Generation of Induced Pluripotent Stem Cells From the Peripheral Blood of a Pyruvate Kinase Decifient Patient

Abstract 4713

The generation of induced pluripotent stem cells (IPSCs) from patients with specific diseases has improved the possibilities of dissecting the etiology of human pathological conditions and has opened potential new therapeutic options for the treatment of human diseases. One of the major safety concerns in the generation of hiPSCs is the persistence of copies of the reprogramming genes in the genome of iPSCs. Additionally, the use of cells from accessible tissues, like peripheral blood, constitutes preferential options for cell reprogramming, compared to other cells whose collection would require a more aggressive intervention. Here we have selected Pyruvate Kinase Deficiency (PKD) as a human disease affordable to be modeled and genetically corrected using hiPSC technology. PKD is an autosomal recessive disease that affects the erythrocyte R-type Pyruvate Kinase (RPK) enzyme by mutations in the PKLR gene, causing chronic nonspherocytic hemolytic anemia. Deficiency in the RPK, the last enzyme of the glycolisis, reduces the energetic balance of red blood cells and therefore affects the viability and function of the erythrocytes. Clinical symptoms of PKD can progress from mild to severe anemia and can be lethal in the most severe cases. Peripheral blood mononuclear cells (PB-MNC) from healthy donors and from a PKD patient were infected with five independent recombinant non integrative RNA Sendai viruses (SeV) encoding four human reprogramming factors (OCT3/4, SOX2, KLF4, c-MYC) and the Azami green protein as reporter. After infection with SeVs, no differences in the reprogramming efficiency of healthy and PKD PB-MNC were observed (1 hES-like colony per 6,000 transduced healthy or PKD PB-MNC). Integrated SeVs were not found in PBMNC-hiPS lines, as deduced from the lost of Azami green expression and also from RT-PCR analyses of the reprogramming factors. Healthy and PKD PBMNC-hiPSCs could be cultured for more than 15 passages, indicating their self-renewal ability. The pluripotent characteristics of these PBMNC-hiPS were confirmed by the analysis of expression of endogenous pluripotent genes by Q-RT-PCR and immunofluorescence. This study confirms the feasibility to generate integration-free hiPS lines from healthy and PKD PB-MNC. These hiPSC lines constitute an invaluable tool for the development of a human model of PKD and for applying new gene therapy strategies.