Reduced hypertrophy in vitro after chondrogenic differentiation of adult human mesenchymal stem cells following adenoviral SOX9 gene delivery

Abstract

Background: Mesenchymal stem cell (MSC) based-treatments of cartilage injury are promising but impaired by high levels of hypertrophy after chondrogenic induction with several bone morphogenetic protein superfamily members (BMPs). As an alternative, this study investigates the chondrogenic induction of MSCs via adenoviral genedelivery of the transcription factor SOX9 alone or in combination with other inducers, and comparatively explores the levels of hypertrophy and end stage differentiation in a pellet culture system in vitro. Methods: First generation adenoviral vectors encoding SOX9, TGFB1 or IGF1 were used alone or in combination to transduce human bone marrow-derived MSCs at 5 × 10 2 infectious particles/cell. Thereafter cells were placed in aggregates and maintained for three weeks in chondrogenic medium. Transgene expression was determined at the protein level (ELISA/Western blot), and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy. Results: SOX9 cDNA was superior to that encoding TGFB1, the typical gold standard, as an inducer of chondrogenesis in primary MSCs as evidenced by improved lacuna formation, proteoglycan and collagen type II staining, increased levels of GAG synthesis, and expression of mRNAs associated with chondrogenesis. Moreover, SOX9 modified aggregates showed a markedly lower tendency to progress towards hypertrophy, as judged by expression of the hypertrophy markers alkaline phosphatase, and collagen type X at the mRNA and protein levels.Conclusion: Adenoviral SOX9 gene transfer induces chondrogenic differentiation of human primary MSCs in pellet culture more effectively than TGFB1 gene transfer with lower levels of chondrocyte hypertrophy after 3 weeks of in vitro culture. Such technology might enable the formation of more stable hyaline cartilage repair tissues in vivo.

Different types of cartilage neotissue fabricated from collagen hydrogels and mesenchymal stromal cells via SOX9, TGFB1 or BMP2 gene transfer

“…SOX9 led to chondrogenic differentiation but also different levels of chondrogenic hypertrophy in MSCs. This proved that our earlier findings regarding the different effects of gene transfer of TGFB1 and SOX9 to MSCs could not only be observed when using pellet cultures but also when culturing MSCs in type I collagen hydrogels which are already in clinical use for CTE [ 30 ]. This technology may be used to overcome the search for effective delivery methods of cells and chondrogenic growth factors in vivo while offering possibilities to regenerate specific zones of native cartilage upon damage.…”

“…We and other researchers previously examined the use of gene therapy as a possibility to enable an autonomous, long-lasting production of chondrogenic growth factors by MSCs [ 14 , 16 , 30 ]. The single or combined adenoviral gene transfer of TGFB1 and BMP2 promoted chondrogenic differentiation in MSCs when using pellet culture [ 15 ].…”

“…The single or combined adenoviral gene transfer of TGFB1 and BMP2 promoted chondrogenic differentiation in MSCs when using pellet culture [ 15 ]. Later we and others found that the gene transfer of SOX9 not only led to chondrogenic differentiation in MSCs but also reduced chondrogenic hypertrophy in comparison to delivery of TGFB1 or BMP2 [ 23 , 30 ].…”

“…Recently, the transcription factor sex-determining region Y-type high-mobility-group-box (SOX) 9 (encoded by SOX9 ) has been employed for chondrogenic induction of MSCs via gene delivery using adeno-associated virus (AAV) [ 16 ] or adenoviral vectors [ 30 ], which elicited significantly reduced levels of hypertrophy in pellet cultures in vitro . Because SOX9 is an intracellular protein, delivery by means other than gene transfer is difficult.…”

MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes

“…MicroRNAs have emerged as potent therapeutic targets in the treatment of multiple diseases and injuries. To effectively improve intercellular miRNA levels and enhance their therapeutic efficacy, multiple exogenous microRNA delivery systems have been developed, including lentiviral and adenoviral vectors [ 71 ], poly (lactide-co-glycolide) [ 72 ], and liposomes [ 73 ]. In this study, we selected lentiviruses as our vector for delivering exogenous miR-27b.…”