A simplified approach for derivation of induced pluripotent stem cells from Epstein-Barr virus immortalized...

Given the limited availability of tissue, especially brain tissue, for neurological diseases and disorders research, the development of alternative biological tools for investigations of underlying molecular and genetic mechanisms is imperative. One important resource for this task is the large repositories that bank immortalized blood cells (i.e. lymphoblastoid cell lines; LCLs) from affected individuals and their unaffected family members. These repositories document demographic, phenotypic, and, in some cases, genotypic information about the donors and thus provide a ready-made sample source for hypothesis testing. Importantly, patient-specific LCLs can be used to generate induced pluripotent stem cells (iPSC) that, in turn, can be used to create specific cell types for use in mechanistic studies. To investigate this concept further, LCLs from two males (proband and sibling) were obtained from one such repository, the Autism Genetics Resource Exchange (AGRE), and iPSCs were generated by transfection with Epi5 Episomal iPSC reprogramming plasmids. Characterization of the resultant cell lines by PCR, RT-PCR, immunocytochemistry, karyotyping, and the Taqman® human pluripotent stem cell Scorecard™ Panel, was used to provide evidence of endogenous pluripotency and then to evaluate the trilineage potential of four representative clones. Results indicated that all four iPSC lines were initially pluripotent and displayed the trilineage potential predictive for successful differentiation to mesoderm, endoderm, or ectoderm-derived cell types. Compared to other published protocols, this study details a somewhat simplified approach, used here specifically for the generation and characterization of induced pluripotent stem cells from well-characterized and banked LCLs.