Abstract
Numerous investigators have described chondrogenic differentiation of bone marrow stromal cells obtained from both murine and human sources over the past decade. The ease of access and large available quantity of adipose tissue, however, makes Adipose-Derived Stem Cells (ADSC) a far more practical alternative for clinical applications.
Therefore, the primary goal of this research endeavor is to achieve chondrogenic differentiation of ADSC. Previous work had also demonstrated that bone morphogenetic protein receptor 1A (BMP receptor 1A) signaling is required for postnatal maintenance of articular cartilage. In fact, cartilage within the joints of transgenic mice deficient in BMP receptor 1A rapidly degenerates after birth in a process resembling accelerated human osteoarthritis. Based on this evidence, we used a lentiviral vector to increase expression of BMP receptor 1A by our isolated stem cells in order to direct their differentiation into the chondrocyte lineage.
We harvested subcutaneous adipose tissue intraoperatively from consenting patients undergoing elective lipoplasty and panniculectomy procedures. The stromal vascular fraction was isolated from this tissue and further refined by passaging in selective media to yield a stable population of ADSC in primary culture. Both the identity and homogeneity of this stem cell population was confirmed using adipogenic induction media and differentiation cocktails. In addition, we subcloned an expression plasmid containing the BMP receptor 1A locus in tandem with green fluorescent protein (GFP) under the transcriptional control of a single promoter. This plasmid was packaged into a lentiviral vector to provide a reliable method of achieving both genomic integration and long-term expression of the BMP receptor 1A gene. Hence, transduction of ADSC using this vector resulted in overexpression of BMP receptor 1A by these multipotent cells.
The GFP was then utilized to screen and enrich the ADSC population for stem cells with a robust expression of BMP receptor 1A. The ADSC that overexpressed BMP receptor 1A were found to achieve chondrogenic differentiation after 13 to 16 days of in vitro culture, as revealed by immunohistochemistry assays for the bio-markers of articular cartilage (type II collagen and the proteoglycan aggrecan).
Our results demonstrate that stem cells derived from the adipose tissue of a patient represent a viable means of culturing autologous chondrocytes in vitro for future implantation at the site of osteochondral defects. This method of attaining cartilaginous regeneration is intuitively appealing, given the minimal donor site morbidity associated with removing subcutaneous fat. By transducing the ADSC with a lentiviral vector, we have also collected further evidence implicating the critical importance during chondrogenesis of signaling mediated by the BMP receptor 1A. Further tissue engineering studies are now in progress to evaluate the ability of ADSC to differentiate into chondrocytes after seeding onto poly-caprolactone polymer scaffolds.
Correspondence should be addressed to Diane Przepiorski at ISTA, PO Box 6564, Auburn, CA 95604, USA. Phone: +1 916-454-9884; Fax: +1 916-454-9882; E-mail: ista@pacbell.net
