A. Luan1, C. Duldulao1, A. McArdle1, K. J. Paik1, M. T. Chung1, D. A. Atashroo1, E. R. Zielins1, R. Tevlin1, K. Senarath-Yapa1, T. Wearda1, S. Menon1, S. Shailendra1, M. Lee2, G. C. Gurtner1, D. C. Wan1, M. T. Longaker1 1Stanford University,Hagey Laboratory For Pediatric Regenerative Medicine, Department Of Surgery, Plastic And Reconstructive Surgery Division,Palo Alto, CA, USA 2University Of California – Los Angeles,Laboratory Of Biomaterials And Bioengineering, Division Of Advanced Prosthodontics, Department Of Bioengineering, Weintraub Center For Reconstructive Biotechnology,Los Angeles, CA, USA
Introduction: The reduced ability of bone to regenerate past the age of 2 years in humans poses significant challenges for reconstructive surgeons. An ability to drive differentiation of adult stem cells isolated from human fat towards an osteogenic lineage (together with the use of biomimetic scaffolds) has resulted in techniques that offer great promise for treating bony defects, which present a significant biomedical burden. Human adipose-derived stromal cells (hASCs), however, are a heterogeneous population of multipotent cells. The aim of this study was to enrich for an osteogenic subpopulation within the heterogeneous hASC pool based on cell surface marker expression. This will ultimately offer a novel cell-based strategy for treating bone loss.
Methods: We used a combination of single cell transcriptional analysis and Boolean implication networks to identify cell surface markers expressed on hASCs that are associated with upregulation of osteogenic gene expression. Using a bioinformatics analysis of our data, we identified CD13 as a potential target to enrich for osteogenic subpopulations of hASCs. To assess the osteogenic potential of CD13+ hASCs, we compared them in vitro to CD13– and unsorted hASCs. To evaluate osteogenesis in vivo, unsorted, CD13+, and CD13– hASCs were seeded onto a hydroxyapatite-poly(lactic-co-glycolic acid) scaffold and placed into critical sized calvarial defects of immunocompromised mice. Healing was measured over an eight week period using micro-computed tomography analysis. After 8 weeks, calvaria were harvested for histological assessment by pentachrome staining.
Results: Our results show that CD13+ hASCs demonstrate increased osteogenic differentiation in vitro as demonstrated by alkaline phosphatase staining at day 7 (**p<0.01) and extracellular matrix mineralization with Alizarin Red staining at day 14 (*p<0.05). In addition, gene expression analysis using qRT-PCR revealed an upregulation of RUNX2 and osteocalcin, early and late markers of osteogenesis, respectively. In vivo bone quantification showed increased bone formation in all three groups, with CD13+ hASCs demonstrating the greatest amount of healing. Bone formation was confirmed histologically.
Conclusion: The isolation of hASCs that express the CD13 surface marker enriches for a highly osteogenic subpopulation of hASCs. An ability to isolate an osteogenic subpopulation based on a single surface marker would allow for a clinically translatable method to isolate osteogenic populations for bone regeneration.
