Nassor Ferid, Jarray Rafika, Biard Denis S F, Maïza Auriane, Papy-Garcia Dulce, Pavoni Serena, Deslys Jean-Philippe, Yates Frank
Service d'Etude des Prions et des Infections Atypiques (SEPIA), Institut François Jacob, Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Université Paris Saclay, Fontenay-aux-Roses, France.
CellTechs Laboratory, Sup'Biotech, Villejuif, France.
Front Cell Neurosci. 2020 Feb 11;14:14. doi: 10.3389/fncel.2020.00014. eCollection 2020.
Human brain organoids (mini-brains) consist of self-organized three-dimensional (3D) neural tissue which can be derived from reprogrammed adult cells and maintained for months in culture. These 3D structures manifest substantial potential for the modeling of neurodegenerative diseases and pave the way for personalized medicine. However, as these 3D brain models can express the whole human genetic complexity, it is critical to have access to isogenic mini-brains that only differ in specific and controlled genetic variables. Genetic engineering based on retroviral vectors is incompatible with the long-term modeling needed here and implies a risk of random integration while methods using CRISPR-Cas9 are still too complex to adapt to stem cells. We demonstrate in this study that our strategy which relies on an episomal plasmid vector derived from the Epstein-Barr virus (EBV) offers a simple and robust approach, avoiding the remaining caveats of mini-brain models. For this proof-of-concept, we used a normal tau protein with a fluorescent tag and a mutant genetic form (P301S) leading to Fronto-Temporal Dementia. Isogenic cell lines were obtained which were stable for more than 30 passages expressing either form. We show that the presence of the plasmid in the cells does not interfere with the mini-brain differentiation protocol and obtain the development of a pathologically relevant phenotype in cerebral organoids, with pathological hyperphosphorylation of the tau protein. Such a simple and versatile genetic strategy opens up the full potential of human organoids to contribute to disease modeling, personalized medicine and testing of therapeutics.
人类大脑类器官（微型大脑）由自组织的三维（3D）神经组织组成，该组织可从经重编程的成体细胞中获得，并在培养中维持数月。这些3D结构在神经退行性疾病建模方面具有巨大潜力，为个性化医疗铺平了道路。然而，由于这些3D大脑模型能够表达人类完整的遗传复杂性，因此获得仅在特定且可控的遗传变量上存在差异的同基因微型大脑至关重要。基于逆转录病毒载体的基因工程与这里所需的长期建模不兼容，并且存在随机整合的风险，而使用CRISPR-Cas9的方法仍然过于复杂，无法应用于干细胞。我们在本研究中证明，我们基于源自爱泼斯坦-巴尔病毒（EBV）的附加体质粒载体的策略提供了一种简单而稳健的方法，避免了微型大脑模型的其余缺陷。为了进行这个概念验证，我们使用了带有荧光标签的正常tau蛋白和导致额颞叶痴呆的突变基因形式（P301S）。获得了同基因细胞系，它们在表达任何一种形式的情况下都能稳定传代超过30次。我们表明，细胞中质粒的存在不会干扰微型大脑的分化方案，并在脑类器官中获得了与病理相关的表型，tau蛋白出现病理性过度磷酸化。这样一种简单且通用的遗传策略充分发挥了人类类器官在疾病建模、个性化医疗和治疗测试方面的潜力。
