Effect of LIF concentration on the maintenance of undifferentiated state on ES cells.

<div><p>(A) R1 cells were cultured for 5 days in the presence of various doses of LIF (0–1,000 units/ml), on E-cad-Fc-coated plates (open bars) or gelatin-coated plates (closed bars).</p> <p>Then cells were replated onto gelatin-coated plates and three days later, the ratios of ES cell colonies with high ALP activity were estimated.</p> <p>*:<i>P</i><0.05 vs. gelatinized plate in the presence of 1,000 units/ml LIF.</p> <p>#:<i>P</i><0.05 for ES cells cultured on E-cad-Fc-coated plates versus gelatinized plate in the presence of same concentration of LIF.</p> <p>(B) The maintenance of the pluripotent efficiency of ES cells, which was cultured on an E-cad-Fc-coated surface at a low concentration of LIF (100 units/ml), was assessed by the characterisation of teratomas.</p> <p>ES (EB3) cells were maintained on E-cad-Fc-coated surface in the presence of 100 units/ml of LIF and then transplanted into mouse testis.</p> <p>H&E staining of teratomas showed the differentiation into ectoderm (epidermis: top right, bar: 100 µm), mesoderm (striated muscle cells: top left, and cartilage: top centre, bar: 100 µm, inset: 10 µm) and endoderm (ciliated columnar epithelium, possibly bronchial epithelium: bottom left, bar: 50 µm, inset: 10 µm).</p> <p>Differentiation into ectoderm was confirmed by specific staining for the neural markers GAP-43 (bottom centre) and Neurofilament-M (bottom right).</p></div

Pluripotency of ES cells on E-cad-Fc-coated surface.

<div><p>(A) R1 cells were maintained on gelatin or E-cad-Fc for 26 days, and then were cultured to form embryoid bodies.</p> <p>After 14 days culture of embryoid bodies, expression of marker genes was analyzed by RT-PCR.</p> <p>Lane 1: undifferentiated cells; lane 2: on gelatin; lane 3: on E-cad-Fc.</p> <p>(B and C) Characterization of teratomas from ES cells (EB3) cultured on an E-cad-Fc-coated surface.</p> <p>(B) H&E staining of teratomas showed the differentiation into ectoderm (epidermis), mesoderm (cartilage, and striated muscle cells) and endoderm (ciliated columnar epithelium, possibly bronchial epithelium).</p> <p>Differentiation into ectoderm was confirmed by specific staining for the neural markers βIII-tubulin, GFAP, Neurofilament-M and GAP-43 (C).</p> <p>Scale bar indicates 50 µm.</p> <p>(D) EB3 cells cultured on gelatin- or E-cad-Fc-coated dishes for 15 days were introduced into approximately 100 blastocysts of C57BL/6 (B6) mice in each group, which yielded 4 and 7 heads of chimera pups, respectively.</p> <p>Furthermore, by mating with wild-type B6 females, 2/4 chimera males from the gelatin-coated group and 3/5 chimera males from the E-cad-Fc-treated group produced offspring with ES cell-derived coat colors, suggesting comparable chimera formation and germ-line transmission abilities in E-cad-Fc-treated ES cells.</p> <p>Germ-line transmission was also verified genetically by DNA microsatellite marker analysis.</p> <p>PCR-based microsatellite marker analysis was performed on a litter mate.</p> <p>The primer sequences for D4Mit72 and D4Mit116 microsatellite markers were obtained from Mouse Microsatellite Data Base of Japan (<a href="http://shigen.lab.nig.ac.jp/mouse/mmdbj/top.jsp" target="_blank">http://shigen.lab.nig.ac.jp/mouse/mmdbj/top.jsp</a>).</p></div

ES cells show higher proliferation and higher transfection efficiency on the E-cad-Fc-coated surface.

<div><p>(A) The proliferative activity of ES cells on a gelatin- or E-cad-Fc-coated surface was evaluated.</p> <p>EB3 cells were seeded on gelatin-coated (open square) or E-cad-Fc-coated (filled square) dishes and the cell number was counted after staining with alamar Blue reagent.</p> <p>The data indicate means±SD of experiments (n = 3). **:<i>P</i><0.01 versus gelatinized plates.</p> <p>(B) BrdU incorporation of EB3 cells under colony-forming (on gelatin) or scattering conditions (on E-cad-Fc).</p> <p>Relative BrdU incorporation value was evaluated.</p> <p>The data indicate means±SEM. §:<i>P</i><0.001.</p> <p>(C) Transfection efficiency of ES3 cells cultured on gelatin- or E-cad-Fc-coated surface.</p> <p>Relative expression of GFP was evaluated.</p> <p>The data indicate means±SEM. §:<i>P</i><0.001 versus gelatinized plates.</p></div

Cell adhesion, morphology of ES cells on the E-cad-Fc fusion protein-immobilized surface.

