As a preclinical study, many researchers have been attempted to convert the porcine PSCs into several differentiated cells with transplantation of the differentiated cells into the pigs. Here, we attempted to derive neuronal progenitor cells from pig embryonic germ cells (EGCs). As a result, neuronal progenitor cells could be derived directly from pig embryonic germ cells through the serum-free floating culture of EB-like aggregates (SFEB) method. Treating retinoic acid was more efficient for inducing neuronal lineages from EGCs rather than inhibiting SMAD signaling. The differentiated cells expressed neuronal markers such as PAX6, NESTIN, and SOX1 as determined by qRT-PCR and immunostaining. These data indicated that pig EGCs could provide valid models for human therapy. Finally, it is suggested that developing transgenic pig for disease models as well as differentiation methods will provide basic preclinical data for human regenerative medicine and lead to the success of stem cell therapy.

The deleted in azoospermia like (DAZL) gene has been identified in many vertebrate species. DAZL shows high homology with deleted in azoospermia (DAZ) genes that identified only in humans, great apes and Old World monkeys, and boule homolog (BOLL) that identified in many vertebrate species. These genes encode RNA binding proteins (RBP), which regulate the post-transcriptional functions of several genes. In humans, DAZ copies are linked to Y chromosome, while DAZL and BOLL are linked to chromosomes 3 and 2, respectively. DAZ copies has been reported to express in prenatal and postnatal germ cells, particularly in the premeiotic spermatogonia. BOLL has been reported to express during the meiotic G2/M transition in germ cells. DAZL has been reported to express in all stages of germ cells. Compared to humans and mice, the detailed functionalities of DAZL is not clear in many vertebrate species. In our studies, we use chickens as an animal model to examine the expression profiling of DAZL gene in germ cells right from the early embryonic development to the adult. Also, we are studying the effects of small interfering RNA (siRNA) mediated knockdown of DAZL and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) mediated knockout of DAZL in the chicken primordial germ cells (PGCs). In the chicken, DAZL is linked to chromosome 2 (2p1.3-p1.2), and encodes a 289 amino acids protein. By in situ hybridization, we detected a strong expression of DAZL in the germ plasm of chicken oocytes. Later, the expression of DAZL was strongly detected in all stages of intrauterine development and post-ovipositional development especially in the PGC specifying cells. Moreover, the expression of DAZL was strong and constant in the male and female germ cells until adult stage. The siRNA mediated knockdown of DAZL significantly reduced the PGCs proliferation and increased the apoptosis in vitro. We examined the knockout efficiency of DAZL using CRISPR/Cas9 technique in chicken DF1 fibroblast cell line, prior to test in the PGCs. The results of T7 endonuclease I (T7E1) assay and subsequent sequencing indicates clear mutations on the DAZL gene in DF1 cells, and the method could be applicable to cause mutations on the DAZL gene in PGCs. In conclusion, chicken DAZL express in all stages of germ cells as a germ line marker, and alteration in the gene expression causes germ cells impairment.

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

Pluripotent cells are categorized as either "naive" or "primed" based upon their pluripotent status. According to previous studies, embryonic stem cells and embryonic germ cells are identified as naive pluripotent stem cells and epiblast stem cells are identified as primed pluripotent stem cells. In a permissive species such as the mouse, naive and primed pluripotent stem cells can be derived from embryos without genetic manipulations. In non-permissive species such as humans and pigs, primed pluripotent cells are only established from embryos. However, previous studies have shown that the embryonic germ cells of non-permissive species share similar morphology and features with naive pluripotent cells. For these reasons porcine embryonic germ cells (pEGCs) may provide a useful cell source for comparative studies on naive pluripotent cells in non-permissive species. In this study, we attempted to establish and characterize porcine embryonic germ cells. Consequently, an embryonic germ cell line was derived from the genital ridges of a porcine dpc 30 fetus in media containing LIF and bFGF. After establishment, this cells were cultured and stabilized in LIF or bFGF contained media. This cell lines displayed a dome-shaped colony morphology in both culture condition. The cell lines were maintained in both condition over an extended time period and were able to differentiate into the three germ layers in vitro. Interestingly, cell lines cultured in LIF or bFGF expressed different pluripotency markers. LIF-dependent pEGCs expressed naive-pluripotency markers such as OCT4, SOX2, NANOG and SSEA1, while bFGF-dependent pEGCs expressed primed-pluripotency markers such as OCT4, SOX2, NANOG and SSEA4. However, as a result of analysis of XCI, two cell lines showed hemi-methylated pattern similarly in XIST promoter regions. In conclusion, we were able to successfully derive embryonic germ cells from genital ridges of a porcine fetus. Pluripotent state of pEGCs were regulated by modulation of culture condition. In LIF supplement, pEGCs showed naive-pluripotency expressing SSEA1, while pEGCs show primed-pluripotency expressing SSEA4 in bFGF condition. This cell line could potentially be used as naive pluripotent cell source for comparative study with porcine embryonic stem cells and other pluripotent cell lines. As porcine pluripotent cells, pEGCs could be useful candidates for preliminary studies of human disease as well as a source for generating transgenic animals.

In the present study, embryoid bodies (EBs) obtained from induced pluripotent stem cells (iPSCs) were induced to differentiate into germ lineage cells by treatment with bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). The results were compared to the results for embryonic stem cells (ESCs) and multipotent spermatogonial stem cells (mSSCs) and quantified using immunocytochemical analysis of germ cell-specific markers (integrin-, GFR-, CD90/Thy1), fluorescence activating cell sorting (FACS), and real time-RT-PCR. We show that the highest levels of germ cell marker-expressing cells were obtained from groups treated with 10 ng/ BMP4 or 0.01 RA. In the BMP4-treated group, GFR- and CD90/Thy-1 were highly expressed in the EBs of iPSCs and ESCs compared to EBs of mSSCs. The expression of Nanog was much lower in iPSCs compared to ESCs and mSSCs. In the RA treated group, the level of GFR- and CD90/Thy-1 expression in the EBs of mSSCs Induced pluripotent stem cells, Mouse embryonic stem cells, Multipotent spermatogonial stem cells, Germ cell lineage, Differentiation potential. was much higher than the levels found in the EBs of iPSCs and similar to the levels found in the EBs of ESCs. FACS analysis using integrin-, GFR-, CD90/Thy1 and immunocytochemistry using GFR- antibody showed similar gene expression results. Therefore our results show that iPSC has the potential to differentiate into germ cells and suggest that a protocol optimizing germ cell induction from iPSC should be developed because of their potential usefulness in clinical applications requiring patient-specific cells.