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

肿瘤、创伤或手术可能导致严重的骨缺损，需要广泛的重建。自体或异体移植或假体材料用于骨重建有相当大的缺点，需要探索其他选择。转化生长因子β (TGFβ)在骨代谢中起重要作用。据推测，抑制tgf - β信号传导可促进骨再生。小分子SB431542是一种TGFβ抑制剂，可导致成骨分化增加，因此为骨缺损的替代治疗提供了一种有效的方法。利用培养扩增间充质干细胞（MSC）进行组织工程在骨再生的实验研究中已被证明是非常有效的。然而，干细胞在损伤部位的植入和分化表现不佳。有人提出，间充质干细胞分泌生物活性因子，其作用直接通过促进细胞内信号传导或间接通过刺激邻近细胞。间充质干细胞释放的外泌体是一种生物分子纳米结构，可传递miRNA、蛋白质和其他分子。我们假设外泌体作为自分泌和/或旁分泌信号因子的载体，来自人类脂肪组织来源的干细胞（前hascs），因此可以从细胞中分离出来并直接应用于骨缺损。我们认为SB431542预处理可以增强衍生外泌体的骨修复能力。我们提出的项目旨在研究SB431542处理的前hASCs的骨再生能力，与未处理的hASCs分离的前hASCs进行比较。此外，我们将表征前hascs的货物含量，目的是确定在增强颅骨骨骨再生中起关键作用的特定因素。首先，我们将从未经处理和SB431542处理的hASCs中分离外泌体。通过体外实验，我们将利用RT-PCR、qPCR和免疫印迹技术分析前hascs对tgf - β信号的抑制作用。利用显微ct、组织学和免疫荧光染色在颅骨缺损模型中分析前hascs的骨骨再生能力。分离的外泌体的货物含量将通过成像技术、免疫印迹、蛋白质组学分析和miRNA分析来表征tgf - β信号与未经处理的hASCs外泌体的抑制。最后，我们将对候选蛋白和/或mirna进行功能测试，我们之前通过功能增益和功能丧失实验确定了这些候选蛋白和/或mirna。我们相信外泌体可以作为靶向药物递送的载体，并且可以通过基因预处理来改善靶部位的治疗。因此，外泌体有可能取代基于细胞的治疗，并消除MSC来源细胞移植的肿瘤转化风险。

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.