Lentiviral Transduced Fluorescent Adipose Derived Stem Cells (Adscs) Undergo Spontaneous Osteogensis On Low-Oxygen Content Graphene (Log) Surfaces

Background Traditionally, severe bone defects caused by trauma or disease are treated with autologous bone grafts, but can result in additional pain and discomfort for the patient. Bone tissue engineering strategies involving the use of osteoprogenitor cells and novel scaffolds offer hope as an alternative therapy for the treatment of bone defects. We have demonstrated that adipose derived stem cells (ADSCs) grown on graphene nanoparticles with low oxygen content (LOG) results in spontaneous osteoblast differentiation in vitro. Due to the dark nature of the LOG surface, ADSCs cannot be monitored by traditional light microscopy. Hence, the goal of this work was to create fluorescent ADSCs that can be used for studies on LOG surfaces. Objective Determine if fluorescent ADSCs transduced by lentivirus undergo osteogenesis on low-oxygen content graphene (LOG) surfaces. Hypothesis Fluorescently transduced ADSCs behave similarly to non-transduced ADSCs on LOG coated surfaces. Methods Adipose tissue was obtained from human patients undergoing pannulectomies via an approved IRB and patient consent. In vitro expanded ADSCs were transduced at a multiplicity of infection (MOI) of 5 (>90% transduction efficiency) with lentivirus particles encoding green fluorescent protein (GFP), expanded in vitro, characterized by trilineage staining/flow cytometry and used for characterization on LOG surfaces. Results Our data show that ADSCs, obtained from two different human patients, undergo trilineage differentiation normally by histological staining and express positive surface markers (CD29, CD44, CD73, CD90, CD105) and lack expression of negative surface markers (CD34, CD45, CD106, HLA-DR) indicative of stem cells, by flow cytometry. Moreover, these same ADSCs transduced by lentivirus particles, encoding GFP, undergo trilineage differentiation normally and expression of fluorescence is not reduced/lost upon differentiation. GFP expressing ADSCs seeded, grown and visualized on LOG coated surfaces demonstrated spontaneous osteogenesis in the absence of exogenous inducing factors compared to control ADSCs on polystyrene dishes via alizarin red staining and quantitation. Furthermore, addition of dexamethasone, beta-glycerophosphate and ascorbic acid resulted in higher osteogenesis on LOG coated surfaces. Conclusions Based on these data we conclude the following: 1) lentivial transduction of ADSCs does not alter trilineage differentiation potential; 2) GFP expressing ADSCs can be used to continuously monitor cells on LOG coated surfaces; 3) Transduced ADSCs undergo osteogenesis similar to non-transduced ADSCs on LOG coated surfaces; and 4) ADSCs obtained from different human patients behaved similarly on LOG coated surfaces suggesting LOG can be used for regenerative medicine purposes with any human ADSCs.