Schematic of PCR strategy for template preparation for 454 sequencing of L1Hs family members (adapted from Ewing et al) [33].

<p>L1Hs libraries were prepared as previously described, except that the 454 primers A and B were used instead of Illumina adapters and that high throughput sequencing was performed by using the primer A instead of the primer B, thus allowing the detection of the polyA (pA) sequence followed by the sequence of the new locus of insertion. The sequences were then processed for mapping on the genome to detect reference as well as non-reference L1Hs insertions. L1Hs reference insertion sequences would match the reference genome from their 3′UTR sequence to the end of their flanking sequence in one location only while non-reference insertion sequences will have their 3′UTR sequence and flanking sequence match the genome on two distinct locations.</p

L1 transcriptional up-regulation in human iPSC clones is independent of donors.

<p>L1 expression was evaluated by quantitative real-time RT-PCR on total RNA extracted from iPSC clones derived from (A) NHDF1 (B) HFF (C) IMR90 cell line. To evaluate the respective basal level of L1 expression, total RNA extracts from the respective parental cells were subjected to real-time PCR. Real-time RT-PCR results were normalized with respect to GAPDH content. Fold increase of L1 expression was then calculated with respect to the result obtained from the parental cells. Results are shown as average ± standard deviation. RNA extracts from the H1 human embryonic stem cell line was used a positive control. Asterisks denote statistical significant increase in L1 expression when compared to the reference parental cells as assessed by the Wilcoxon rank sum test (p<0.05).</p

L1 up-regulation is observed during the reprogramming process and is independent of the transducing vector.

<p>HFF were transduced with either the FRh11 lentiviral vector or the pMX murine γ-retroviral vector encoding <i>OCT4, C-MYC, SOX2</i> and <i>KLF4</i>. Three days post-transduction, the cells were then seeded onto a feeder layer of iMEFs and cultured under hESC conditions. Total cells were collected at 8, 14, 21 and 28 days post-seeding and iMEFs were removed by positive selection. Total RNA extracts were obtained from the remaining human cells which were then subjected to quantitative real-time RT-PCR to assess L1 expression. Total RNA extracts obtained from H1-hESC and iMEFs were used as positive and negative controls, respectively. Quantitative real-time RT-PCR results were normalized with respect to GAPDH content. Fold increase of L1 expression was then calculated with respect to the results of HFF. Results are shown as average ± standard deviation. Asterisks denote statistical significant increase in L1 expression when compared to the reference parental cells as assessed by the Wilcoxon rank sum test (p<0.05).</p

Deep Sequencing Reveals Low Incidence of Endogenous LINE-1 Retrotransposition in Human Induced Pluripotent Stem Cells

<div><p>Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3′ end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.</p></div

Summary of the germline insertion results.

<p>Summary of the germline insertion results.</p

Protection from R5- and X4- tropic HIV-1-mediated CD4⁺ T-cell loss in hu-BLT mice by Dual sh1005/sh516.

<p>hu-BLT mice transplanted with Dual sh1005/sh516-transduced HSPC were challenged intravenously with either HIV-1_NFNSX or HIV-1_NL4-3 ∼12 weeks post-transplantation. Levels of marked CD4⁺ T-cells, shown as percentages within total CD3⁺ cells in peripheral blood (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053492#pone.0053492.s003" target="_blank">Table S2</a>), were normalized to that of the time point preceding decline of percentages of control-vector transduced cells to determine relative fold change over time. Values above and below 1.0 on the y-axis depict increases or decreases, respectively, in CD4⁺ T-cell levels. Closed circles: EGFP-marked. Open circles: mCherry-marked. Error bars: mean + SEM in n = 5 and n = 4 for HIV-1_NFNSX- and HIV-1_NL4-3- infected mice, respectively. Statistical significance calculated by two-way ANOVA/Bonferroni post-test. *  =  p<0.05, **  =  p<0.01, ***  =  p<0.001.</p

Down-regulation of CCR5 in systemic lymphoid tissues and inhibition of HIV-1 replication <i>ex vivo</i> by Dual sh1005/sh516.

<p>Tissues were isolated from a Dual sh1005/sh516-transduced HSPC-transplanted mouse at eleven weeks post-transplantation. <b>A.</b> Representative data showing CCR5 expression in EGFP- and mCherry- marked human CD4⁺CD3⁺CD45⁺CD19⁻ T-cells within a gated lymphocyte population. <b>B.</b> Splenocytes were depleted of human CD8⁺ cells and murine CD45⁺ cells and then stimulated with PHA/IL-2 for two days. Five days later, EGFP⁺ and mCherry⁺ cells were then sorted by FACS at >97% purities. Sorted cells (5×10⁴) were then infected with either HIV-1_NL4-3 at MOI 0.5, HIV-1_{NFNSX SL9} at MOI 5.0, or HIV-1_JR-CSF at MOI 1.0 for four hours. Cells were then washed five times before culturing. HIV-1 replication was monitored by p24 ELISA analysis of culture supernatants at four and seven days post-infection. Error bars: mean + SD. Representative data of three independent experiments.</p

Vector stability and the effects of sh1005/sh516 co-expression on cell viability and HSPC differentiation potential.

