Osteopetrotic induced pluripotent stem cells derived from patients with different disease-associated mutations by...

Autosomal recessive osteopetrosis is a genetically and phenotypically heterogeneous disease, caused by defects in osteoclast formation and function. The only available treatment is allogeneic stem cell transplantation that has still high morbidity and mortality. The goal of the present study was to generate iPSCs from bone marrow-derived MSCs of osteopetrosis patients with three most common mutations by using two different integration-free gene transfer methods and compare their efficiencies. The secondary objective was to select the most appropriate integration-free production method for our institutional iPSC bank using this rare disease as a prototype.

Two different integration-free gene transfer methods (episomal and Sendai viral vectors) were tested and compared on the same set of patient samples exhibiting three different mutations associated with osteopetrosis. Generated iPSCs were characterized by standard assays, including immunophenotyping, immunocytochemistry, RT-PCR, 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 by immunofluorescence staining. Over 90% of the cells were found positive for SSEA-4 and OCT3/4 and negative/weak positive for CD29 by flow cytometry. Immunohistochemical staining of teratoma and spontaneously differentiated embryoid body sections confirmed their trilineage differentiation potential. All iPSC lines expressed pluripotency-related genes. Karyotype analyses were found normal. Direct sequencing of PCR-amplified DNA showed that disease-related mutations were retained in the patient-specific iPSCs.

Generation of iPSC using SeV and episomal DNA vectors have several advantages over other methods like the ease of production, reliability, high efficiency, and safety, which is required for translational research. Furthermore, owing to the pluripotency and self-renewal capacity, patient-specific iPSCs seem to be ideal cell source for the modeling of a rare genetic bone disease like osteopetrosis to identify osteoclast defects, leading to clinical heterogeneity in osteopetrosis patients, especially among those with different mutations in the same gene.