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Thursday, October 7, 2010, 10:20-10:50Session B3: Cartilage Repair/RegenerationOsteogenic Differentiation of Adult Human Adipose-Derived Stem Cells IsAchieved by Overexpression of the BMP Receptor 1A*Farhang Raaii - Detroit Medical Center - Detroit, United States of America*Email: raaii.farhang@gmail.comNumerous investigators have described osteogenic differentiation of bone marrow stromal cellsobtained from both murine and human sources over the past decade. The ease of access andlarge available quantity of adipose tissue, however, makes Adipose-Derived Stem Cells (ADSC)a far more practical alternative for clinical applications, such as operative treatment of nonunionsand regeneration of critical bone defects. Therefore, the primary goal of this researchendeavor is to achieve osteogenic differentiation of ADSC. Previous work has alreadydemonstrated that bone morphogenetic protein receptor 1A (BMP receptor 1A) signaling isrequired for healing critical bone defects. Based on this evidence, we used a lentiviral vector toincrease expression of BMP receptor 1A by our stem cell population in order to direct theirdifferentiation into the osteoblastic lineage.We harvested subcutaneous adipose tissue intraoperatively from consenting patients undergoingelective lipoplasty and panniculectomy procedures. The stromal vascular fraction was isolatedfrom this tissue and further refined by passaging in selective media to yield a stable populationof ADSC in primary culture. Both the identity and homogeneity of this stem cell populationwas confirmed using adipogenic induction media and differentiation cocktails. In addition, wesubcloned an expression plasmid containing the BMP receptor 1A locus in tandem with greenfluorescent protein (GFP) under the transcriptional control of a single promoter. This plasmidwas packaged into a lentiviral vector to provide a reliable method of achieving both genomicintegration and long-term expression of the BMP receptor 1A gene. Hence, transduction ofADSC using this vector resulted in overexpression of BMP receptor 1A by these multipotentcells. The GFP was then utilized as a reporter gene to screen and enrich the ADSC populationfor only those stem cells with a robust expression of BMP receptor 1A. The ADSC thatoverexpressed BMP receptor 1A were found to achieve osteogenic differentiation after 18 to 20days of in vitro culture, as revealed by immunohistochemistry assays for osteocalcin.Osteogenic differentiation was further confirmed by alizarin red staining and quantitative PCRfor alkaline phosphatase gene expression as a biomarker for the osteoblastic lineage.Our results demonstrate that stem cells derived from the adipose tissue of a patient represent aviable means of culturing autologous osteoblasts in vitro for future implantation at the site ofcritical bone defects. This method of attaining osseous regeneration is intuitively appealing,given the minimal donor site morbidity associated with removing subcutaneous fat. Bytransducing the ADSC with a lentiviral vector, we have also collected further evidenceimplicating the critical importance of signaling mediated by the BMP receptor 1A duringosteogenesis. Further tissue engineering studies are now in progress to evaluate the osteogenicdifferentiation potential of these stem cells under hydrostatic and fluid flow shearingmechanical loads.file:///E|/ISTA2010-Abstracts.htm[12/7/2011 3:15:47 PM]