers are listed in Section 2.13.3). Ct values were obtained for each target and for GAPDH as a control for normalization, and fold inductions (relative to control aggregates) were measured by using the 2^−ΔΔCt method. * depicts p < 0.05 when compared P80PX/psox9 and P80PX/LPF groups with control group.

Figure 6

Biochemical analyses of hMSC aggregates cultured in chondrogenic medium (control; negative control: grey) and transfected with psox9 via P80PX (P80PX/psox9: dark purple) or LPF (LPF/psox9: white) complexes after 3 weeks in vitro. (A) Proteoglycan contents standardized to the protein contents and (B) Proteoglycan contents standardized to the DNA contents. * depicts p < 0.05 and ** p < 0.01 when compared P80PX/psox9 and P80PX/LPF groups with control.