Metabolic Activities and Chondrogenic Differentiation of Human Mesenchymal Stem Cells Following Recombinant Adeno-Associated Virus–Mediated Gene Transfer and Overexpression of Fibroblast Growth Factor 2

Abstract

The genetic manipulation of bone marrow-derived mesenchymal stem cells (MSCs) is an attractive approach to produce therapeutic platforms for settings that aim at restoring articular cartilage defects. Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated overexpression of human fibroblast growth factor 2 (hFGF-2), a mitogenic factor also known to influence MSC differentiation, upon the proliferative and chondrogenic activities of human MSCs (hMSCs) in a three-dimensional environment that supports chondrogenesis in vitro. Prolonged, significant FGF-2 synthesis was noted in rAAV-hFGF-2-transduced monolayer and aggregate cultures of hMSCs, leading to enhanced, dose-dependent cell proliferation compared with control treatments (rAAV-lacZ transduction and absence of vector administration). Chondrogenic differentiation (proteoglycans, type-II collagen, and SOX9 expression) was successfully achieved in all types of aggregates, without significant difference between conditions. Most remarkably, application of rAAV-hFGF-2 reduced the expression of type-I and type-X collagen, possibly due to increased levels of matrix metalloproteinase-13, a key matrix-degrading enzyme. FGF-2 overexpression also decreased mineralization and the expression of osteogenic markers such as alkaline phosphatase, with diminished levels of RUNX-2, a transcription factor for osteoblast-related genes. Altogether, the present findings show the ability of rAAV-mediated FGF-2 gene transfer to expand hMSCs with an advantageous differentiation potential for future, indirect therapeutic approaches that aim at treating articular cartilage defects in vivo.

Benefits of Recombinant Adeno-Associated Virus (rAAV)-Mediated Insulinlike Growth Factor I (IGF-I) Overexpression for the Long-Term Reconstruction of Human Osteoarthritic Cartilage by Modulation of the IGF-I Axis

“…pAd8 contains the AAV-2 replication and encapsidation functions (48). rAAV-lacZ carries the lacZ gene for β-galactosidase (β-gal) under the control of the cytomegalovirus immediate-early (CMV-IE) promoter (20,27,31,45,49,50). rAAV-red fluorescent protein (rAAV-RFP) carries a 776-bp Discosoma sp.…”

“…rAAV-red fluorescent protein (rAAV-RFP) carries a 776-bp Discosoma sp. RFP cDNA fragment (45,49,50). A hIGF-I cDNA (51) was generated by polymerase chain reaction (PCR) using the primers 5′-I-A (A 5 ctgcag[Pst I]G 17 CTTCA GAAGC A) and 3′-I-A (A 5 aagctt[Hind III]TGCGG TGGCA TGTCA CTCTT CAC) with pCMVhIGF-I (52) as a template for amplification.…”

“…rAAVs were packaged as conventional (not self-complementary) vectors in the 293 cell line, an adenovirus-transformed human embryonic kidney cell line, using adenovirus 5 and pAd8 for helper functions. The preparations were purified by dialysis and titered by real-time PCR (20,27,31,45,49,50), averaging 10 10 transgene copies/mL (ratio virus particles to functional vectors = 500/1) (49).…”

Gene therapy for chondral and osteochondral regeneration: is the future now?

“…SOXs regulate chondrocyte differentiation, improve ECM production and reduce the levels of hypertrophy and osteogenic/ adipogenic markers. ZNF145 regulates the differentiation of 3-lineages of MSCs, as observed in in vitro studies [54,59,65,77], while in vivo it increases osteochondral and full-thickness defects healing [54,59,75]. The co-infection of SOX9 and TGF-β potentiates chondrogenesis of MSCs in vitro, in comparison to their separate use [64].…”

“…On the other hand, sc-AAV produces higher levels of protein more quickly and, for this reason, it could become the preferred choice for gene therapy trials [60]. rAAV is used for the delivery of IGF-1 [61][62][63], TGF-β [2,64], FGF-2 [65], SOXs [59,64,66] and IL1ra genes [67]. scAVV is used for the delivery of IL1ra [68] (Table 1).…”