Reprogramming human fibroblasts – CELL REPROG

The ordinary fate of a cell is differentiation with concomitant loss of potency, and later in the cell life, ageing with the biological stigma of senescence. Differentiation and senescence are both associated with profound modifications in chromatin organization and replication timing. Somatic cell nuclear transplantation (SNCT) has indisputably demonstrated that cell differentiation is a process that can be reversed by the oocyte cytoplasm. This cell fate modification is called cell reprogramming. We propose that cell reprogramming might in fact as well reverse senescence. Thus cell reprogramming would be at the center of two major stakes for medicine: (1) dedifferentiation of any adult cell to yield versatile drug cells, and (2) rejuvenate cells of prematurely aged organs. Alternative strategies to SNCT have shown very encouraging results. Somatic cells can be reprogrammed by transient permeabilization followed by application of Xenopus egg or ES cell extract. In this setting, Lemaître et al. recently showed that prior mitotic remodeling is essential for reprogramming differentiated nuclei for DNA replication, and thus likely for dedifferentiation toward a proliferating pluripotent state. Another strategy is the simultaneous expression of several key transcription factors (OCT3/4, SOX2, CMYC and KLF4) to dedifferentiate mice embryonic or adult fibroblasts towards a pluripotent stem cell fate. All these results have not been reproduced in human cells yet. Our aim is to develop and evaluate strategies to modify the fate of a fully differentiated human cell. We will study the human fibroblast as a paradigm of a somatic differentiated cell and our objectives are to (1) reverse senescence (LEMAITRE TEAM), (2) dedifferentiate human fibroblasts towards pluripotency (DE VOS TEAM). To this end, we will reprogram normal and senescent human fibroblasts either with human embryonic stem cell extracts on permeabilized fibroblasts samples, or expression of selected human transcription factors. We will assess the extent of the reprogramming first by simple morphological, immunofluorescence, flow cytometry, QPCR on the reprogrammed cells. A selection procedure to be able to select a rare reprogramming event by expressing EGFP or a neo cassette under the human POU5F1/OCT3/4 promoter will also be used. The reprogrammed fibroblasts clones will be further evaluated by high definition transcriptome analysis (Exon ST 1.0 microarrays), replication analysis by DNA combing, chromatin modifications by ChIP chip and finally by simple differentiation capacities evaluation. Of note, we will study whether imposing dedifferentiation constraints on a senescent cell erases senescence marks. Finally, we will identify the cell extract fraction that is effective in reprogramming (LEHMANN TEAM). This project is based on our complementary skills in stem cells biology, cell extract based reprogramming, lentiviral vector construction and handling, as well as replication, transcriptome, and proteome analysis. Our three teams are part of three large Research Institutes (Institut de Génomique Fonctionnelle, Institut de Génétique Humaine and Institut de Recherche en Biothérapie) and our project will be backed up by the very large skills and resources available in these Institutes. Furthermore, we already have a large body of preliminary tools and results such as the reversing of senescence with Xenopus egg extracts, several lentiviruses expressing pluripotency transcription factors and a collection of very high definition transcriptome of human samples including hESC and fibroblasts. We have the authorization to handle human embryonic stem cells. To our knowledge, this research program is the first one to combine these approaches and study simultaneously genome plasticity in the reversal of cellular senescence and cellular differentiation. Overall, our three teams have access to all the skills and tools to successfully step into this international competition in which France must me present.