Hyaline articular cartilage has a poor intrinsic healing capacity, and damage to this tissue can initiate the progression to osteoarthritis. Unfortunately there are few treatment options for repairing cartilage, and none is capable of permanently restoring the articular surface. Mesenchymal stem/progenitor cells (MSCs) are adult stem cells that offer exciting possibilities for clinical cell-based repair of cartilage lesions. However, desite their widespread research interest, preclinical studies have yet to fulfill their considerable potential. We believe that this can be attributed to the wide variation in model systems used among laboratories, and the lack of standardized methods to quantify efficacious cell-based repair. To facilitate the clinical advancement of MSC- based cartilage repair, this proposal aims to develop broadly applicable tools that can be reliably used to compare chondrogenic potential among various MSC populations and distinguish chondrogenic and terminally differentiating cell phenotypes during differentiation. Lentiviral based reporter constructs containing up to three independent expression cassettes under constitutive (EF-1?) and conditionally active (chondrocyte-lineage specific, or terminal differentiation-associated) promoters will be used to label MSC populations and track cellular differentiation in vitro and in vivo. Each cassette will encode either fluorescent or luciferase reporters to enable quantification of cellular responses by luciferase assay, flow cytometry and fluorescence microscopy to reveal 1) the proportion and distribution of undifferentiated, chondrogenic and terminally differentiating cells within an MSC population and 2) the overall chondrogenic potency of MSC populations derived from bone marrow, synovium and adipose tissue.
In Aim 1, a COL2A1-specific transcriptional reporter will be used to quantify the chondrogenic responses of disparate MSC populations in aggregate and PEG-HA cultures in vitro.
In Aim 2, the same culture systems will be employed to investigate a novel transcriptional reporter for quantitation of terminally-differentiation following chondroinduction.
In Aim 3, we will validate the utility of these reporter systems to track chondrogenic and terminally differentiating cells in vivo, using a mouse xenotransplantation model. If successful, these studies should establish the reporter constructs as recognized tools that provide sensitive and unbiased quantification of MSC-based chondrogenesis which can be used to improve cartilage repair protocols.
Mesenchymal Stem Cells (MSCs) offer much hope for repairing damaged articular cartilage resulting from joint injury, or osteoarthritis. This proposalis directed toward harnessing their therapeutic potential by establishing chondrogenic reporter gene constructs as tools that can quantitatively assess the cartilage-forming ability of MSCs in vitro and in vivo. If successful, these tools should aid in the development of more effective MSC- based strategies for therapeutic repair of cartilage defects.
| Martin-Pena, A; Porter, R M; Plumton, G et al. (2018) Lentiviral-based reporter constructs for profiling chondrogenic activity in primary equine cell populations. Eur Cell Mater 36:156-170 |