<p>IL-2/PHA-stimulated PBMCs (4×10⁵) were transduced with lentiviral vectors at MOI 0.6–1.0. <b>A.</b> Representative data showing EGFP expression in vector-transduced PBMCs over time. <b>B.</b> Vector-transduced cells were subjected to CytoTox-Glo^TM Cytotoxicity Assay (Promega) four and six days post-transduction. Relative cytotoxicities were calculated by dividing the dead cell count by the total cell count and normalizing to that of mock-transduced cells. 1 μM Staurosporine (STS) and U6 promoter-driven sh1005 expression (U6-sh1005) served as positive controls. Error bars: mean + SD. <b>C.</b> OAS1 mRNA expression relative to β-actin was assessed by qRT-PCR analysis of total RNA isolated from vector-transduced PBMCs. PBMCs harvested two days post-electroporation with 500 pg/μL poly(I:C) served as positive control. Values were normalized to those of mock-transduced cells. Error bars: mean + SD. <b>D.</b> Cytokine-pre-stimulated mPB-CD34⁺ cells were transduced with lentiviral vectors at MOI 10. HSPC differentiation potential was assessed by counting colony forming units produced from mock- and vector- transduced mPB-CD34⁺ cells plated on semi-solid methylcellulose plates one day after transduction. CFU-GEMM: granulocyte/erythrocyte/macrophage/megakaryocyte colony forming units. C/BFU-E: erythroid colony/burst forming units. CFU-GM: granulocyte/monocyte colony forming units. Error bars: range of values between duplicate samples. Representative data of three independent experiments.</p

Down-regulation of CCR5 and inhibition of HIV-1 replication in MOLT4-CCR5 cells and PBMCs by Dual sh1005/sh516.

<p><b>A.</b> MOLT4/CCR5 (1×10⁵) cells were transduced with lentiviral vectors at MOI 0.5. EGFP and CCR5 expression was assessed three days post-transduction. CCR5 MFIs shown below plots as “MFI: MFI_EGFP−/MFI_EGFP+.” Representative data of three independent experiments. <b>B.</b> Sorted EGFP⁺ cells (2×10⁵) were infected with either HIV-1_NL4-3 at MOI 0.5 and HIV-1_{NFNSX SL9} at MOI 5.0. Levels of p24 antigen in culture supernatants were measured by ELISA four and seven days post-infection. Errors bars: mean + SD. <b>C.</b> IL-2/PHA stimulated PBMCs (4×10⁵) were transduced with lentiviral vectors at MOI 0.6–1.0. EGFP and CCR5 expression was measured at seven days post-transduction. CCR5 MFIs shown as in <b>A</b>. Representative data showing CCR5 expression in vector-transduced PBMCs. <b>D.</b> Sorted EGFP⁺ cells (5×10⁴) were infected with either HIV-1_{NFNSX SL9} at MOI 5.0, HIV-1_JR-CSF at MOI 1.0, or HIV-1_NL4-3 at MOI 0.1. p24 production was measured as in <b>B</b>.</p

Reconstitution of Dual sh1005/sh516-transduced HSPCs in humanized BLT mice.

<p><b>A.</b> Schematic of generating vector-transduced HSPC-transplanted hu-BLT mouse. NSG mouse is treated with Busulfan 24 hours pre-transplantation. CD34⁺ and CD34⁻ cells are isolated from human fetal liver (FL). CD34⁺ cells are transduced with either therapeutic (EGFP-marked) or control (mCherry-marked) vectors. Therapeutic vector- and control vector- transduced CD34⁺ cells are mixed at a 50 50 ratio. The cell mixture is then 1) combined with CD34⁻ cells, solidified with Matrigel, and implanted under the kidney capsule with a human fetal thymus segment (FT) and also 2) intravenously injected. <b>B.</b> EGFP and mCherry reporter gene expression was monitored in human CD45⁺, CD3⁺CD45⁺, and CD19⁺CD45⁺ cells within a gated lymphocyte population in peripheral blood at twelve weeks post-transplantation. CD4/CD8 ratios were analyzed in mCherry- and EGFP- marked CD3⁺CD45⁺ cells. Data were generated from n = 13 mice for both Mono sh1005- and Dual sh1005/sh516- HSPC-transplanted animals from an aggregate of three donors. Error bars: mean + standard error of mean (SEM) in n = 13 per group.</p