Lentiviral-based reporter constructs for profiling chondrogenic activity in primary equine cell populations

Abstract

Successful clinical translation of mesenchymal stem cell (MSC) based therapies for cartilage repair will likely require the implementation of standardized protocols and broadly applicable tools that facilitate 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 of 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.

Specific, Sensitive, and Stable Reporting of Human Mesenchymal Stromal Cell Chondrogenesis

“…For example, similar COL2-GF activity has been observed for equine MSCs differentiated in pellet culture. 57 Because a very similar sequence was used successfully in murine ATDC5 cells, 26 it is likely that the 4eCOL2A1 promoter can also be used with cells of rodent origin; however, to date this has not been explicitly tested.…”

“…To overcome these limitations, the 407 kb 4eCO-L2A1 promoter can be inserted into alternative constructs that incorporate selectable markers. 57 A related limitation to this approach is that it is likely more adaptable to studying culture-expanded cells than minimally-manipulated cells (i.e., without culture expansion) that are also being examined for tissue repair, such as bone marrow concentrate 72 or the stromal vascular fraction of adipose tissue. 73 Finally, the use of an immune-compromised rat may have limited the physiological relevance of the defect microenvironment on chondrogenic activity by implanted MSCs.…”

Nanoliposomes from Agro-Resources as Promising Delivery Systems for Chondrocytes

“…The low transfection effectiveness also appears to be typical of the human chondrocytes since transfection above 40% has been reported with non-cationic lipids in bovine or rabbit chondrocytes [11,[13][14][15]. This explains why studies requiring a prolonged expression of a transgene in chondrocytes increasingly involve infections with retroviruses, adenoviruses, adeno-associated viruses, or more recently lentiviruses [16][17][18][19][20][21].…”