Analysis of LCL-iPSCs for reprogramming and viral elements and trilineage differentiation of LCL-iPSCs. (A) RT-PCR analysis of H1 (hESC line), LCL-1, LCL-2, and 2 representative LCL-iPSC clones from each donor line at passage 25 for expression of EBV genes: EBNA-1, EBNA-2, LMP-2A, and BZLF-1. GAPDH was used as a positive loading control for each sample, and cDNA made in the absence of reverse transcriptase (no RT) was used to verify that genomic DNA did not contaminate the RNA samples. (B) Immunohistochemistry was performed on LCL-1, LCL-2, and 2 representative LCL-iPS clones from each donor line using an anti–EBNA-1 antibody to detect EBNA-1 protein expression (brown). Images were captured using original magnification ×100. (C) EBV loss during reprogramming is confirmed by quantitative PCR. The viral load during reprogramming was evaluated by harvesting genomic DNA harvested at various passages during the reprogramming. Quantitative PCR was performed using primers, and TaqMan probes targeted 3 EBV gene segments: BamH1W, EBNA-1, and EBER1. The human APOB gene was used for normalization, and the loss of EBNA-1 (i), EBER1 (ii), or BamH1W (iii) was fitted to a general exponential function y = b*fn, where y refers to the copies of viral elements (EBNA-1, EBER1, or BamH1W) per cell for passage n, f is the fraction of daughter cells that contain the virus per passage, and both b and f were chosen to fit the data. (D) H&E staining of teratomas derived from immunodeficient mice injected with LCL-iPS2a shows tissues representing all 3 embryonic germ layers: endoderm (goblet cells, left panel), ectoderm (neural rosettes, middle panel), and mesoderm (cartilage, right panel). All H&E images were captured using original magnification ×40. (E) Trilineage in vitro differentiation. (i) Quantification of α1-antitrypsin-expressing hepatocyte precursors derived from LCL-iPSCs at day 33 by flow cytometry. (ii) Quantification of cardiac troponin T (cTNT)-positive cardiomyocytes derived from LCL-iPSCs at day 14 by flow cytometry. (iii) Neural cultures from LCL-iPS2b immunostained for the presence of β3-tubulin (original magnification × 20). (iv) Quantification of β3-tubulin and nestin-coexpressing neural precursors from neural induced LCL-iPSCs by flow cytometry. (v) Megacult cultures of HPCs derived from LCL-iPS2a cells stained for the presence of megakaryocytes and proplatelets (original magnification ×40; left panel). HPC-derived LCL-iPS2b expanded in the presence of granulocyte-macrophage colony-stimulating factor and stained for the presence of monocytes, neutrophils, and macrophages (original magnification ×20; right panel). (vi) Quantification of colony-forming units (CFU) to demonstrate the presence of erythroid (CFU-E/BFU-E), myeloid composing macrophage (CFU-M), granulocyte (CFU-G), and granulocyte-macrophage (CFU-GM), and multipotent composing granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) colonies from all 4 LCL-iPSCs. (vii) In vitro differentiation into LCL-iPSCs to cells to HPCs via a defined serum-free embryoid body differentiation protocol for 12 days. The percentage of single-positive CD34, CD45, CD43, CD41, and CD235a and double-positive CD34/CD43, CD45/CD43, and CD34/45 cells was quantified by flow cytometry. All images were captured on a Olympus 1×71 microscope equipped with a Olympus DP70 camera and DP Controller Version 3 software
