Autosomal Recessive Osteopetrosis caused by mutations in the TCIRG1 gene, is a severe bone disorder characterised by bone marrow fibrosis and consequent pancytopenia, multiple spontaneous fractures, blindness and hearing loss. The oc/oc mouse well recapitulates the clinical signs of the disease. To date, haematopoietic stem cell (HSC) transplantation is the unique possible treatment, however the chance of cure is limited by the need for a matched donor. With the final aim to exploit novel therapeutic strategies allowing the use of corrected autologous HSC, we evaluated the feasibility and potentiality of induced pluripotent stem cells (iPSc), as alternative and unlimited source of autologous stem cells. To this end, we reprogrammed murine wild-type (wt) and oc/oc fibroblasts into iPSc, to genetically correct the Tcirg1 mutation by homologous recombination, and to generate haematopoietic stem and progenitor cells able to give rise to functional osteoclasts. We employed a third generation polycistronic lentiviral vector carrying the reprogramming genes Oct4, Sox2 and Klf4, subsequently excisable by the Cre recombinase. After reprogramming, iPS clones with low vector copy number and normal numerical distribution of chromosomes were treated with Cre and sub-cloned. Obtained iPSc showed normal karyotype and pluripotency tested by teratoma formation assay, in vitro embryonic germ layers differentiation, and expression of stemness markers by immunocytochemistry and RT-PCR. Importantly, iPSc were successfully derived from oc/oc fibroblasts, and then corrected through homologous recombination upon transfection with a BAC containing the wt gene. iPSc were guided to differentiate towards haematopoietic belonging to different lineages. We obtained differentiation towards osteoclasts, the relevant cells in our model, which were functional as demonstrated by the dentine resorption assay. In conclusion, we provided the first evidence of targeted gene correction in osteopetrotic iPSc, supporting the rationale of using iPSc as future source of donor cells in the clinical setting.
Disclosure: The authors declared no competing interests. The research has received funding from the FP7 n.602300 (SYBIL), PRIN Project 20102M7T8X_003,and RF-2009-1499,542 to A.V.