Project 1:        

Differentiation of adult bone marrow-derived mesenchymal stem cells (MSCs)

The major aim of this research project is to determine the regulatory mechanisms that control differentiation of MSCs toward the chondrogenic or osteoblastic lineage. In particular, we are interested in generating the type of chondrocytes found in articular cartilage rather than growth plate cartilage in order to benefit the field of cartilage tissue engineering as a therapy for conditions such as osteoarthritis. Here, we apply techniques involving stem cell isolation and culture, in vitro differentiation assays as well as viral expression systems. Currently, we are focusing on manipulating MSC differentiation by modulating expression of specific transcription factors to attempt to generate a chondrocyte phenotype similar to that found in hyaline / articular cartilage. In addition we also plan to establish an effective differentiation assay system based on utilizing 3D biomaterial scaffolds.

Project 2:

Regulation and function of precursor mRNA alternative splicing of cartilage genes in development and disease    

Primarily, this research project has involved analysis of the type II collagen alternative splicing event that occurs during cartilage development. We have generated a type II collagen (COL2A1) mini-gene which has proven to be an effective model system to decipher important cis elements and trans-acting splicing factor proteins that regulate this important splicing event. Recently, our laboratory, in collaboration with Dr Thomas Hering (Case Western Reserve University) and Dr Brian Johnstone (Oregon Health and Sciences University) discovered two additional spliced isoforms of type II collagen. We hypothesize that one of these new isoforms is functional only at the level of mRNA while the other is translated to protein. The functional role of these isoforms with respect to cartilage development is currently being investigated.

Project 3:

Generation of a novel knock-in mouse model that will synthesize only one isoform of the extracellular matrix protein, type II collagen

From our ongoing studies on alternative splicing regulation, we have developed a unique strategy to generate an in vivo mouse model that will express only the embryonic alternatively-spliced isoform of type II collagen. In doing so, we will inhibit the developmentally-regulated splicing switch that normally occurs during chondrogenesis. This will directly address the question of whether this alternative splicing event is indeed necessary for production of correct, healthy cartilage in vivo.  It is also worth noting that this novel recombinant mouse strategy will also have more generalized appeal as a means to study other alternatively-spliced genes during tissue development.

Project 4:

Viability and function of epiphyseal growth plates following ex vivo storage and heterotopic transplantation