Figure 2.

Characterization of LCL-derived iPSCs. (A) PCR-based detection of episomal plasmids in clonal iPSC lines derived from EBV-LCLs at passage 16 (P16). Total DNA was isolated from two clonal iPSC lines derived from each of two parent LCLs (Line 1 & Line 2) at multiple passages using the DNeasy Blood & Tissue kit. A 665bp PCR product is present in the agarose gel if any of the plasmids are still present in the cells. Plasmid DNA from the reprogramming kit was used as the positive control (+); the negative control had nuclease-free water in the place of DNA (-). β-actin was used as a positive loading control for each sample. (B) RT-PCR analysis of one LCL and two representative LCL-iPSC clones from each LCL at passage 18 for expression of EBV genes: EBNA-2, BZLF-1, and LMP-2. β-actin was used as a positive loading control for each sample. Negative control for each primer set consisted of using nuclease-free water in place of cDNA. Results from a single LCL is displayed, however both LCLs tested positive for all EBV genes and B-actin. (C) Validation of iPSC pluripotency by PCR. RT-PCR analysis of original LCLs and two representative LCL-iPSC clones from each LCL at passage 18 for expression of pluripotency genes: Oct3/4, Nanog, DMNT3B and PODXL. β-actin was used as a positive loading control for each sample. Negative control for each primer set consisted of using nuclease-free water in place of cDNA (-). (D) Validation of iPSC pluripotency by immunocytochemistry on passage 23 LCL-derived IPSCs. ICC was conducted on LCL-derived iPSCs following reprogramming with Epi5™ episomal reprogramming plasmids and propagation to passage 23 (i & v, brightfield). Extracellular markers for Tra-1-60 (ii) and SSEA4 (vi) appear green. Nanog was used for intracellular staining and appear red (iii, vii). Pairings of Tra-1-60/Nanog and SSEA4/Nanog indicate proper cell localization of these markers (iv, viii). All images were taken at 40X. Scale bars are 200μm.