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

肿瘤、创伤或手术可导致需要广泛重建的临界尺寸骨缺损。自体或同种异体移植或假体材料用于骨重建具有相当大的缺点，需要探索其他选择。转化生长因子β（Transforming growth factor beta，TGFβ）在骨代谢中起重要作用。假设抑制TGFβ信号传导促进骨再生。小分子SB 431542是一种TGFβ抑制剂，可导致成骨分化增加，从而为骨缺损的替代治疗提供了一种有效的方法。 利用培养扩增的间充质干细胞（MSC）的组织工程已被证明是非常有效的骨再生的实验研究。然而，MSC在损伤部位的移植和分化已经显示出较差。已经提出MSC分泌生物活性因子，其通过促进细胞内信号传导直接起作用或通过刺激邻近细胞间接起作用。从骨髓间充质干细胞中释放的外泌体是一种递送miRNA、蛋白质和其他分子的生物分子纳米结构，我们假设外泌体作为来自人脂肪组织来源的干细胞（ex-hASCs）的自分泌和/或旁分泌信号因子的载体，因此可以从细胞中分离并直接应用于骨缺损。我们认为，用SB 431542预处理增强了衍生的外泌体的骨骨骼修复能力。我们提出的项目旨在研究用SB 431542处理的ex-hASC的骨再生能力，与从未处理的hASC分离的ex-hASC相比。此外，我们将表征ex-hASCs的货物含量，目的是鉴定在增强颅骨的骨再生中起关键作用的特定因素。通过体外实验，我们将使用RT-PCR、qPCR和免疫印迹分析ex-hASC在抑制TGFβ信号传导后的骨诱导活性。将使用显微CT、组织学和免疫荧光染色在颅骨缺损模型中分析ex-hASC的骨骨骼再生能力。分离的外来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分析在抑制TGFβ-信号传导后相对于来自未处理的hASC的外来体进行表征。最后，我们将对我们先前使用功能获得和丧失实验鉴定的候选蛋白质和/或miRNA进行功能测试。我们认为，外泌体可以用作靶向药物递送的载体，并且可以通过遗传预处理来改善靶位点的治疗。 因此，外泌体可以潜在地取代基于细胞的治疗，并消除MSC衍生的细胞移植的肿瘤转化的风险。

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.