Pluripotent stem cells could self-renew and differentiate into various cells. In particular, porcine pluripotent stem cells are useful for preclinical therapy, transgenic animals, and agricultural usage. These stem cells have naïve and primed pluripotent states. Naïve pluripotent stem cells represented by mouse embryonic stem cells form chimeras after blastocyst injection. Primed pluripotent stem cells represented by mouse epiblast stem cells and human embryonic stem cells. They could not produce chimeras after blastocyst injection. Populations of embryonic stem cells are not homogenous; therefore, reporter systems are used to clarify the status of stem cells and to isolate the cells. For this reason, studies of the OCT4 reporter system have been conducted for decades. This review will discuss the naïve and primed pluripotent states and recent progress in the development of porcine OCT4 reporter systems.

Induced pluripotent stem cells (iPSCs) can be generated from adult cells. Somatic cells can be reprogrammed to form iPSCs by overexpressing transcription factors such as Oct4, Sox2, cMyc, and Klf4. To maintain undifferentiated state of iPSCs in vitro, cells have traditionally been maintained on mouse embryonic fibroblast feeders and passaged by enzymatic or mechanical dissociation methods. In this study, we compared the morphology and pluripotency of porcine iPSCs (piPSCs) after subsequent passaging using enzymatic and mechanical dissociation methods. Enzymatically and mechanically passaged piPSCs showed embryonic stem cell-like morphologies with compact cell adhesion and clear colony borders. In addition, alkaline phosphatase staining was positive for both enzymatically and mechanically passaged piPSCs. However, visual observation revealed that some colonies of enzymatically passaged piPSCs were spontaneously differentiated more than those of piPSCs mechanically passaged from 5 passage. Quantitative real-time RT-PCR demonstrated that enzymatically and mechanically passaged piPSCs expressed pluripotent genes such as Oct4, Sox2 and Nanog well at early passage. Immunofluorescent staining also confirmed that pluripotent markers such as Oct4, Sox2, and Nanog were positively expressed at early passage. However, expression levels of pluripotent genes in mechanically passaged piPSCs were also higher than those in enzymatically passaged piPSCs at early passage. Collectively, we found that mechanical passage method was better than enzymatic passage in terms of morphology and pluripotency of piPSCs at early passage. Further studies are needed to compare these dissociation methods with those obtained after more passages of piPSCs.

Characteristics of induced pluripotent stem (iPS) cells are consistent with those of embryonic stem (ES) cells. However, exogenous genes integrated by using retrovirus delivery systems cannot be completely removed from the cells. In a recent report, activation-induced cytosine deaminase (AID) and thymine DNA glycosylase (TDG) can induce pluripotency state in mouse differentiated cells through the process of DNA demethylation. Thus, we hypothesized that the two reprogramming factors may convert efficiently bovine differentiated cells into pluripotency state. So, genes of AID and TDG were integrated into pCMV6-AC-IRES-GFP-Puro expression vector, which was transfected into bovine differentiated cells. As results, the colonies derived from AID+TDG-induced bovine cells were formed on day 7 after culture. The number of AP positively colonies in AID+TDG-induced bovine cells was significantly higher than in AID-induced bovine cells (p<0.05). Additionally, expression of pluripotent genes (OCT-3/4, NANOG, SOX2) was slightly increased in AID+TDG-induced bovine cells, as compared to AID-induced bovine cells. Protein expressions of OCT-3/4, NANOG and SOX2 in AID+TDG-induced bovine cells were slightly increased rather than AID-induced bovine cells. Finally, DNA demethylation in the promoter regions of pluripotent markers in AID+TDG-induced bovine cells was increased than that of AID-induced bovine cells. In conclusion, pluripotent stem cells could be efficiently produced from bovine differentiated cells by using non-integrating delivery system with the reprogramming factors (AID and TDG).

The estrogen-mediated effect of mesenchymal stem cells (MSCs) is a highly critical factor for the clinical application of MSCs. However, the present study is conducted on MSCs derived from adult donors, which have different physiological status with steroid hormonal changes. Therefore, we explores the important role of 17β-estradiol (E2) in MSCs derived from female and male newborn piglets (NF- and NM-pBMSCs), which are non-sexually matured donors with steroid hormones. The results revealed that in vitro treatment of MSCs with E2 improved cell proliferation, but the rates varied according to the gender of the newborn donors. Following in vitro treatment of newborn MSCs with E2, mRNA levels of Oct3/4 and Sox2 increased in both genders of MSCs and they may be correlated with both estrogen receptor α (ERα) and ERβ in NF-pBMSCs, but NM-pBMSCs were only correlated with ERα. Moreover, E2-treated NF-pBMSCs decreased in β-galactosidase activity but no influence on NM-pBMSCs. In E2-mediated differentiation capacity, E2 induced an increase in the osteogenic and chondrogenic abilities of both pBMSCs, but adipogenic ability may increased only in NF-pBMSCs. These results demonstrate that E2 could affect both genders of newborn donor-derived MSCs, but the regulatory role of E2 varies depending on gender-dependent characteristics even though the original newborn donors had not been affected by functional steroid hormones.

