Generation of induced pluripotent stem cells from mesenchymal stem cells of osteopetrosis patients by two different integration-free methods under feeder-free conditions

Aim of this study is to generate induced pluripotent stem cells (iPSC) from bone marrow-derived mesencyhmal stem cells (BM-MSC) of children with malignant infantile osteopetrosis (MIOP), a rare autosomal recessive disorder caused by defects in osteoclast formation/function, and establish an institutional human iPSC bank by using MIOP as a prototype disease. Osteopetrotic and healthy donor BM-MSCs which were used as control were reprogrammed by using CytoTune-IPS 2.0 Sendai (SeV) and Epi5 Episomal reprogramming kits according to the given instructions. iPSC colonies which started to appear around day 18 for SeV and day 21 for episomal vector were picked, manually passaged and expanded in matrigel-coated plates. Assesment of colony morphology, immunofluorescence (IF) staining and flow cytometry besides live staining with alkaline phosphatase were performed for characterization (≥3 clones/line). Expression of pluripotency genes, detection of SeV and episomal vectors were evaluated with PCR. In vitro and in vivo pluripotency were assessed by embryoid body and teratoma assays. Karyotype analyses were performed to evaluate genetic stability. iPSC lines exhibiting typical ESC-like colony morphology were shown to express pluripotency markers (OCT4, SSEA-4, SOX2,TRA-1- 60) by IF staining. 85–95% of the cells were found positive for SSEA-4 and Oct3/4 and negative for CD29 with flow cytometry. Immunohistochemical staining of teratoma sections confirmed the trilineage differentiation potential. All iPSC lines expressed pluripotency related genes (Nanog, DNMT3B, CDH1, UTF1, REX1, TERT). Loss of SeV genome was observed at late passages. Karyotype analyses were found normal. Analysis of DNA methylation profile is in progress. In conclusion, iPSC were succesfully derived from osteopetrotic BM-MSC using two different methods. Generation of iPSC using non-integrating SeV and episomal vectors have several advantages over other methods such as ease of production, reliability, high efficiency and safety profile, which will be important in translation into clinic. Furthermore patient-spesific iPSC because of pluripotency and self renewal capacity seem to be ideal for disease modeling and investigate new therapeutic targets which will pave the way for the development of cellular replacement therapies.