Embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocyst-embryo stage. The potential of ES cells to differentiate in all kind of tissues, opens the possibility for future promising approaches, as genetic therapy and/or cell replacement, to treat many diseases, including type1 diabetes. Although several scientific groups have reported the successful differentiation of mouse ES cells to insulin producing cells, the therapeutic benefits have yet to be achieved. Recently, in convincing experiments, it has been suggested for the first time that the adult pancreas contains “Endocrine Progenitor Cells (EPC)” that differentiate in beta cells. The lack of “signature genes”
is a main limitations to isolate and to culture pure populations of EPCs. Our study aims to find new molecular markers specific of beta cells progenitor cells. To systematically identify gene candidates as markers expressed in EPC, we will screen the enormous source of expression data from cDNA library and microarray public databases (like MIAMI, NCBI) of mouse ES cells differentiation toward insulin producing cells and definitive endoderm derivatives. The evaluation of such genes will prominently performed in vivo using various techniques (like in situ hybridization, immunostaining). To study the specification of EPCs and characterize in vitro plasticity, we aim to generate stable modified (knocked-in) mES cell lines reporting expression of new identified EPCs specific marker through GFP (green fluorescent protein). We aim to isolate EPCs homogeneous population both in vivo and in vitro by Fluorescence-Activated Cell Sorting (FACS) and Laser Capture microdissection (LCM). RNA obtained from such cells will be amplified and hybridized to Affymetrix mouse gene chip MOE430_2 to conduct a comprehensive portrait of the transcriptional profile of EPCs. Our research will provide valuable information on EPCs biology and on basic phenomena about definitive endoderm development.
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