Lentiviral-mediated multiple gene transfer to chondrocytes promotes chondrocyte differentiation and bone formation in rabbit bone marrow-derived mesenchymal stem cells

Abstract

Abstract. The aim of the present study was to provide a theoretical and experimental foundation on the differentiation of stem cells through the induction of multiple genes. The lentiviral vector carrying TGF-β1 and IL-10 genes was transfected to bone marrow-derived mesenchymal stem cells (BMSCs) which differentiated into chondrogenesis. Healthy New Zealand white rabbits, 2-3 months of age were used in the present study. A 6-8 ml of bone marrow was isolated from the iliac and tibial shaft of each rabbit. The BMSCs suspension was aspired following centrifugation of the bone marrow by percoll separating medium. The BMSCs were primarily cultured and subcultured in vitro, then divided into four groups according to the difference of lentivirus vectors: group A, receiving transforming growth factor β1 (TGF-β1); group B, receiving TGF-β1 and Interleukin-10 (IL-10); group C, empty vector transfection; and group D, receiving no cell growth factor. Fluorescence expression was detected 12 h after transfecting the lentiviral vector carrying the TGF-β1 and IL-10 gene to BMSCs. The transfection efficiency was approximately 70% with a MOI=100 after 96 h. Expression of SOX-9 aggrecan and Type Ⅱ collagen in groups A-E on day 7 and 14 was detected by RT-PCR and western blot analysis. The expression level of three genes expressed in groups A and C were higher compared to the expression in groups B, D and E. The expression level of the three genes expressed in group B was higher compared to the expression in group D. The expression level of three genes expressed in group A and C showed no statistical difference. Cytokines therefore play an important role in cell proliferation and chondrogenic differentiation. TGF-β1 has a synergistic effect in the differentiation. In addition, IL-10 may have a protective role in the restoration of cartilaginous tissue.

Microfibrillar-associated protein 5 regulates osteogenic differentiation by modulating the Wnt/β-catenin and AMPK signaling pathways

“…Dysfunctional osteogenic differentiation of BMSCs is associated with osteoporosis initiation and development, accompanied by a significant decline in osteogenic differentiation and increased adipogenic differentiation (Chen et al 2016 ; Li et al 2018 ). BMSCs can develop into cells of different types; the mechanism that determines the direction of differentiation remains unclear (Guo et al 2020 ; Liu et al 2015 ). A better understanding of BMSC osteogenesis is needed.…”

Co-overexpression of TGF-β and SOX9 via rAAV gene transfer modulates the metabolic and chondrogenic activities of human bone marrow-derived mesenchymal stem cells

“…Various gene sequences have been tested thus far as potential chondroregenerative candidates, including the cartilage oligomeric matrix protein (COMP), bone morphogenetic proteins (BMPs), transforming growth factor beta (TGF-β), insulin-like growth factor I (IGF-I), basic fibroblast growth factor (FGF-2), the SOX family of transcription factors, zinc finger protein 145 (ZNF145), Indian hedgehog (Ihh), and Wnt11 [ 13 – 24 ]. Yet reports from diverse groups showed that multiple therapeutic gene transfer might be more valuable to stimulate the repair activities in these cells relative to independent treatments [ 13 , 17 , 21 , 25 – 27 ], a finding also described by us in human articular chondrocytes [ 28 ].…”

Effects of combined rAAV-mediated TGF-β and sox9 gene transfer and overexpression on the metabolic and chondrogenic activities in human bone marrow aspirates

“…In this regard, genetic modification of bone marrow concentrates by transfer of sequences coding for chondrogenic and/or chondroreparative factors prior to implantation in sites of cartilage injury might be a powerful tool to overcome such critical issues (Frisch et al 2015 ; Johnstone et al 2013 ). Various therapeutic sequences have been tested to achieve this goal, including the cartilage oligomeric matrix protein (COMP), bone morphogenetic proteins (BMPs), transforming growth factor beta (TGF-β), insulin-like growth factor I (IGF-I), basic fibroblast growth factor (FGF-2), the sex-determining region Y-type high-mobility group box (SOX) family of transcription factors, zinc finger protein 145 (ZNF145), Indian hedgehog (Ihh), Wnt11, and small interfering RNA (siRNA) against p53 and runt-related transcription factor 2 (Runx2) (Babister et al 2008 ; Cucchiarini et al 2009 ; Cucchiarini et al 2011 ; Frisch et al 2014 ; Haleem-Smith et al 2012 ; Huang et al 2010 ; Ikeda et al 2004 ; Jeon et al 2012 ; Kim and Im 2011 ; Lee et al 2011 ; Liu et al 2011 ; Liu et al 2013 ; Liu et al 2014 ; Liu et al 2015 ; Neumann et al 2013 ; Pagnotto et al 2007 ; Steinert et al 2009 ; Steinert et al 2012 ; Tao et al 2016 ; Venkatesan et al 2012 ). Interestingly, previous reports from diverse groups, among which ours, demonstrated that multiple therapeutic gene transfer might be necessary to promote optimal reparative activities in various cells of the musculoskeletal system (articular chondrocytes, MSCs) (Cucchiarini et al 2009 ; Ikeda et al 2004 ; Kim and Im 2011 ; Liu et al 2013 ; Liu et al 2015 ; Steinert et al 2009 ; Tao et al 2016 ).…”