Multilineage differentiation of MSCs.

<p>Representative images of MSCs differentiated towards adipogenic (a), chondrogenic (b) or osteogenic (c) lineages.</p

Multilineage differentiation of MSCs.

<p>Representative images of MSCs differentiated towards adipogenic (a), chondrogenic (b) or osteogenic (c) lineages.</p

<i>In vitro</i> and <i>in vivo</i> development of clone horse embryos reconstructed with iPSCs or after microinjection of recipient oocytes with pluripotent gene sequences.

<p><i>In vitro</i> and <i>in vivo</i> development of clone horse embryos reconstructed with iPSCs or after microinjection of recipient oocytes with pluripotent gene sequences.</p

Microinjection of non-integrative pluripotency-inducing genes in horse oocytes before enucleation.

<p>a) Plasmid construct containing the <i>OCT4</i>, <i>SOX2</i>, <i>MYC</i> and <i>KLF4</i> human sequences. b) Photomicrograph showing the plasmid microinjection procedure.</p

<i>In Vitro</i> and <i>In Vivo</i> Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors - Fig 2

<p>a) Cloned polo pony derived from 1-2h group in experiment 1, born on October 2013, b) Jumping horse cloned derived from 2-3h group in experiment 1, born on November 2014, c) Thoroughbred horse cloned derived from the control group in experiment 2, born on June 2014.</p

EDA–treated mouse ESC can differentiate to the three germ layers.

<p>Ainv15 mES cells were cultured for three days on gelatin coated dishes in the presence of EDA+ peptide. Then, 10⁶ cells were injected subcutaneously into nude mice, as described in Material and Methods Section. Four-micrometer sections from teratoma tissue were stained with hematoxylin and eosin. (A–C) Representative histology of teratoma (A) Neuroepithelium (arrow, ectoderm) and mesenchymal tissue (star, mesoderm); (B) Neuroepithelium (arrow, ectoderm) and glial tissue (ectoderm); Glandular tissue (arrow, endoderm).</p

MEF conditioned medium that contains FN EDA⁺ increases the proliferation rate of mESC.

<p>Ainv15 mES cells were cultured in the media conditioned by wild type MEF (wt), EDA^+/+ MEF (^+/+) or EDA^-/- MEF (^-/-) for 2 days. The proliferation was evaluated by MTT assay. A representative experiment of at least three replicates is shown. Bars represent mean ± SD. Statistical analysis was done by an unpaired t-test. * indicate significant differences between treatments (<i>P</i> < 0.01).</p

EDA –treated ES cells express pluripotency markers.

<p>WA09 human ES cells and Ainv15 mouse ES cells were cultured for three days on feeder free conditions in the indicated medium (untreated cells, C; EDA^+/+ MEF-CM, CM; EDA+ including peptide, PEP). RNA was extracted and the expression of Oct4, Sox2 and Nanog pluripotency gene markers was analyzed by RT-PCR. The expression of the housekeeping GAPDH gene was used as control.</p

EDA⁺ but not EDA^- peptide increases the proliferation rate of mESC.

<p>(A) Ainv 15 mouse ES cells were cultured in the standard media supplemented by the corresponding peptide preparation to a final dose of 10 µg of protein per ml of medium for 72 hours. (EDA-containing recombinant peptide, EDA⁺; EDA-lacking recombinant peptide, EDA^-). Representative brightfield pictures are shown. Bars correspond to 50 µm. (B) The proliferation of Ainv15 mES cells cultured in the media described in B for 48 or 72 hours, as indicated, was evaluated by crystal violet. A representative experiment of at least three replicates is shown. Bars represent mean ± SD. Statistical analysis was done by One way ANOVA with Bonferroni test for multiple comparisons. * indicate significant differences between treatments (<i>P</i> < 0.05); ** indicate significant differences between treatments (<i>P</i> < 0.005).</p

FN EDA⁺ but not FN EDA^- increases the proliferation of human ESC.

<p>WAO9 human ES cells were plated in standard proliferation medium. 24 hours later, when the cells were attached, medium was replaced by fresh medium containing the corresponding peptide preparation to a final dose of 10 µg of protein per ml of medium. (A) Representative brightfield pictures of the wound-healing assay. The scratch was made the same day that the peptide was added to the medium. Left panel, linear scratch; right panel, circular scratch. The references are the same than in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080681#pone-0080681-g002" target="_blank">Figure 2</a>. Bars correspond to 200 µm. (B) Quantification of the filled areas of a representative experiment of three replicates. Proliferation was calculated from the decrement in damaged area. The areas were quantified with the ImageJ software. The filled area in each condition was referred as the amount of filled area in the control, considered as 100%. The graph is representative of a scratch wound healing assay of three replicates.</p