2018 Volume No 36 – pages 156-170
Title: Lentiviral-based reporter constructs for profiling chondrogenic activity in primary equine cell populations |
Authors: A Martin-Pena, RM Porter, G Plumton, TM McCarrel, AJ Morton, MV Guijarro, SC Ghivizzani, B Sharma, GD Palmer |
Address: Department of Orthopaedics and Rehabilitation, University of Florida, 1600 SW Archer Rd, MSB, M2-235, Gainesville, FL, 32610, USA. |
E-mail: glynpal at gmail.com
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Abstract: Successful clinical translation of mesenchymal stem cell (MSC)-based therapies for cartilage repair will likely require the implementation of standardised protocols and broadly applicable tools to facilitate the comparisons among cell types and chondroinduction methods. The present study investigated the utility of recombinant lentiviral reporter vectors as reliable tools for comparing chondrogenic potential among primary cell populations and distinguishing cellular-level variations of chondrogenic activity in widely used three-dimensional (3D) culture systems. Primary equine MSCs and chondrocytes were transduced with vectors containing combinations of fluorescent and luciferase reporter genes under constitutive cytomeglavirus (CMV) or chondrocyte-lineage (Col2) promoters. Reporter activity was measured by fluorescence imaging and luciferase assay. In 3D cultures of MSC aggregates and polyethylene glycol-hyaluronic acid (PEG-HA) hydrogels, transforming growth factor beta 3 (TGF-β3)-mediated chondroinduction increased Col2 reporter activity, demonstrating close correlation with histology and mRNA expression levels of COL2A1 and SOX9. Comparison of chondrogenic activities among MSC populations using a secretable luciferase reporter revealed enhanced chondrogenesis in bone-marrow-derived MSCs relative to MSC populations from synovium and adipose tissues. A dual fluorescence reporter – enabling discrimination of highly chondrogenic (Col2-GFP) cells within an MSC population (CMV-tdTomato) – revealed marked heterogeneity in differentiating aggregate cultures and identified chondrogenic cells in chondrocyte-seeded PEG-HA hydrogels after 6 weeks in a subcutaneous implant model – indicating stable, long-term reporter expression in vivo. These results suggested that lentiviral reporter vectors may be used to address fundamental questions regarding chondrogenic activity in chondroprogenitor cell populations and accelerate clinical translation of cell-based cartilage repair strategies. |
Key Words: Mesenchymal stem cell, chondrogenesis/cartilage repair, lentiviral reporter construct, Col2 promoter, fluorescence imaging, live-cell monitoring, tissue engineering/regenerative medicine, 3D culture. |
Publication date: October 12th 2018 |
Article download: Pages 156-170 (PDF file) |
