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, 85 (8), 348-62

Efficient Induction of Transgene-Free Human Pluripotent Stem Cells Using a Vector Based on Sendai Virus, an RNA Virus That Does Not Integrate Into the Host Genome

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Efficient Induction of Transgene-Free Human Pluripotent Stem Cells Using a Vector Based on Sendai Virus, an RNA Virus That Does Not Integrate Into the Host Genome

Noemi Fusaki et al. Proc Jpn Acad Ser B Phys Biol Sci.

Abstract

Induced pluripotent stem cells (iPSC) have been generated from somatic cells by introducing reprogramming factors. Integration of foreign genes into the host genome is a technical hurdle for the clinical application. Here, we show that Sendai virus (SeV), an RNA virus and carries no risk of altering host genome, is an efficient solution for generating safe iPSC. Sendai-viral human iPSC expressed pluripotency genes, showed demethylation characteristic of reprogrammed cells. SeV-derived transgenes were decreased during cell division. Moreover, viruses were able to be easily removed by antibody-mediated negative selection utilizing cell surface marker HN that is expressed on SeV-infected cells. Viral-free iPSC differentiated to mature cells of the three embryonic germ layers in vivo and in vitro including beating cardiomyocytes, neurons, bone and pancreatic cells. Our data demonstrated that highly-efficient, non-integrating SeV-based vector system provides a critical solution for reprogramming somatic cells and will accelerate the clinical application.

Figures

Fig. 1
Fig. 1
Expression of exogenous genes in human fibroblasts by SeV vectors. A. Efficient induction of GFP cDNA by TSΔF/SeV in BJ and HDF at an MOI of 3. BC: bright contrast. B. Schematic presentation of SeV vector genomes. Reprogramming genes were inserted at 18+, PM, HN, HNL and Leis (L), respectively. The expression levels of inserted genes decreased depending on the inserted site (polar effect: Refs. 21) as shown by Western blotting on day 3 after infection. Anti-SeV blot was performed to confirm equal infection efficiency of the vectors.
Fig. 2
Fig. 2
Efficient generation of human iPSCs by non-integrating SeV vectors. A. The reprogramming efficiency with SeV vectors. iPS colonies were determined by ALP positive and ES-like morphology. Lane numbers correlate with the conditions listed in the column under the figure. Each dot represents one experiment. The bars represent the average efficiency for each condition. B. ALP-staining of growing cells on 100 mm dishes. C. Typical ALP-positive colonies (scale bar: 100 μm). Numbering of C and B are correlated with the numbers in the column in A. Colonies in conditions 1,2 and feeder free were similar to those of 3 (data not shown).
Fig. 3
Fig. 3
Expression of hES markers and telomerase activity in SeV-iPSC. A. RT-PCR anaysis of human ES cell-marker genes. Primers used for Oct3/4, Sox2, Klf4 and c-Myc were designed to detect the expressions of endogenous genes, but not of transgenes. Cont: PCR without cDNA. B. Telomerase activity of human SeV-iPSC. Telomerase activity was detected by the TRAP method. Heat-inactivated samples (+) were used as negative controls. C. Immunofluorescence staining of established clones with human ES cell-markers (Tra-1-60, Tra-1-81, SSEA-4 and Nanog). SeV-iPS colonies were positive for ALP and negative for SSEA-1 as in hES cells. Nuclei were stained with TO-PRO3 (blue).
Fig. 4
Fig. 4
Genomic Southern blot, karyotyping and fingerprinting of SeV-iPSC. A. DNA fingerprinting of SeV-iPS clones. PCR analysis of three variable number of tandem repeats (VNTR) loci of D17S1290, MCT118 and ApoB-100 using genomic DNA from the SeV-iPS clones confirmed that these clones were originated from human fibroblasts BJ or HDF. B1, HNL1 and HNL5 were derived from BJ; XH1, 7H5, 7H8 and 7H10 were from HDF. B. Viral transgenes were not detected from the host genome as analyzed by genomic Southern blot. C. Karyotyping of SeV-iPSC. Viral-free SeV-iPSC HNLs at passage 34 were used for karyotyping.
Fig. 5
Fig. 5
SeV vectors were diluted and lost during cell growth. A. Kinetics of transgene expression determined by RT-PCR using combination of specific primers for SeV and transgenes. cont: no template. B. Kinetics of SeV genome expression during cell growth by real time quantitative PCR. Pn means passage numbers. C. SeV-derived protein expression determined by Western blotting with anti-SeV polyclonal antibody. Passage numbers are correlated with A. cont: positive control from SeV-infected LLC-MK2 cells. Viral proteins in HNLs were slightly existed at this time (P8), but those of HNL1 were completely lost later at P17 as well as HNL1 at P9 (B). D. Anti-SeV-immunostaining revealed that SeV distribution was heterologous in iPS colonies (Upper). SeV could be removed by anti-HN-antibody mediated negative selection using anti-mouse IgG1-conjugated IMag-beads. Anti-HN antibody separated SeV-negative population (−) and SeV-enriched population (+).
Fig. 6
Fig. 6
DNA methylation and global gene expression profiles of SeV-iPSC. A. Methylation analysis of Oct3/4 and Nanog promoter regions in SeV-iPSC. B. The global gene-expression patterns were compared between SeV-iPSC (HNL1) and BJ, human ES cells (H9) and HDF-iPSC with microarrays. The lines indicate the diagonal and 5-fold changes between two samples.
Fig. 7
Fig. 7
In vitro differentiation of transgenes-free SeV-iPSC. A.In vitro differentiation of mononuclear cells (mesoderm: shown with Wright-Giemsa staining) via embryoid bodies, putative dopaminergic neurons co-expressing tyrosine hydroxylase (TH)(ectoderm), definitive endoderm (Sox17), and pancreatic cells (PDX1).B.In vitro differentiation of cardiomyocytes and mononuclear cells from SeV-iPSC via embryo bodies using cytokine cocktails (SCF, Flt3L, TPO, G-CSF, IGF-2, and VEGF). In the adherent culture, hematopoietic sac-like structures filled with mononuclear cells (left, upper) and beating colonies (left, middle) emerged. FACS analysis shows differentiation of SeV-iPSC to mononuclear cells (neutrophil, monocyte, and macrophage), expressing CD34, CD45, CD33, and neutrophil-specific marker CD66b (right). RT-PCR analysis shows that pluripotent marker (Nanog) was decreased and various cardiomyocytes-specific differentiation markers (TnTc, MEF2C, MYHCB) were increased after differentiation (left, lower). U: undifferentiated; EB: embryoid body; D: differentiated.
Fig. 8
Fig. 8
In vivo pluripotency of transgenes-free SeV-iPSC. Hematoxylin and eosin staining of teratoma sections of SeV-iPS clones (6 weeks post-injection into SCID mice). Tissues were differentiated from Tg-free human neonatal fibroblast BJ-derived HNLs (A to C), HNL1 (D to I), and adult fibroblast HDF-derived XH1 (J to L) containing multiple tissues derived from three germ layers: glandular structures (A, G, K), cartilage (B, J), bone (C, F, white arrows) and bone marrow-like structure (F), epithelium (J, D), transitional epithelium (E), population of secreting-like cells (B, D, indicated by black arrows), muscle (C, I, K, L), and glomerulus of kidney-like tissue (H).

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