Long Term Gene Expression in Human Induced Pluripotent Stem Cells and Cerebral Organoids to Model a Neurodegenerative Disease

Ferid Nassor,Frank Yates,Dulce Papy-Garcia,Denis S F Biard,Jean-Philippe Deslys,Auriane Maïza,Serena Pavoni,Rafika Jarray

doi:10.3389/fncel.2020.00014

Abstract

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.

Highlights

The prevalence of neurodegenerative diseases has amplified continuously over the years, notably due to an increasingly elderly population, and has become a major concern for many facets of society
A different approach applicable to stem cells has been described by several authors with the use of episomal plasmid vectors derived from the Epstein-Barr virus (EBV; Ren et al, 2006; Thyagarajan et al, 2009)
We developed a model based on a 4R tauopathy, the fronto-temporal dementia, which can be triggered with a mutation in the exon 10 of the MAPT gene, resulting in the conversion of a proline in a serine at the amino acid 301 in the tau protein (P301S; Hutton et al, 1998; Bugiani et al, 1999)

Summary

Introduction

The prevalence of neurodegenerative diseases has amplified continuously over the years, notably due to an increasingly elderly population, and has become a major concern for many facets of society. Gene editing techniques based on CRISPR-Cas in human stem cells (embryonic or induced pluripotent), have been applied to create isogenic cell lines by adding or correcting a mutation (Grobarczyk et al, 2015; Li et al, 2015; Paquet et al, 2016). This novel approach circumvents the difficulty of finding isogenic controls, but the method requires both time and major resources, preventing a wide application of this technology. Existing approaches using EBV-based plasmids have been limited so far to the establishment of proofs of concept in stem cell lines, expressing fluorescent proteins, and do not show long term modeling approaches or validation for disease modeling

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Results

Conclusion