Human Adipose-derived Stem Cells as a Potential Source of Endothelial Cells in Clinical Tissue Engineering Applications
Hakan Orbay, MD, PhD1, Derek B. Asserson, BS2, Kamaljit Devi, BS1, Priscilla A. Williams, BS3, Tima Dehghani, BS1, Eduardo A. Silva, PhD 3, David E. Sahar, MD1
1University of California Davis Department of Surgery, Division of Plastic Surgery, Sacramento, CA, 2California Northstate University College of Medicine, Elk Grove, CA, 3University of California Davis, Department of Biomedical Engineering, Davis, CA
PURPOSE: Seeding the tissue-engineered constructs with endothelial cells (ECs) may accelerate neovascularization and prevent the resorption of the constructs. However, the use of autologous ECs is hampered by the need to harvest a blood vessel from the patient and the technical challenges of EC culture. Our aim in this study is to determine whether human adipose-derived stem cells (ASCs) can be an alternative EC source for clinical tissue engineering applications.
METHODS: We harvested human ASCs from adipose tissue samples via enzymatic digestion and characterized them with flow cytometry and tri-lineage differentiation. We fed ASCs from PIII-V with EGM-2MV endothelial cell differentiation medium (Lonza Pharmaceuticals, Basel, Switzerland) for up to three weeks. We harvested the cells after 1, 2 and 3 weeks, and evaluated endothelial differentiation with Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), flow cytometry, and angiogenic sprouting assays.
RESULTS: ASCs were CD 90+, CD 44+, and CD 31- prior to differentiation. They differentiated into adipogenic, osteogenic, and chondrogenic lineages as detected by Oil Red O, Alizarin red and Alcian blue staining, respectively. The expression of EC specific genes in ASCs made a peak at the second week of differentiation. The fold changes in expression of CD31, vascular endothelial growth factor receptor-1, nitric oxide synthase, and von Willebrand Factor genes at week 2 were 0.4 ± 0.1, 34.7 ± 0.3, 2.03 ± 0.25 and 12.5 ± 0.3 respectively. The percentage of CD 31+ cells in total ASCs population as detected by flow cytometry was 0.2, 0.64, and 1.6 at weeks 1, 2, and 3, respectively. ASCs formed an average of 4.0±0.4, 1.8±0.6, and 0.7±0.1 sprouts/bead at weeks 1, 2, and 3 of differentiation, respectively. Moreover, human ASCs derived ECs displayed enhanced sprouting capability as compared to human microvascular endothelial cells (p<0.05) (Fig.1).
CONCLUSION: Human ASCs have the potential to become a clinical source of ECs. Further refinement of the growth factor concentrations in the differentiation medium may increase the endothelial differentiation of human ASCs.
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