<div><p>(A) ES cells (EB3) adhered to E-cad-Fc-coated dishes with equivalent efficiency as to 0.1% gelatin-coated dishes after 3 hours of incubation.</p> <p>(B) ES cells (EB3) were cultured on E-cad-Fc-coated or fibronectin-coated dishes without serum.</p> <p>EGTA (5 mM) was added to the culture medium at 3 hours after seeding (open bar).</p> <p>Detached cells were removed and remaining cells were counted using alamar Blue reagent.</p> <p>*:<i>P</i><0.05, §:<i>P</i><0.001 vs. no treated condition (closed bar).</p> <p>(C and D) Morphological observation of ES cells (EB3) on the two different matrices.</p> <p>ES cells were cultured on polystyrene surfaces coated with 0.1% (wt/vol) gelatin (C), or 10 µg/ml E-cad-Fc (D) in the presence of LIF for 2 days.</p> <p>High magnification images are shown in (C′) and (D′).</p> <p>(E) ES cells (EB3 and R1 cells) were cultured on the plates coated with gelatin or E-cad-Fc and differentiation was induced by the withdrawal of LIF.</p> <p>Morphological characteristics were observed as phase contrast images.</p> <p>Bar indicates 100 µm.</p> <p>The data indicate means±SD of 3 separate experiments.</p></div

Derivation of Transgene-Free Human Induced Pluripotent Stem Cells from Human Peripheral T Cells in Defined Culture Conditions

<div><p>Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.</p></div

In vitro and in-TiPSCs.

<p>(A): Immunofluorescence staining for Sox17 (endodermal marker), αSMA (mesodermal marker), and Nestin (ectodermal marker) in each TiPSCs1-derived differentiated cell in vitro. (B): Gross morphology of representative teratomas derived from TiPSCs1 in vivo (hematoxylin and eosin staining).</p

Characterization of M-TiPSCs generated under a defined culture condition.

<p>(A): ALP staining in M-TiPSCs. (B): QT-PCR analyses of M-TiPSCs for the ESC marker genes <i>OCT3/4</i>, <i>NANOG</i>, <i>SOX2</i>, <i>KLF4</i>, <i>c-MYC</i>, and <i>TERT1</i>. (C): QT-PCR analyses of M-TiPSCs for the transgenes, <i>OCT3/4</i>, <i>SOX2</i>, <i>KLF4</i>, and <i>c-MYC</i>. (D): Immunofluorescence staining for pluripotency and surface markers (NANOG, OCT3/4, SSEA3, SSEA4, TRA-1–60, and TRA-1–81) in M-TiPSCs1. (E): Heat map analyses of M-TiPSCs, ESCs, and the parental human T cells. (F): Scatter plots comparing the global gene expression profiles of M-TiPSCs with those of T cells and ESCs.</p

Analysis of TiPSCs genome modification and karyotype.

<p>(A): Bisulfite sequencing analysis of the <i>NANOG</i> and <i>OCT3/4</i> promoter regions in peripheral T cells, ESCs, and M-TiPSCs. Each row of circles for a given amplicon represents the methylation status of the CpG dinucleotides in one bacterial clone for that region. Open circles represent unmethylated CpGs and closed circles represent methylated CpGs. (B): G-band analysis for karyotypes of M-TiPSCs generated under a defined culture condition. M-TiPSCs1 and M-TiPSCs2 at passages 6 and 15, respectively, were used for G-band analysis. (C): Analysis of TCR rearrangements. V, D, and J segment usages in the <i>TCRB</i> gene locus were sequenced and identified by comparison to the international ImMunoGeneTics information system database. M-TiPSCs showed rearrangements of Vβ/Dβ1,2 and Dβ1,2/Jβ2.</p

Generation of human TiPSCs under defined conditions.

<p>(A): Strategy used in the present study for reprogramming T cells. (B): Typical ESC-like TiPSC colony on day 25 after blood sampling under the defined culture condition. (C): Comparison of reprogramming efficiencies between the culture system using a feeder cell layer and that using defined culture conditions. Data show the mean ± s.d. (D): Comparison of representative 10-cm dishes stained for ALP (red spots) between feeder layer condition and defined culture condition (Matrigel) on day 25. (E): Comparison of reprogramming efficiencies between a culture system using a feeder cell layer and one using Matrigel and mTeSR1 medium for samples from five donors. Data show the mean ± s.d.</p

Additional file 1: of Selective modulation of local linkages between active transcription and oxidative demethylation activity shapes cardiomyocyte-specific gene-body epigenetic status in mice

Figure S1. K-means 12 clustering summary. Figure S2. Strict gene length restriction in constitutive genes. Figure S3. Comparison of gene length and promoter features observed among the 12 cluster populations. Figure S4. Cell-type-specific gene body DNA hypomethylation in cardiomyocytes (validation of the HELP tagging method). Figure S5. Comparison gene body and promoter DNA methylation patterns. Figure S6. Validation of 5hmC enrichment by BGT-qPCR assay. Figure S7. Dynamic CTCF binding sites in promoter and gene body regions