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Optimal Construction and Delivery of Dual-Functioning Lentiviral Vectors for Type I Collagen-Suppressed Chondrogenesis in Synovium-Derived Mesenchymal Stem Cells

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ABSTRACT

Purpose

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.

Conclusion

LV-1 vector is competent to promote Col I-suppressed chondrogenesis in SMSCs.

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Abbreviations

Col I:

type I collagen

Col II:

type II collagen

LV-dual:

recombinant lentiviral vector that expresses both shRNA targeting type I collagen and TGF- β3

LV-T:

recombinant lentiviral vector that specifically expresses TGF-β3

shRNA:

short hairpin RNA

SMSC:

synovium-derived mesenchymal stem cell

TGF-β3:

transforming growth factor-β3

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ACKNOWLEDGEMENTS

This research was financially supported by AcRF Tier 1 Grant RG64/08, Ministry of Education (MoE), and NMRC/EDG/1001/2010, Singapore.

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Authors and Affiliations

  1. 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 Zhang, Yongchang Yao, Ruijie Zhou, Kai Su, Fudiman Citra & Dong-An Wang

  2. School of Materials Science and Engineering, South China University of Technology, Guangzhou, 510641, China

    Yongchang Yao

  3. Key Laboratory of Specially Functional Materials and Advanced Manufacturing Technology, Ministry of Education, South China University of Technology, Guangzhou, 510641, China

    Yongchang Yao & Dong-An Wang

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  1. Feng Zhang
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  2. Yongchang Yao
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Correspondence to Dong-An Wang.

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Zhang, F., Yao, Y., Zhou, R. et al. Optimal Construction and Delivery of Dual-Functioning Lentiviral Vectors for Type I Collagen-Suppressed Chondrogenesis in Synovium-Derived Mesenchymal Stem Cells. Pharm Res 28, 1338–1348 (2011). https://doi.org/10.1007/s11095-010-0305-5

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  • DOI: https://doi.org/10.1007/s11095-010-0305-5

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