Abstract
Numerous investigators have described osteogenic 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, such as operative treatment of non-unions and regeneration of critical bone defects. Therefore, the primary goal of this research endeavor is to achieve osteogenic differentiation of ADSC. Previous work has already demonstrated that bone morphogenetic protein receptor 1A (BMP receptor 1A) signaling is required for healing critical bone defects. Based on this evidence, we used a lentiviral vector to increase expression of BMP receptor 1A by our stem cell population in order to direct their differentiation into the osteoblastic 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 as a reporter gene to screen and enrich the ADSC population for only those stem cells with a robust expression of BMP receptor 1A. The ADSC that overexpressed BMP receptor 1A were found to achieve osteogenic differentiation after 18 to 20 days of in vitro culture, as revealed by immunohistochemistry assays for osteocalcin. Osteogenic differentiation was further confirmed by alizarin red staining and quantitative PCR for 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 a viable means of culturing autologous osteoblasts in vitro for future implantation at the site of critical bone defects. This method of attaining osseous 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 of signaling mediated by the BMP receptor 1A during osteogenesis. Further tissue engineering studies are now in progress to evaluate the osteogenic differentiation potential of these stem cells under hydrostatic and fluid flow shearing mechanical loads.
