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Combining genetic engineering and stem cells to obtain tailormade "mini-brains"

Researchers from the SEPIA (CEA)-Sup'Biotech partnership laboratory and the Gly-CRRET (UPEC) laboratory have developed a genetic tool for the long-term expression of proteins in human brain organoids. This work, published in Frontiers in Cellular Neuroscience, concerns the modelling of a genetic form of frontotemporal dementia, a neurodegenerative disease with a tauopathic side similar to Alzheimer's disease. Such a system, simple in design and versatile, opens up new potential in the use of human stem cells and organoids for in vitro modeling and personalized medicine.

Published on 18 March 2020

Due to the progressive ageing of the population and the lack of curative treatments, the number of people suffering from neurodegenerative diseases has increased considerably in recent decades and is expected to grow steadily in the coming years.

The development of new tools to model these pathologies is therefore becoming necessary to help understand their physiopathogenesis and rapidly find molecules of therapeutic interest.

Brain organoids, three-dimensional structures derived from human stem cells, are used in this context to study the development of certain neurodegenerative diseases. Recent publications have presented the relevance of this model for the study of Alzheimer's disease with the identification of specific markers of this disease. However, in order to ensure rigorous in vitro modeling, the question arises as to the use of adequate "control" samples.

Because of the multiplicity of genetic factors when comparing the cells of two individuals, it is necessary to ensure that suitable isogenic 1 controls are available.

In order to create isogenic brain organoid models, genetic editing of stem cells can be used, but with limitations in their applications. The use of retroviral vectors (i.e. lentivirus) is incompatible with the long-term modeling required for this type of study, and includes a risk of random integration into the host genome. Genetic engineering methods using CRISPR-Cas9 are still too complex and expensive to be routinely adapted to stem cells.

It is in this context that the SEPIA (CEA/François Jacob Institute of Biology) - CellTechs (Sup'Biotech) partnership laboratory, in collaboration with the Gly-CRRET laboratory (UPEC), has used an alternative genetic engineering strategy based on the use of episomal plasmid vectors to develop isogenic brain organoids (controls vs. pathological) for the study of a genetic form of frontotemporal dementia.

Frontotemporal dementia (FTD) is a group of neurodegenerative diseases characterized by behavioral and language disorders associated with intellectual deterioration. It is one of the most common neurodegenerative dementias after Alzheimer's disease and is caused by progressive changes in the frontal and temporal areas of the brain.

There are certain genetic forms of FTD, which affect several members of the same family (familial forms). Some of these are due to a mutation in the gene coding for the tau protein. The tau protein accumulates and forms toxic inclusions leading to the neuronal death. The observed tauopathy is all the more interesting as it is close to that observed in Alzheimer's disease.

By developing models of human brain organoids of FTD, it would therefore be possible to summarize the markers of the tau side of Alzheimer's disease which are :

  • synaptic loss,
  • hyperphosphorylation of the tau protein,
  • the onset of neurofibrillary degeneration,
  • neuronal death.

To do this, researchers have genetically modified human induced pluripotent stem cells using episomal plasmids derived from the Epstein-Barr virus. These plasmids allow the expression of a transgene in the cell and are capable of replicating during cell division thanks to the presence of an origin of replication on the episome, without integration into the host genome. The maintenance of the plasmid as an extra-chromosomal element in low copy is possible thanks to the EBNA-1 gene sequence, and its maintenance in the cell is achieved by selection using antibiotics in culture.

Two isogenic stem cell lines have been created from the same control line: a line overexpressing a normal form of the gene coding for the tau protein and a line overexpressing the mutated form (P301S) of the same gene.

In addition, the overexpressed tau protein (normal vs. mutated) is coupled with a fluorescent reporter to monitor the maintenance of the episomal plasmid in the cell. These lines stably express the described genetic forms for more than 30 passages.

Human pluripotent induced stem cells expressing tau protein coupled to a fluorescent reporter by means of an episomal vector, differentiated into embryoid bodies and brain organoids. (photo credit: F. Nassor)

Brain organoids were obtained from these stem cell lines, the presence of the plasmid in the cells did not interfere with the differentiation protocol.

In addition, the organoids carrying the gene mutation show a pathological tau protein hyperphosphorylation profile similar to the early pathological process observed in humans with FTD.

This new approach makes it possible to demonstrate the technical feasibility of expressing a transgene in human stem cells in a simple and efficient way without hindering the differentiation protocols necessary for the further study of pathologies. In this study, the authors were able to show that the brain organoid was able to develop the first pathological hallmarks of FTD in a complex human-based in vitro study model. This methodology could eventually be used for other pathologies to create isogenic models.

Such a system, simple in design and versatile, opens up new potential in the use of human stem cells and organoids for in vitro modelling, personalized medicine and the testing of new therapeutics.

1: In an isogenic lineage, all individuals share the same genetic heritage, making it easy to compare them with each other. Thus, when one gene is replaced by another and a new characteristic can be observed, it is possible to attribute the modification to this new gene.


This research work has been supported by the Programme Investissement d'Avenir PIA2 n° P112331-3422142 (3DNS), the European JPND program (3DMiniBrain) and the ANS foundation.

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