Osteoskeletal regenerative ability of exosomes derived from adipose-derived stem cells upon inhibition of Transforming Growth Factor-β-signaling

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Tumors, trauma or surgery can lead to critical-sized bone defects requiring extensive reconstruction. Autologous or allogenic transplantation or prosthetic material for bone reconstruction comes with considerable drawbacks and other options need to be explored. Transforming growth factor beta (TGFβ) has been identified to play an important role in bone metabolism. It is hypothesized that inhibition of TGFβ signaling promotes bone regeneration. Small molecule SB431542, a TGFβ inhibitor, leads to increased osteogenic differentiation, thus presenting a valid approach in alternate treatment of bone defects. Tissue engineering using culture-expanded mesenchymal stem cells (MSC) has been shown to be highly effective in experimental research of bone regeneration. However, MSC engraftment and differentiation at the injury site has been shown to be poor. It has been proposed that MSCs secrete bioactive factors, which act directly by promoting intracellular signaling or indirectly by stimulating neighboring cells. Exosomes released from MSCs are biomolecular nanostructures delivering miRNA, proteins and other molecules.We hypothesize that exosomes act as vehicles for autocrine and/or paracrine signaling factors emanating from human adipose tissue-derived stem cells (ex-hASCs) and can thus be isolated from the cells and applied directly to the bone defect. We believe that pretreatment with SB431542 enhances the osteoskeletal repair ability of derived exosomes. Our proposed project aims at investigating the bone regeneration capacity of ex-hASCs treated with SB431542, in comparison to ex-hASCs isolated from untreated hASCs. Moreover, we will characterize the cargo content of ex-hASCs with the goal to identify specific factors playing a key role in enhancing the osteoskelatal regeneration of calvarial bones.First we will isolate the exosomes from untreated and SB431542 treated hASCs. Through in vitro experiments we will analyse the osteoinductive activity of ex-hASCs upon inhibition of TGFβ signaling, using RT-PCR, qPCR and immunoblotting. Osteoskeletal regenerative capacity of ex-hASCs will be analysed in a calvarial defect model using micro-CT, histology and immunofluorescence staining. The cargo content of the isolated exosomes will be characterised via imaging techniques, immunoblotting, proteomic analysis and miRNA analysis upon inhibition of TGFβ-signaling versus exosomes from untreated hASCs. Lastly we will perform functional testing of candidate proteins and/or miRNAs that we earlier identified using gain- and loss-of-function experiments. We believe that exosomes can be used as vehicles for targeted drug delivery and can be preconditioned genetically to improve therapy at the target site. Thus, exosomes could potentially replace cell-based therapy and eradicate the risk of neoplastic transformation of MSC derived cell transplantation.