Effects of rAAV-mediated overexpression of sox9 and TGF-### via carbon dots on the chondrogenesis of human bone marrow-derived mesenchymal stem cells

Objectives Gene therapy using rAAV vectors is a promising tool to treat cartilage lesions but the presence of neutralizing antibodies against AAV capsid elements in a majority of patients remains a limitation to the use of these vectors in the clinics. Carbon dots (CDs), a new class of nanocarriers capable of transferring plasmid DNA in several cell lines, may provide effective platforms to deliver rAAV as a means to overcome such a limitation. Here, we examined the ability of CDs to deliver rAAV sox9 and rAAV TFG- β vectors to human bone marrow-derived mesenchymal stem cells (MSCs), a potential source of regenerative cells, as a means to enhance the chondrogenic potential of such cells.

Methods rAAV-lacZ carries the E. coli β -galactosidase (lacZ) reporter gene, rAAV-FLAG-hsox9 a human FLAG-tagged sox9 sequence, and rAAV-hTGF- β a 1.2-kb human transforming growth factor-beta 1 sequence, all controlled by the CMV-IE promoter/enhancer. MSCs were prepared as previously described and seeded at passage 1 in 48-well plates (5,000 cells/well), maintained in DMEM, 10% FBS, 100 U/ml penicillin G, 100 μ l/ml streptomycin, and incubated at 37ºC during 12 h before addition of the CDs. The CD-MC148 compound was generated through pyrolysis of citric acid. CDs were mixed with rAAV-FLAG-hsox9, rAAV-hTGF- β, or rAAV-lacZ in equal parts and applied to the cultures for up to 21 days. Control cultures were treated with CDs lacking rAAV vectors. Transgene expression was monitored by immunohistochemistry (SOX9, TGF- β) and by specific ELISA (TGF- β; R&D Systems). Immunohistochemical analyses were also performed (type-II/-I/-X collagen) . Cell viability was monitored with the Cell Proliferation reagent WST-1 (Roche Applied Science). Alcian blue staining was performed and quantitatively estimated by solubilization in guanidine hydrochloride 6 M. Each condition was performed in duplicate in three independent experiments. A t-test was employed with p ≤ 0.05 considered statistically significant.

Results and Conclusion Successful therapeutic delivery of rAAV was achieved in hMSCs via CD-MC148 as noted by the effective and specific expression of sox9 and of TGF- β in the cultures over time (21 days) relative to the control treatments. Overexpression of sox9 and TGF- β in hMSCs via CD-MC148 led to significant increases in the levels of cell proliferation (21 days) compared with the control conditions. In addition, delivery of rAAV sox9 or rAAV TGF- β from the CD-MC148 compound enhanced the chondrogenic differentiation processes by stronger type-II collagen deposition over time (21 days) versus control conditions and by higher amounts of matrix proteoglycans as noted by Alcian blue staining in the sox9- and TGF- β -treated versus control cultures over time (21 days). Finally, application of the sox9 and TGF- β constructs via CD-MC148 advantageoulsy delayed the hypertrophic differentiation. These results show delivery of rAAV via CD-MC148 effective tools for the furture treatment of cartilage lesions.

Stichwörter Carbon dots, rAAV, hMSCs

Publication History

Article published online:
15 October 2020