French National Research Agency

Several teams of I-Stem joined industrial partners – research contract RIB – and French academic teams to address call for proposals from the National Research Agency. A good number of these requests were successful, making ANR an essential institutional financer, in particular for consumables and temporary recruitment.


ANR program “Blanc” 2011


Induced Pluripotent Stem cells for engineering osteo‑articular tissues.

Program summary:

The treatment of osteoarticular pathologies by tissue engineering faces some hurdles. The cells currently used (chondrocytes, osteocytes, or mesenchymal stem cells (MSC)) have shown the clinical feasibility of this treatment for certain osteoarticular disorders, however they also showed some limitations. Indeed, these cells have a limited potential of proliferation and differentiation. The ability to reprogram adult somatic cells to obtain iPS cells with the cardinal properties of embryonic stem cells: unlimited capacity of self‐renewal and possibility to differentiate in any cell types forming an organism, opens new perspectives for tissue engineering development.

This project proposes to explore the human iPS cells potentials for tissue engineering. However, currently, the production of iPS is largely dependent on the use of recombinant defective viruses that require integration into the host cell genome, which may cause potential drift towards a neoplastic phenotype. Therefore, based on the skills of the partners of this project (osteoarticular engineering, gene transfer, production of iPS), we propose both to develop a nonviral protocol for production of iPS and also to validate the use of iPS cells in the case of osteoarticular tissue engineering.

Electrogenetransfer (EGT, also called electroporation), a method of choice for nonviral gene transfer using modulated electrical pulses (intensity / duration / frequency) will be optimized for non viral iPS generation. Preliminary results have shown the feasibility of this approach, and the need for refinement of the method. The potential of iPS cells for osteoarticular tissue engineering will be assessed both in vitro and in vivo. Special focus will be put on development of the protocols for osteoarticular differentiation of the iPS cells and safety of the overall procedure for future clinical applications, as these are the two main areas where iPS are still lacking. This project will allow:

1) to define optimized protocols for non viral production of iPS cells

2) to define conditions of automated production of iPS cells and to built a bank of qualified iPS cells

3) to define protocols to induce efficient differentiation of iPS cells to treat osteo‐articular disorders

4) to validate differentiated cells derived from iPS for osteoarticular repair in preclinical models




Program summary: 

Huntington’s disease (HD) is a devastating monogenic disease. There is no known treatment for this pathology whose symptoms include progressive motor, psychiatric and cognitive dysfunctions, associated with a massive degeneration of the striatal medium size spiny GABA neurons. Most of HD patients die within 15-18 years after the onset of symptoms. Recent clinical findings have shown that HD would partially respond to treatment by substitutive cell therapy using grafts of fetal origin. However, this grafting technique is limited by logistic and ethical problems that restrict considerably the number of patients that may benefit from it. Potent alternative sources of cells that would be easily accessible to surgeons are therefore urgently needed. Finding such cells is a crucial step towards the development and validation of an efficient clinical application in patients. Because of their extended differentiation potential and their unlimited capacity to self-renew, human pluripotent stem cells are in theory the best candidate cells to be grafted. In fact, we have recently demonstrated that human and monkey embryonic stem cell differentiation can be directed towards striatal neurons both in vitro and in vivo after transplantation in HD rats. This suggests a possible therapeutic potential of hESC for HD. However, a thourough pre-clinical assessment of such pluripotent stem cell-derived grafts is required to ascertain the applicability of this therapeutic strategy in HD patients.

The goal of the HD-SCT (HD-Stem Cell Therapy) proposal is to build on recent achievements in stem cell biology and clinical trials using fetal neural cells in HD patients to evaluate the therapeutic potential of pluripotent stem cells in a pre-clinical setup (allograft) and to develop the protocols needed for the establishment of banks of clinical-grade, ready-to-use, and safe batch striatal progenitor cells.

