GENERAZIONE DI CELLULE STAMINALI PLURIPOTENTI INDOTTE E LORO CORREZIONE IN VITRO IN UN MODELLO MURINO DI OSTEOPETROSI

ABSTRACT The induced pluripotent stem cells (iPSc) entrance in the stem cell landscape has given the scientific community a novel approach for studying human diseases and a new promising tool for regenerative medicine. iPSc generation from patients affected by genetic diseases could allow their site-specific genetic correction followed by differentiation and autologous transplantation for disease cure. Infantile malignant osteopetrosis is a life-threatening recessive bone disease caused by a mutation in the TCIRG1 gene, which severely affects osteoclasts resorbing activity. The resulting increased bone density causes severe growth retardation, thickened bones, and reduced medullary cavity, symptoms recapitulated by the oc/oc mouse. Hematopoietic stem cell (HSC) transplantation is the unique possible treatment, however the chance of cure is strongly limited by the need for a matched donor. Therefore, patients should benefit from the generation of corrected autologous HSCs for a novel approach to therapy. The aim of the present thesis was to generate iPSc from murine wt and affected fibroblasts, to correct the TCIRG1 genetic mutation, to differentiate iPSc into the hematopoietic lineage including HSCs, and to transplant them in vivo to revert the oc/oc phenotype. To generate iPSc lines, as delivery system for the reprogramming genes Oct4, Sox2 and Klf4 we employed a third generation polycistronic lentiviral vector, excisable from the host genome by the Cre recombinase. After reprogramming, iPS clones with low vector copy number and normal numerical distribution of chromosomes were chosen, treated with Cre recombinase and sub-cloned to select lines without integrated vectors. Pluripotency of the obtained iPSc was tested by teratoma formation assay, embryonic germ layers in vitro differentiation, and expression of pluripotency markers through immunocytochemistry and real time PCR. Karyotype analyses showed the presence of normal sets of chromosomes. Importantly, iPSc were successfully derived from oc/oc fibroblasts, and subsequently corrected through homologous recombination upon transfection with a BAC containing wt TCIRG1. In conclusion, with our studies we will provide a proof of principle for the future clinical use of a new tool to treat osteopetrosis and potentially other genetic blood disorders.