Abstract 579: De-differentiation of Somatic Fibroblasts by Mouse Embryonic Stem Cell-free Extracts: Multi-lineage Re-differentiation and Therapeutic Efficacy of Re-programmed Cells in Mouse Models of Hind limb and Acute Myocardial Ischemia
Background: De-differentiation of adult somatic cells into multipotent progenitor cells might provide an attractive, oocyte-independent alternate source for therapeutic cloning to generate pluripotent, autologous stem cells for regenerative medicine. We tested the hypothesis that exposure of reversibly permeabilized NIH3T3 fibroblasts to mouse embryonic stem cell (mES) extracts may provide regulatory molecules required for epigenetic changes leading to re-programming of terminally differentiated somatic cells and that such reprogrammed cells may display multi-lineage differentiation.
Methods and Results: Streptolysin O-permeabilized NIH3T3 cells were exposed to mESC extracts in ATP-regenerating system. Plasma membrane was resealed by culturing in medium 2mM CaCl2. We report that upon exposure to mES extracts 3T3 cells undergo dedifferentiation as evident from morphological changes, induction of mES specific and the loss of lamin A/C, a specific marker of the soma. At epigenetic level, mES extract treatment induced de-methylation of Oct4 promoter and histone3 hyperacetylation at lysine 9 residue, as evident from restriction enzyme mapping, bisulphite genomic sequencing and chromatin immuno-precipitation experiments. Under defined culture conditions in vitro, reprogrammed 3T3 cells can be differentiated into neuronal, adipocyte, cardiomyocyte (CMC) and endothelial cells (EC). Moreover, transplantation of labeled reprogrammed 3T3 cells into mouse hind limb ischemia and acute myocardial infarction models resulted in the gain of physiological functions and showed evidence of acquiring the expression of muscle, EC and CMC specific proteins, in vivo. Moreover, when injected in SCID mice, reprogrammed cells formed teratomas.
Conclusion: Taken together our biochemical, molecular and functional data provide a novel and oocyte- independent approach for the generation of functional autologous stem like cells from terminally differentiated somatic cells which may potentially be of therapeutic application in regenerative medicine.