Fetal graft therapy currently used in HD clinical trials across Europe has greatly benefitted from the results obtained using primate models of HD. Since human striatal grafts mature too slowly to be appropriately assessed in rats, allografts in HD monkeys was the model of choice. Moreover, motor and cognitive behavioral tests are more relevant in primate models than in rodents. In addition, allografting better mimics “clinical transplantation” scenario with regard to immune response of the host to the graft. Accordingly, the first aim of HD-SCT is to assess the therapeutic potential of striatal cells derived from monkey-ESs and iPSCs in a monkey model of HD. Neuro-imaging will be used to monitor graft survival and cell maturation and the potential correction of cortical hypometabolism through striatal repopulation. Behavioral tests aimed at quantifying motor and cognitive deficits reminiscent HD pathology will be used to measure the potential benefit associated with stem cell transplantation. Current differentiation protocols that rely on mouse feeder cells or serum containing media are not compatible with future clinical application. Consequently, HD-SCT’s second aim is to adapt current research-grade differentiation protocols to clinical-grade standard and address the issue of the banking of stem cell derived striatal graft. Furthermore, HD-SCT will address the issue of the generation of safe transgenic pluripotent stem cell lines. Accordingly, HD-SCT’s third aim is to test “meganuclease-driven homologous recombination” technology as a way to produce safer transgenic pluripotent stem cell lines for human use.

HD-SCT expected impact is ultimately to accelerate the development of clinical applications of human pluripotent stem cells for the treatment of Huntington’s disease. To achieve this, HD-SCT relies on a small consortium of three experienced partners in the field of stem cell biology and its pre-clinical/ clinical application, with balanced and complementary skills highly relevant to the subject of the proposal.

Coordinator: Anselme Perrier (I-Stem)



Generation of induced-pluripotent stem (iPS) cells from normal and retinitis pigmentosa patients for phenotype screening of photoreceptor differentiation and studying photoreceptor disease mechanisms.

Program summary:

The retina is part of the central nervous system and contains specialized neurons, photoreceptors that convert light signals into electric signals, further transmitted to the brain by different neurons. Any defect involved in these processes of phototransduction and transmission in the retina lead to visual impairment. Currently, retinal diseases are a major cause of inevitable blindness worldwide and inherited retinal degeneration, particularly retinitis pigmentosa (RP) associated with the loss of photoreceptors, is the leading cause of inherited blindness or visual impairment in the young- adult population. While clinical trials based on gene therapy are imminent for few genetically characterized sub-groups of patients, the overall genetic heterogeneity implies that diverse therapeutic approaches need to be developed. An alternative approach would be to transplant replacement cells thereby returning visual function to the retina. In this context, the recent discovery that is possible to directly reprogram somatic cells to an embryonic stem (ES) cell-like pluripotent state, known as induced pluripotent stem (iPS) cells, offer a great potential use in regenerative medicine. Furthermore, the generation of iPS cells from an individual RP patient would enable the large-scale production of the cell types affected by the patient’s disease. Although traditional cell replacement remains a central goal in applied stem cell research, the derivation of patient-specific iPs cells might be equally useful for disease-related research. Indeed, these cells could in turn be used for disease modelling and drug discovery.

The expected impact of this proposal will be to develop pre-clinical studies required for the development of an iPS cell-derived cellular therapy for the treatment of a number of retinal diseases. We will attempt (i) to reprogram keratinocytes of healthy and RP patients into iPS cells, (ii) to develop specific iPS reporter cell lines allowing the efficient generation and isolation of photoreceptor precursors from iPS cells and (iii) to study photoreceptor dystrophy mechanisms using mutated iPS cells from RP patients. We anticipate that the generation of novel iPS cell-based models of disease will allow us to narrow the gap between pre-clinical and clinical observations.




The “safety switch”: ensuring post-grafting safety riddance of cell therapy products with the TK-suicide gene strategy

Program summary:

The cellular therapy is currently a therapeutic approach in the course of evaluation in a number of pathologies unceasingly growing (neurodegenerative diseases, muscular infarctions, cancer,…) This approach rests on the replacement of non functional cells by relevant exogenous cells. However, whatever the cell type considered, a constant risk exists that the cells injected exert a deleterious effect on the grafted patient. There is no procedure to date allowing elimination of these cells in vivo, in the event of a major side effect. This is the principal safety obstacle with the development of cell therapy and, conversely, potentially represents the possibility of an access to a considerable market. This is the principal objective of this research program. The system suggested rests on a genetic engineering of cells before their injection allowing the expression of a gene in each therapeutic cell that would allow them to “commit suicide” and the possibility of eliminating them “at will” in vivo. LTKfarma, the leader of the project, has exclusive rights of exploitation for the TK suicide gene system, in partnership with the University Paris 6 via 31 patents in Europe, the United States and Japan. Its ambition is to become a world leader in the security of cell therapy.

Coordinator: Laurent de Narbonne (LTKfarma)

Partners: José Cohen (CNRS), Philippe Hantraye (CEA), Anselme Perrier (I-Stem)



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