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

Characterization of hybrid cells.

<p><b>(A)</b> Both ES-pNSC and pES-NSC hybrid cells are positive for alkaline phosphatase staining (100 ×). <b>(B)</b> RT-PCR analysis of <i>Oct4</i>, <i>Nanog</i>, <i>Sox2</i>, and <i>Nestin</i> expression in fusion partner and reprogrammed hybrid cells. Pluripotency markers, <i>Oct4</i> and <i>Nanog</i>, which were not expressed in NSCs and pNSCs were expressed in GFP⁺ fusion hybrid cells. On the other hand, <i>Nestin</i>, which was expressed in NSCs and pNSCs was silenced after forming GFP⁺ fusion hybrid cells. <b>(C)</b> Immunocytochemistry analysis of Oct4 and Nanog in ES-pNSC and pES-NSC hybrid cells (100 ×). <b>(D)</b> <i>In vitro</i> differentiation of ES-pNSC and pES-NSC hybrid cells into ectoderm (Tuj1), mesoderm (SMA), and endoderm (Sox17) lineages (200 ×). <b>(E)</b> In vivo differentiation potential of ES-pNSC and pES-NSC hybrid cells through teratoma assay. These hybrid cells were contributed to secretory epithelium (ectoderm), cartilage (mesoderm) and gut epithelium (endoderm), which were stained with PAS, Asian blue, and hematoxylin eosin, respectively. Each tissue was indicated by arrow head.</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

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

Differentially expressed genes in iPS-NSCs and iPS-cNSCs.

<p><b>(A)</b> Up-regulated or down-regulated genes in iPS-NSCs or iPS-cNSCs, compared with the corresponding expression levels in brain-derived NSCs. <b>(B)</b> GO analysis of genes that were up-regulated in iPS-NSCs, when compared with brain-derived NSCs. <b>(C)</b> GO analysis of genes that were up-regulated in iPS-cNSCs, when compared with brain-derived NSCs. <b>(D)</b> GO analysis of down-regulated genes in iPS-NSCs. <b>(E)</b> GO analysis of down-regulated genes in iPS-cNSCs.</p