Pathological modelling iPS
Team:
Mathilde Girard : Research associate (CECS)
Cécile Denis : Associate engineer (CECS)
Laure Chatrousse : Research engineer (INSERM)
Laetitia Aubry : Assistant Professor (UEVE)
Benjamin Forêt : Technician
Marie André : Ph Student
Aims and background :
Induced Pluripotent Stem (iPS) cells harbour the same characteristics and capabilities as embryonic stem cells without carrying their ethical constraints. They offer the opportunity on the one hand to model diseases for which there are no available embryonic stem cell lines, and on the other hand to represent a new source for cellular therapy.
The iPS cells pathological modeling team was created in order to develop two axes on iPS cell research:
Optimization and standardisation of the reprogramming process, and quality control of the iPS cell clones
The objective is to develop robust protocols for the transduction of somatic cells, the efficient reprogramming of these cells into iPS cells, and the quality control of the iPS cell lines in term of stability and pluripotence.
Pharmaceutical screening on iPS cell lines The objective is to develop tools for screening on pathological models presenting markers that are particularly suitable for screening. These pathologies include for example metabolic diseases, enzymatic defects, and mitochondrial pathologies, which in all cases present markers of the pathology that can be detected by biochemical processes adapted to high throughput screening. The proof of concept of this strategy will be done on the Lesch-Nyhan syndrome and on the Leigh syndrome, for which iPS cells are currently being derived and characterized. Strategy, means and methods :
Technology development on iPS cells
We develop new technologies acting on several aspects of the reprogramming process:
- The origin of the reprogrammed cells,
- The way of delivery of the reprogramming factors,
- The follow-up of the reprogramming process and identification of emerging clones,
- The quality control of the iPS cell clones.
Pharmaceutical screening on iPS cell lines
The strategy is to:
- Reprogram patients cell into iPS cells and make quality control of the obtained clones,
- Differentiate iPS cells into cell types relevant for the pathology and for the screening,
- Perform high throughput screening on pharmaceutical libraries,
- Confirm and validate obtained hits by cellular and molecular protocols,
- Initiate the transfer for clinical application.
Pathological modelling and drug screening on iPS cells
Results and future prospects :
We developed a robust protocol for the reprogramming of fibroblasts, myoblasts and keratinocytes, allowing the reprogramming of cells from more than 15 different donors. We used iPSC as a tool for pathological modelling by reprogramming cells carrying mutations associated with 13 different pathologies, including Myotonic Dystrophy type 1 (DM1), Spinal Muscular Atrophy (SMA) and Hutchinson-Guilford Progeria Syndrome (HGPS). We also developed a stringent quality control of the iPS cells including control of genetic stability, expression of stemness markers, and pluripotency.
Quality control of iPS cell clones. Upper left: morphology of an iPS cell colony. Upper right: normal karyotype of an iPS cell line. Lower left: staining of an iPS cell colony with stemness markers Oct-4 and TRA-1-60. Lower right: pluripotency assessment by embryoid bodies formation from iPS cells.
Collaborations :
Dr. Vincent Petit and Dr. Marc Sitbon, IGMM, Montpellier, France, (development of new stem cell markers)
Dr. Philippe Mangeot and Dr. Vincent Lotteau, INSERM U851, Lyon, France, (protein transfer by engineered microvesicles)
Dr. Nicolas Fortunel and Dr. Michèle Martin, LGRK CEA, Evry, France, (reprogramming on progenitor keratinocytes)
Dr. Yohann Moal and Dr. Pascale Bouillé, Vectalys, Toulouse, France, (non integrative reprogramming vectors)
Dr. Philippe Mangeot and Dr. Vincent Lotteau, INSERM U851, Lyon, France, (protein transfer by engineered microvesicles)
Dr. Nicolas Fortunel and Dr. Michèle Martin, LGRK CEA, Evry, France, (reprogramming on progenitor keratinocytes)
Dr. Yohann Moal and Dr. Pascale Bouillé, Vectalys, Toulouse, France, (non integrative reprogramming vectors)
