Cellular Reprogramming using Pluripotent Stem Cell Derived Exosomes

目标。利用小鼠胚胎干细胞(MESC)模型，我们已经确定了一种使用外体重新编程细胞命运的新方法。外显子介导的重编程(XRP)可能会彻底改变组织工程的细胞来源。我们建议1)通过标准化方法从搅拌悬浮生物反应器中获得和分离具有最大重编程潜力的ESC外切体来优化XRP，以及2)开发一个通过将额外的转录因子(TF)加载到外切体来促进重编程和反式分化的平台。我们将利用我们在多功能干细胞制造、细胞重新编程和基因组工程方面的专业知识。洞察力。早期的细胞重编程步骤包括间充质细胞到上皮细胞的转变(MET)，其中底物依赖的细胞变得黏附依赖。当我们将mESC外切体应用于成纤维细胞时，我们观察到了与MET和诱导的多能干细胞(IPSC)克隆形成一致的形态变化。免疫荧光显微镜证实E-钙粘素的存在，与MET一致。然而，补充Oct4对于促进稳定的IPSC集落形成和多能性是必要的。我们假设ESC外切体促进部分重编程或IPSCs前。而存在于mESC外体中的miR-302a可能控制着我们观察到的部分重编程。科学的方法。利用之前的NSERC资助，我们最近证明了生物反应器流体剪切通过诱导非典型的Wnt信号来促进多能性。由于miR-302a被ç-catenin上调，我们假设在生物反应器中培养的ESCs衍生的外体可能在促进细胞重编程方面更有效。本研究计划的第一个目标是通过利用生物反应器流体剪切来提高XRP的效率。我们的第二个目标是开发一种新的方法来将转录因子负载到mESC外体中，以促进XRP的反式分化。与我们关于ESC外切体促进部分重编程的假设一致，我们可以使用特定的分化鸡尾酒产生外胚层(即神经)、中胚层(即心脏)和内胚层(即肝脏)细胞类型。很像Oct4补充剂(如上)，我们假设XRP的反式分化将通过将组织特异性的TF装载到外体中来实现。我们的方法利用两个特征(即热休克蛋白富含外切体和隔离ERT2融合蛋白)来将ERT2-TF加载到外切体中，以在他莫昔芬存在的情况下完成细胞重新编程和/或转分化。意义重大。XRP是一种新兴的颠覆性平台技术，有可能简化基础研究和应用的细胞重新编程和转分化。通过消除对病毒载体的需求，XRP可以使细胞重新编程和转分化大众化。我们使用生物反应器来规模化生产转基因ESCs的外切体，这将使这一愿景成为可能。

OBJECTIVES. Using the mouse embryonic stem cell (mESC) model, we have identified a new approach to cellular fate reprogramming using exosomes. Exosome-mediated reprogramming (XRP) may revolutionize cell sourcing for tissue engineering. We propose to 1) optimize XRP by standardizing methods for deriving and isolating ESC exosomes with maximal reprogramming potential derived from stirred suspension bioreactors, and 2) develop a platform that promotes reprogramming and trans-differentiation by loading additional transcription factors (TFs) into exosomes. We will leverage our expertise in pluripotent stem cell manufacturing, cellular reprogramming, and genome engineering. INSIGHT. An early cellular reprogramming step involves mesenchymal to epithelial transition (MET) wherein substrate-dependent cells become adhesion-dependent. When we applied mESC exosomes onto fibroblasts, we observed morphological changes consistent with MET and induced pluripotent stem cell (iPSC) colony formation. Immunofluorescence microscopy confirmed the presence of E-cadherin, consistent with MET. However, Oct4 supplementation was necessary to promote stable iPSC colony formation and pluripotency. We hypothesize that ESC exosomes promote partial reprogramming or pre-iPSCs. and that miR-302a, present in mESC exosomes, may govern the partial reprogramming we observe. SCIENTIFIC APPROACH. Using prior NSERC funding, we recently demonstrated that bioreactor fluid shear promotes pluripotency by inducing non-canonical Wnt signalling. Since miR-302a is upregulated by ß-catenin, we hypothesize that exosomes derived from ESCs cultured in the bioreactor may be more effective at promoting cellular reprogramming. The first objective of this research program is to improve the efficiency of XRP by exploiting bioreactor fluid shear. Our second objective will develop a novel approach to load TFs into mESC exosomes to promote XRP trans-differentiation. Consistent with our hypothesis that ESC exosomes promote partial reprogramming, we can generate ectodermal (i.e., neural), mesodermal (i.e., cardiac) and endodermal (i.e., hepatic) cell types using specific differentiation cocktails. Much like Oct4 supplementation (above), we hypothesize that XRP trans-differentiation will be enabled by loading tissue specific TFs into exosomes. Our approach leverages two features (i.e., that heat shock proteins are rich in exosomes and sequester ERT2 fusion proteins) to load exosomes with ERT2-TFs to complete cellular reprogramming and/or trans-differentiation in the presence of tamoxifen. SIGNIFICANCE. XRP is a nascent disruptive platform technology that has the potential to simplify cellular reprogramming and trans-differentiation for basic research and application. By eliminating the need for viral vectors, XRP could democratize cellular reprogramming and trans-differentiation. Our use of bioreactors to scale the manufacture of exosomes from genetically modified ESCs will enable this vision.