Cellular Reprogramming using Pluripotent Stem Cell Derived Exosomes

使用多能干细胞衍生的外泌体进行细胞重编程

• 批准号: RGPIN-2022-03166
• 负责人: Rancourt, Derrick
• 金额: $ 2.48万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744274
• 关键词: Cellular; Reprogramming; using; Pluripotent; Stem

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.

目标.使用小鼠胚胎干细胞（mESC）模型，我们已经确定了一种使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）通过标准化方法来优化XRP，用于衍生和分离具有来自搅拌悬浮生物反应器的最大重编程潜力的ESC外泌体，以及2）通过将额外的转录因子（TF）加载到外泌体中来开发促进重编程和转分化的平台。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.早期细胞重编程步骤涉及间充质向上皮转化（MET），其中基质依赖性细胞变为粘附依赖性细胞。当我们将mESC外泌体应用于成纤维细胞时，我们观察到与MET和诱导的多能干细胞（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Oct 4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iPSC。mESC外泌体中的miR-302 a可能控制我们观察到的部分重编程。科学的方法。利用先前的NSERC资金，我们最近证明了生物反应器流体剪切通过诱导非经典Wnt信号传导促进多能性。由于miR-302 a被β-连环蛋白上调，我们假设来源于在生物反应器中培养的ESC的外泌体可能在促进细胞重编程方面更有效。该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将TF加载到mESC外泌体中以促进XRP转分化。与我们的ESC外泌体促进部分重编程的假设一致，我们可以产生外胚层（即，神经的），中胚层的（即，心脏的）和内胚层的（即，肝）细胞类型。与Oct 4补充（上文）非常相似，我们假设XRP转分化将通过将组织特异性TF加载到外泌体中来实现。我们的方法利用了两个特征（即，热休克蛋白富含外泌体并螯合ERT 2融合蛋白），以用ERT 2-TF装载外泌体，从而在他莫昔芬存在下完成细胞重编程和/或转分化。意义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可以使细胞重编程和转分化民主化。我们使用生物反应器来规模化地从转基因ESC制造外泌体将实现这一愿景。