Optimal construction and delivery of dual-functioning lentiviral vectors for type I collagen-suppressed chondrogenesis in synovium-derived mesenchymal stem cells.
OBJECTIVE
This study aims to deliver both transforming growth factor β3 (TGF-β3) and shRNA targeting type I collagen (Col I) by optimal construction and application of various dual-functioning lentiviral vectors to induce Col I-suppressed chondrogenesis in synovium-derived mesenchymal stem cells (SMSCs).


METHODS
We constructed four lentiviral vectors (LV-1, LV-2, LV-3 and LV-4) with various arrangements of the two expression cassettes in different positions and orientations. Col I inhibition efficiency and chondrogenic markers were assessed with qPCR, ELISA and staining techniques. Among the four vectors, LV-1 has two distant and reversely oriented cassettes, LV-2 has two distant and same-oriented cassettes, LV-3 has two proximal and reversely oriented cassettes, and LV-4 has two proximal and same-oriented cassettes. Col I and chondrogenic markers, including type II collagen (Col II), aggrecan and glycosaminoglycan (GAG), were examined in SMSCs cultured in 3-D alginate hydrogel.


RESULTS
All of the four vectors showed distinct effects in Col I level as well as diverse inductive efficiencies in upregulation of the cartilaginous markers. Based on real-time PCR results, LV-1 was optimal towards Col I-suppressed chondrogenesis.


CONCLUSIONS
LV-1 vector is competent to promote Col I-suppressed chondrogenesis in SMSCs.
Division of Bioengineering School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, N1.3-B2-13, Singapore 637457, Republic of Singapore.
Feng
F
Zhang
Yongchang
Y
Yao
Ruijie
R
Zhou
Kai
K
Su
Fudiman
F
Citra
Dong-An
DA
Wang
D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000187:drug effects* / Q000235:genetics; D024042:Collagen Type I / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D006025:Glycosaminoglycans / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D013552:Swine; D013583:Synovial Membrane / Q000378:metabolism; D053782:Transforming Growth Factor beta3 / Q000008:administration & dosage / Q000235:genetics; D015854:Up-Regulation
D053674:Aggrecans; D024042:Collagen Type I; D024043:Collagen Type II; D006025:Glycosaminoglycans; D034741:RNA, Small Interfering; D053782:Transforming Growth Factor beta3