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, 9 (12), e113052
eCollection

New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived From Chimpanzee Blood

Affiliations

New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived From Chimpanzee Blood

Yasumitsu Fujie et al. PLoS One.

Abstract

Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.

Conflict of interest statement

Competing Interests: HB is an employee of DNAVEC Corporation. M. Hasegawa is a founder and adviser of DNAVEC Corporation. NF was an employee of DNAVEC Corporation until January 2013 but not now. The commercial product developed by DNAVEC Corporation is similar to the vectors described in this paper but the component is different. The patent of the Sendai virus vectors to generate iPS cells that was applied by and of DNAVEC Corporation is pending (WO/2010/008054). NF and HB have waived the right of the patent. These do not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Generation of a new temperature-sensitive Sendai virus vector, TS12KOS.
(a) Comparison of schematic structures among the newly constructed Sendai virus (SeV) vector, TS12KOS, and previous vectors. The TS12KOS vector contains three point mutations in the RNA polymerase–related gene (P) and carries the coding sequences of KLF4 (K), OCT3/4 (O), and SOX2 (S) in the KOS direction. In comparison, the HNL/TS15 c-Myc vector carries two additional mutations, L1361C and L1558I, in the large polymerase (L) gene and an exogenous c-MYC cDNA sequence inserted between the hemagglutinin-neuraminidase (HN) and L genes, and the conventional vectors individually carry three reprogramming factors as indicated. (b) iPS cell generation from human skin-derived fibroblasts. The efficiency of iPS cell generation was significantly higher using the TS12KOS vector than with the conventional vectors at all multiplicities of infection (MOI) tested. iPSC colonies were identified on day 28 of induction by the appearance of alkaline phosphatase-positive (AP+) colonies with embryonic stem (ES) cell-like colony morphology. N1, N2, and N3 represent individual healthy volunteers. Experiments were conducted in triplicate (mean ± SD). *P<0.01, TS12KOS vector versus conventional vectors, Student's t-test. (c) Temperature shift from 37°C to 36°C for the indicated periods in iPSC generation. Data are means ± SD of three independent experiments. # P<0.05, Experiment 2, 3 and 4 versus Experiment 1. Student's t-test. (d) Nested RT-PCR analysis of SeV vector elimination after the temperature shift from 37°C to 38°C in human fibroblast-derived iPSCs. The elimination of TS12KOS vector was faster than the conventional vectors.
Figure 2
Figure 2. Characterization of human iPSCs generated by the TS12KOS vector.
(a) iPSC generation from human peripheral blood cells. Experiments were conducted in triplicate (mean ± SD). N1, N2, and N3 indicate individual healthy volunteers. *P<0.01, TS12KOS vector versus conventional vectors, Student's t-test. (b) Nested RT-PCR analysis of the elimination of SeV vectors after the temperature shift from 37°C to 38°C. (c) Phase contrast images, immunofluorescence for pluripotency markers, and alkaline phosphatase (AP) staining of iPSC lines. The iPSC lines N2-1 and BN2-1 and BN2-2 were derived from the skin-derived fibroblasts and blood cells of N2 healthy volunteer, respectively. Scale bars, 200 µm. (d) RT-PCR analysis of Sendai virus and human ES cell markers. SeV, first RT-PCR for SeV; nested, nested RT-PCR for SeV; 201B7, control human iPSC line; SeV(+), Day 7 SeV-infected human fibroblasts. (e) Chromosomal analyses of iPSC lines generated with the TS12KOS vector. (f) Tissue morphology of a representative teratoma derived from iPSC lines generated with the TS12KOS vector. G, glandular structure (endoderm); C, cartilage (mesoderm); MP, melanin pigment (ectoderm). Scale bars, 100 µm.
Figure 3
Figure 3. Generation of chimpanzee iPSCs with the TS12KOS vector.
(a) Summary of chimpanzee iPSC generation. iPSCs were generated from the blood cells of two chimpanzee individuals with TS12KOS or the conventional SeV vectors. (b) Effect of the T lymphocyte stimulation on iPSC generation. Experiments were conducted in triplicate (mean ± SD). *P<0.01, PHA versus anti-CD3 antibody or Con A stimulations, Student's t-test. (c) Colony morphology and AP staining of iPSCs from stimulated T lymphocytes. (d) Phase contrast images, immunofluorescence for pluripotency markers, and alkaline phosphatase (AP) staining of chimpanzee iPSC lines. C101, C201, C205, and C402 are described in Fig. 3a. Scale bars, 200 µm. (e) RT-PCR analysis of SeV and human ES cell markers. SeV, first RT-PCR for SeV; nested, nested RT-PCR for SeV; 201B7, control human iPSC line; SeV(+), Day 7 SeV-infected human fibroblasts. (f) PCR products with primers that can distinguish chimpanzee and human genomes. Chimpanzee PCR products; 782, 472 and 504 bps, Human PCR products; 203, 245, 278 bps. (g) Chromosomal analyses of chimpanzee iPSC lines generated with the TS12KOS vector. (h) TCR gene recombination. Genes from the chimpanzee iPSC lines were digested with the indicated enzymes and hybridized with the TCR probes by Southern blotting. Arrows indicate the germ bands of TCR genes. HeLa and 201B7: human cell lines, MT4: human T cell line, HSP-239: chimpanzee T cell line.
Figure 4
Figure 4. Characterization of chimpanzee iPSCs.
(a) Differentiation into three germ layers in vitro. The chimpanzee iPSC lines can generate SOX17+ (endoderm), BRACHYURY+ (mesoderm), and βIII-tubulin+ (ectoderm) cells. Scale bars, 100 µm. (b) Tissue morphology of a representative teratoma derived from the chimpanzee iPSC lines generated with TS12KOS vector. G, glandular structure (endoderm); C, cartilage (mesoderm); CE, Cuboidal Epithelium structure (ectoderm); MP, melanin pigment (ectoderm). Scale bars, 100 µm. (c) Principal Component Analysis. All data sets were classified into three principal components, PC1 (47.62%), PC2 (29.81%), and PC3 (22.56%), and then simplified into three-dimensional scores. Percentage shows the portion of variance in each component. The position of chimpanzee iPSC lines is closely placed to that of human ESCs and iPSCs. (d) Hierarchical clustering of chimpanzee iPSCs, human iPSCs and ESCs. The data sets of all genes investigated were clustered according to Euclidean distance metrics. The data sets of chimpanzee iPSCs, human ESC and iPSC lines, and various human tissues were classified into separate branches. The datasets of human ESCs and various tissues referred for Gene Expression Omnibus datasets, GSE22167 and GSE33846, respectively.

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Grant support

This study was supported in part by grants from the Ministry of Health, Labor, and Welfare of Japan, Precursory Research for Embryonic Science and Technology (PRESTO), and Core Research for Evolutional Science and Technology (CREST), Japan Science and Project of the Primate Research Institute, Kyoto University. DNAVEC Corporation provided support in the form of salaries for authors MH & HB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. NF was provided with salary by JST grant (PRESTO). The specific roles of these authors are articulated in the 'author contributions' section.

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