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1.
Figure 7

Figure 7. Regenerative medicine strategy using HAC vectors.. From: Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors.

Our proposed strategy to generate patient-specific iPS cells with therapeutic transgenes carried by HAC vectors is depicted. Integration-free iPS cells are created from patient-specific somatic cells by HAC-mediated reprogramming (iHAC). Subsequently, correction of a gene lesion is achieved by the addition of a HAC with a therapeutic gene and a suicide gene.

Masaharu Hiratsuka, et al. PLoS One. 2011;6(10):e25961.
2.
Figure 3

Figure 3. Characterization of iPS cells generated by iHAC vectors.. From: Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors.

(A, C) Quantitative RT-PCR analysis of total and endogenous reprogramming factor (Klf4, c-Myc, Sox2 and Oct4) expression in iHAC1- and iHAC2-iPS cells. Transcript levels for these genes relative to levels in B6ES cells are shown, after normalization to Nat1 expression. (B, D) Quantitative RT-PCR analyses of pluripotency markers.

Masaharu Hiratsuka, et al. PLoS One. 2011;6(10):e25961.
3.
Figure 2

Figure 2. Reprogramming MEFs with iHAC1 or iHAC2.. From: Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors.

(A) Flowchart of iPS cell generation after MMCT. We screened for cells reprogrammed by iHAC vectors using two criteria: ES-like morphology and EGFP fluorescence. Packed domed colonies emerged 10 days after MMCT. After expansion, EGFP-negative fractions were sorted from isolated clones to isolate iHAC-free cells. (B) Representative bright-field and fluorescence images of iHAC1- and iHAC2-iPS clones obtained by MMCT. (C) FISH analyses of three lines, iHAC1-iPS 1-D6, iHAC2-iPS 2-N and iHAC-free iPS F-C5.

Masaharu Hiratsuka, et al. PLoS One. 2011;6(10):e25961.
4.
Figure 5

Figure 5. Global gene expression analysis of iHAC1-, iHAC2-, and iHAC-free iPS cells.. From: Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors.

(A) Unsupervised hierarchical clustering of global gene expression profiles from ES cells, iPS cells generated by retroviral or iHAC vectors, and MEFs. (B) Scatter plots of microarray data comparing ES cell line B6ES to iHAC1-iPS 1-B, iHAC-free iPS F-B1 and retroviral iPS 20D17 cells. Highlighted marks indicate core pluripotent markers, Nanog, Oct4 and Sox2. Red lines indicate two-fold changes in expression between samples.

Masaharu Hiratsuka, et al. PLoS One. 2011;6(10):e25961.
5.
Figure 4

Figure 4. Establishment of iHAC-free iPS cells.. From: Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors.

(A) Genomic PCR of individual transgenes in cells from an EGFP-negative fraction of iHAC2-iPS cells was compared with that of the parental clones. D8Mit224 was used as an internal control. (B) Cytogenetic analysis of iHAC-free iPS cells. Representative karyotypes of iHAC-free iPS F-C5 (Q-banding) and F-N7 (M-FISH) show normal karyotypes after loss of iHAC2. (C) Quantitative RT-PCR of total and endogenous reprogramming factor expression. (D) Quantitative RT-PCR of pluripotency markers. Transcript levels of 10 pluripotent genes were normalized to Nat1; transcript levels in iHAC-free iPS cells were compared with those in B6ES cells.

Masaharu Hiratsuka, et al. PLoS One. 2011;6(10):e25961.
6.
Figure 6

Figure 6. Pluripotency of iHAC-free iPS cells and a HAC safeguard system.. From: Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors.

(A) in vitro differentiation mediated by EB formation. (B) Representative images of various tissues present in teratomas derived from iHAC-free iPS F-C5 cells. (C) Live-born chimeras from iHAC-free iPS clones. (D, E) Confirmation of the HAC safeguard system. Growth of iHAC-free iPS clones re-transformed with 21HAC2 or with a retroviral iPS clone harboring DYS-HAC was inhibited by GCV in vitro (D). Parental iHAC-free iPS F-C5 and F-C5/21HAC2 were subcutaneously transplanted into nude mice. 30 mg/kg GCV was then administered intraperitoneally for 24 consecutive days (E).

Masaharu Hiratsuka, et al. PLoS One. 2011;6(10):e25961.
7.
Figure 1

Figure 1. Construction of iHAC vectors.. From: Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors.

(A) A map of the reprogramming cassette constructed in a PAC vector, pPH3-9. The expression cassette comprised cDNAs for Klf4, c-Myc, Sox2 and Oct4, each under the control of a CAG promoter and flanked with HS4 insulators. The reprogramming cassette was followed by exon 3 to exon 9 of the human HPRT gene and then by a lox P site. (B) Schematic maps of the iHAC1 and iHAC2 inserts. iHAC1 has a duplicated reprogramming cassette. iHAC2 has this duplicated cassette and an additional cassette with two copies of Oct4 and a p53 shRNA construct. (C) FISH analyses of CHO/iHAC1 and CHO/iHAC2. Digoxigenin-labeled human COT-1 DNA (red) was used to detect the HAC backbone. Biotin-labeled pPAC-KMSO (green) was used to detect the reprogramming cassette in the iHACs. Chromosomal DNA was counterstained with DAPI. White arrows indicate iHAC vectors and the insets show enlarged images of the iHACs.

Masaharu Hiratsuka, et al. PLoS One. 2011;6(10):e25961.

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