Degree of methacrylation as determined by TNBS assay.

<p>(A) Various volume percentages of methacrylic anhydride (0.25%, 1.25% and 20%) were analyzed to investigate the degree of methacrylation of the synthesized fish GelMA. (B) Comparison of a high degree of methacrylation (20% MA) according to the origin of gelatin (fish GelMA vs. porcine GelMA); there was no significant difference. The percentage of incorporated substitution was calculated by comparing the amount of remaining amino groups (-NH₂) in GelMA to that in pristine gelatin. Error bars represent standard deviations (SDs) of measurements performed on six samples.</p

Behavior of cells encapsulated in fish GelMA hydrogels.

<p>(A-F) NIH3T3 cells embedded in medium and high degree of methacrylation fish GelMA containing 5% (w/v) GelMA were stained using calcein-AM/ethidium homodimer at 3 h and 24 h after encapsulation to evaluate the cell viability compared to high degree of methacrylation porcine GelMA. (G-I) After 2 days of culture, cells proliferated and elongated in fish and porcine GelMA hydrogel. (J-L) Representative images of the cells stained with phalloidin for the actin filaments (green) and nuclei counterstained with DAPI (blue) at 72 h. (P) Viability of the encapsulated cells. (Q) Quantification of cell proliferation in GelMA hydrogel until 5 days. Cell proliferation rate was not significantly different at all condition.</p

Synthesis of fish gelatin methacryloyl (GelMA) and fabrication of photocrosslinked GelMA hydrogel.

<p>(A) Gelatin was reacted with methacrylic anhydride (MA) to introduce a methacryloyl substitution group on the reactive amine and hydroxyl groups of the amino acid residues. (B) GelMA photocrosslinking to form A hydrogel matrix under UV irradiation. The free radicals generated by the photoinitiator initiated chain polymerization with methacryloyl substitution. (C) Schematic of formation of patterned hydrogels using photolithography.</p

Fabrication of micropatterned fish GelMA hydrogel and viability of cells on micropatterned gel surfaces.

<p>NIH3T3 cells readily adhered to fish GelMA surfaces irrespective of macromer concentration. (A-C) Pattern fidelity of fish GelMA using 5%, 10% and 15% macromer (scale bar = 800 μm). (D-F) LIVE/DEAD assay at 24 h after adhesion (scale bar = 200 μm). (G) Quantification of cell viability demonstrated high cell survival under all conditions and there was no significant difference between GelMA conditions. Error bars represent SDs of averages obtained from five images per condition.</p

Degradation characteristics of fish GelMA hydrogels.

<p>(A) Degradation profiles of fish GelMA hydrogels with various degrees of methacrylation (low, medium and high) and GelMA concentrations (5%, 10% and 15%) upon exposure to collagenase type II. (B) Comparison of fish and porcine GelMA hydrogels with high degree of methacrylation and 10% gel concentration. Error bars represent SDs of measurements performed on three samples. Representative cross-sectional SEM images of fish GelMA (C~F) and porcine GelMA (G~J) hydrogels reveal different gel morphologies after degradation with collagenase type II. (K) Pore size distribution of GelMA hydrogels (Pore size frequency obtained from 5 SEM images per condition).</p

Changes in Parthenogenetic Imprinting Patterns during Reprogramming by Cell Fusion

<div><p>Differentiated somatic cells can be reprogrammed into the pluripotent state by cell-cell fusion. In the pluripotent state, reprogrammed cells may then self-renew and differentiate into all three germ layers. Fusion-induced reprogramming also epigenetically modifies the somatic cell genome through DNA demethylation, X chromosome reactivation, and histone modification. In this study, we investigated whether fusion with embryonic stem cells (ESCs) also reprograms genomic imprinting patterns in somatic cells. In particular, we examined imprinting changes in parthenogenetic neural stem cells fused with biparental ESCs, as well as in biparental neural stem cells fused with parthenogenetic ESCs. The resulting hybrid cells expressed the pluripotency markers <i>Oct4</i> and <i>Nanog</i>. In addition, methylation of several imprinted genes except <i>Peg3</i> was comparable between hybrid cells and ESCs. This finding indicates that reprogramming by cell fusion does not necessarily reverse the status of all imprinted genes to the state of pluripotent fusion partner.</p></div

Bisulfite genome sequencing analysis of imprinted genes.

<p>DNA methylation patterns of paternally (<i>H19</i> and <i>Igf2</i>), and maternally imprinted genes (<i>Peg1</i> and <i>Peg3</i>) in ESCs, pESCs, NSCs, pNSCs, ES-pNSC, and pES-NSC hybrid cells. Black and white circles represent methylated and unmethylated CpGs, respectively.</p

Generation of parthenogenetic ESCs (pESCs) from parthenogenetically activated embryos.

<p><b>(A)</b> Preimplantation development of parthenogenetically activated embryos from one-cells to blastocyst stage embryos (200 ×). <b>(B)</b> Efficiency of development of parthenogenetic embryos. About 83% of oocytes were successfully activated, of which about 62% progressed to blastocyst stage. <b>(C)</b> Embryonic stem cells derived from parthenogenetic blastocysts (pESCs) were positive for the alkaline phosphatase staining (100 ×). <b>(D)</b> Immunocytochemistry of pESCs using Oct4 and Nanog antibodies (200 ×). pESCs were stained positive for key pluripotency markers, Oct4 and Nanog.</p

Quantitative RT-PCR analysis of imprinted gene expression.

<p>The expression profiles of paternal and maternal imprinted genes were analyzed by real-time RT-PCR. All data are normalized to <i>ACTB</i> expression and calibrated on the ESCs, whose gene expression was considered 1 for all genes. Error bars represent mean values ± SEM of three independent experiments. Student’s t-test: ***, p<0.001; **, p<0.01; *, p<0.05.</p

Generation of fusion hybrid cells between parthenogenetic and biparental cells.

<p><b>(A)</b> GFP fluorescence images of fusion between biparental ESCs and parthenogenetic neural stem cells (ES-pNSC), and between pESCs and biparental neural stem cells (pES-NSC) at day 3 after fusion (200 ×). <b>(B)</b> GFP fluorescence images of ES-pNSC and pES-NSC hybrids after FACS sorting (100 ×). <b>(C)</b> Representative tetraploid karyotype of the hybrid cells.</p