Neuromuscular diseases

 

 

The global objective of our project is to develop new therapeutic strategies for the cure of neuromuscular diseases.
This research program is based on the original scientific concept that human embryonic stem cell lines (hES) which express a disease-related mutant gene could represent a relevant cellular model allowing analytic and therapeutic research for the disease. Thus, pathological modelling using hES cell lines should provide a valuable in vitro tool to understand the molecular mechanisms of monogenic diseases and then, to investigate the efficacy of drug therapies.
To evaluate the feasibility and validate this scientific concept, we took advantage of the existence of three available derived hES cell lines which express the mutant gene causing the Myotonic Dystrophy type 1 (DM1) or Steinert disease. By using these hES cell lines, we are working out the molecular mechanisms implicated in the development of this pathology and identify potential therapeutic compounds.

The experimental approach is based on the comparison of normal and mutated hES cell lines, differentiated and not, in order to identify biomarkers, defined by a quality or quantity expression modified specifically by the presence of the mutation. The analysis of these laters should lead to the obtention of instructive phenotypes.

We hypothesize that biomarkers specifically related to the mutation will most likely be identifiable in cells that have reached a certain stage of differentiation. Although, in the case of DM1, various cell types are affected by the mutation, we decided to focus on two of them mostly affected by the disease, the nervous system and the skeletal muscle. However, as transcriptomic studies require the use of a homogeneous cell population, these differentiation protocols need to be adapted in order to obtain near-homogeneous cell populations.

We currently develop protocols for differentiation of hES cells into neural fates. Neural precursors were chosen as homogenous cell populations which can be obtained relatively easily from hES cell lines.
In parallel, the specification of hES cells towards mesodermal lineage was developed which allows the obtention, after 21 days under specific culture condition, of mesodermal progenitors with a phenotype of mesenchymal stem cells (MSC).

Our results validate the model by demonstrating that the cell progeny of an ES cell line derived from an embryo with DM1 displayed two of the features observed in DM1 patients.
One is the presence the nuclear foci containing the mutated mRNA in both the neural and mesodermal progenitors as illustrated below. The other one is the identification of abnormal alternate splicing of the insulin receptor (INSR).
The obtention of cell populations at near-homogeneity allows to perform a large-scale transcriptomic approach which should identify genes with an altered expression in DM1 compared to control cells. The differential transcriptomic analysis revealed abnormal expression of 89 genes, among which 48 were down-regulated and 39 over-expressed in DM1 cells. Among the differentially modulated genes, some bear particular relevance to the known molecular and clinical features of the disease. Two genes, the already mentioned INSR and SERCA2/ATPA2 (sarcoplasmic/endoplasmic reticulum CA2+ ATPase 2), belong to the genes in which abnormal mRNA splicing has been associated to DM1. Among the down-regulated genes, some may impact heavily either the formation of the neuromuscular junction (see next page) or the development of skeletal muscles. Further analysis of abnormally expressed genes may lead to the identification of new pathological mechanisms.