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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.
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
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 :
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.
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
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.
CEA is a French government-funded technological research organisation in four main areas: low-carbon energies, defense and security, information technologies and health technologies. A prominent player in the European Research Area, it is involved in setting up collaborative projects with many partners around the world.