Cellular Reprogramming using Pluripotent Stem Cell Derived Exosomes

目标。利用小鼠胚胎干细胞（mESC）模型，我们发现了一种利用外泌体进行细胞命运重编程的新方法。外泌体介导的重编程（XRP）可能会彻底改变组织工程的细胞来源。我们建议：1)通过标准化从搅拌悬浮生物反应器中提取和分离具有最大重编程潜力的ESC外泌体的方法来优化XRP； 2)开发一个平台，通过在外泌体中加载额外的转录因子（tf）来促进重编程和反式分化。我们将利用我们在多能干细胞制造、细胞重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到上皮的转变（MET），其中依赖底物的细胞变得依赖粘附。当我们将mESC外泌体应用于成纤维细胞时，我们观察到与MET和诱导多能干细胞（iPSC）集落形成一致的形态学变化。免疫荧光显微镜证实了e -钙粘蛋白的存在，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外泌体促进部分重编程或前ipscs。我们观察到，存在于mESC外泌体中的miR-302a可能控制了部分重编程。科学的方法。利用先前的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Wnt信号传导来促进多能性。由于miR-302a被ß-catenin上调，我们假设生物反应器中培养的ESCs衍生的外泌体可能更有效地促进细胞重编程。本研究计划的第一个目标是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，将tf加载到mESC外泌体中，以促进XRP的转分化。与ESC外泌体促进部分重编程的假设一致，我们可以使用特异性分化鸡尾酒生成外胚层（即神经）、中胚层（即心脏）和内胚层（即肝脏）细胞类型。就像Oct4补充（上文）一样，我们假设通过将组织特异性tf加载到外泌体中可以实现XRP的反式分化。我们的方法利用两个特征（即，热休克蛋白富含外泌体和隔离ERT2融合蛋白），在他莫昔芬存在的情况下，用ERT2- tf装载外泌体来完成细胞重编程和/或反式分化。的意义。XRP是一种新兴的颠覆性平台技术，有可能简化细胞重编程和反分化的基础研究和应用。通过消除对病毒载体的需求，XRP可以使细胞重编程和反式分化民主化。我们利用生物反应器从转基因ESCs中大规模生产外泌体将实现这一愿景。

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.