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Three-dimensional multipotent progenitor cell aggregates for expansion, osteogenic differentiation and ‘in vivo’ tracing with AAV vector serotype 6

Gene Therapy volume 20, pages 158–168 (2013)Cite this article

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Abstract

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived stem cells with a high growth rate suitable for therapeutical applications as three-dimensional (3D) aggregates. Combined applications of osteogenically differentiated MAPC (OD-MAPC) aggregates and adeno-associated viral vectors (AAV) in bone bioengineering are still deferred until information with regard to expansion technologies, osteogenic potential, and AAV cytotoxicity and transduction efficiency is better understood. In this study, we tested whether self-complementary AAV (scAAV) can potentially be used as a gene delivery system in an OD-MAPC-based ‘in vivo’ bone formation model in the craniofacial region. Both expansion of rat MAPC (rMAPC) and osteogenic differentiation with dexamethasone were also tested in 3D aggregate culture systems ‘in vitro’ and ‘vivo’. rMAPCs grew as undifferentiated aggregates for 4 days, with a population doubling time of 37 h. After expansion, constant levels of Oct4 transcripts, and Oct4 and CD31 surface markers were observed, which constitute a hallmark of undifferentiated stage of rMAPCs. Dexamethasone effectively mediated rMAPC osteogenic differentiation by inducing the formation of a mineralized collagen type I network, and facilitated the activation of the wnt/β-catenin, a crucial pathway in skeletal development. To investigate the genetic modification of rMAPCs grown as 3D aggregates before implantation, scAAV serotypes 2, 3 and 6 were evaluated. scAAV6 packaged with the enhanced green fluorescent protein expression cassette efficiently mediated long-term transduction (10 days) ‘in vitro’ and ‘vivo’. The reporter transduction event allowed the tracing of OD-rMAPC (induced by dexamethasone) aggregates following OD-rMAPC transfer into a macro-porous hydroxyapatite scaffold implanted in a rat calvaria model. Furthermore, the scAAV6-transduced OD-rMAPCs generated a bone-like matrix with a collagenous matrix rich in bone-specific proteins (osteocalcin and osteopontin) in the scaffold macro-pores 10 days post-implantation. Newly formed bone was also observed in the interface between native bone and scaffold. The collective work supports future bone tissue engineering applications of 3D MAPC cultures for expansion, bone formation and the ability to alter genetically these cells using scAAV vectors.

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Acknowledgements

We thank Sandra Horton at North Carolina State University for processing ‘in vivo’ tissue specimens for immunohistochemistry and Victoria Madden at UNC Microscopy Services Laboratory for processing in vitro cell matrices for electron microscopy. Also, we would like to acknowledge Dr Ganokon Urkasemsin for her contribution towards the statistical analysis of this project. João Ferreira was supported by the doctoral fellowship of FCT-Portuguese Foundation for Science and Technology (SFRH/BD/36841/2007). This work was supported in part by grants from the: NIH/NIDCR (K08DE018695), NC Biotech Center, American Association for Orthodontist Foundation awarded to C-CK; Northwest Genome Engineering Consortium pilot awarded to MLH; and Wellstone (5U54AR056953) and NIH (5R01AI072176) awarded to RJS.

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Authors and Affiliations

  1. School of Dentistry, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA

    J R Ferreira & C-C Ko

  2. Matrix and Morphogenesis Section, National Institute of Dental and Craniofacial Research, Bethesda, MD, USA

    J R Ferreira

  3. Gene Therapy Center, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA

    M L Hirsch & R J Samulski

  4. Faculty of Stomatology, Capital Medical University, Beijing, China

    L Zhang

  5. Interdepartmental Stem Cell Institute, Catholic University of Leuven, Leuven, Belgium

    Y Park

  6. Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN, USA

    W-S Hu

  7. Materials Sciences and Engineering, North Carolina State University, Raleigh, NC, USA

    C-C Ko

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  1. J R Ferreira
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Correspondence to J R Ferreira.

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Ferreira, J., Hirsch, M., Zhang, L. et al. Three-dimensional multipotent progenitor cell aggregates for expansion, osteogenic differentiation and ‘in vivo’ tracing with AAV vector serotype 6. Gene Ther 20, 158–168 (2013). https://doi.org/10.1038/gt.2012.16

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