To investigated the mechanism, induced pluripotent stem cells(iPSC) is important for clinical application and stem cell research. It is well known that hMAGEA2 expression pattern and effect on differentiation in embryonic stem cell but their specific role in iPS cells are unclear. The present study was schemed to understand the function of hMAGEA2 gene in iPS cells and to elucidate its characteristic. Although overexpression of hMAGEA2 in iPS cells are not different on morphology, their pluripotency and self-renewal capacity are significantly strengthened. And hMAGEA2 contributed to promote the cell cycle progression, this cell cycle changes induced proliferation acceleration. Through embryoid body formation in vitro and teratoma formation in vivo, we found that hMAGEA2 critically decreases the differentiation ability in iPS cells. Our results demonstrate that hMAGEA2 intensified the self-renewal, pluripotency, proliferation degree but efficiency of differentiaton is significantly repressed. Our findings provided that hMAGEA2 play a key role of iPS cells.

To have a better understanding of pluripotency, whole gene expression of embryo-derived stem cells (EdSCs) in bovine species was investigated. EdSCs were established from the embryos produced by in vitro fertilization, parthenogenesis and somatic cell nuclear transfer. Then, the microarray was performed and analyzed. Differently expressed genes (DEGs) were also confirmed by Real-time PCR. Among 10,203 DEGs, little difference was found in gene expression among three kinds of EdSCs. Conversely, all EdSCs have an immensely different gene expression when compared with somatic cells, consistent with scatter plat results. To investigate shared pathways for pluripotency in all EdSCs, 2,415 co-DEGs were identified which compared with somatic cells. By KEGG database, there were 54 signaling pathways in co-DEGs and some of them were related with pluripotency maintenance such as TGFβ, WNT and JAK-STAT signaling. In TGFβ signaling, BMP family and SMAD family were involved in co-up-regulated DEGs. In WNT signaling, WNT family and receptors were included in co-up-regulated DEGs, while inhibitors of WNT signaling were associated with co-down-regulated DEGs. In JAK-STAT signaling, STAT3 belonged to co-down-regulated DEGs. These DEGs were also confirmed by Real-time PCR. Taken together, BMP and WNT pathways may be activated and paly central roles to retain pluripotency in bovine EdSCs, whereas the LIF/STAT3 pathway may not be operated well. This study was supported by a grant from the National Research Foundation of Korea (NRF-2006-2004042, and No. 2015048003 through the Oromaxillofacial Dysfunction Research Center for the Elderly at Seoul National University) and the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture, Food and Rural Affairs (MAFRA; 111160-04), Republic of Korea.

Skin-derived precursor cells (SKPs) are multipotent, sphere-forming and embryonic neural crest‐related precu- rsor cells that can be isolated from dermis. It is known that the properties of porcine SKPs can be enhanced by leuke- mia inhibitory factor (LIF) which is an essential factor for the generation of embryonic stem cells in mice. In our pre- sent study, to enhance or maintain the properties of murine SKPs, LIF was added to the culture medium. SKPs were treated with 1,000 IU LIF for 72 hours after passage 3. Quantitative real time RT‐PCR was then performed to quantify the expression of the pluripotent stem cell specific genes Oct4, Nanog, Klf4 and c‐Myc, and the neural crest specific genes Snai2 and Ngfr. The results show that the expression of Oct4 is increased in murine SKPs by LIF treatment whereas the level of Ngfr is decreased under these conditions. Interestingly, LIF treatment reduced Nanog exp- ression which is also important for cell proliferation in adult stem cells and for osteogenic induction in mesenchymal stem cells. These findings implicate LIF in the maintenance of stem- ness in SKPs through the suppression of lineage differen- tiation and in part through the control of cell proliferation.

Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cell (iPS) experiments have generally demonstrated that a differentiated cell directly converts into a undifferentiated or pluripotent state. In SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor cell nuclei to the recipient cytoplasm of matured oocytes. Although nuclear reprogramming of cells by the ex-ovo methods is not always consistent or efficient, it has been suggested that a combination of nuclear reprogramming technique may improve the efficiency or frequency of normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from GV stage sturgeon's oocytes prior to their use as nuclear donors for SCNT will improve subsequent development. We reported a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte exteact (SOE) to porcine fetal fibroblast cell nuclei ex ovo. Porcine fibroblasts were permeabilized by 4 μg/ml of digitonin for 2 min at 4℃ and then incubated in SOE for 7h at 15 18℃ followed by resealing of cell membrane. As results, no difference was observed in the number of fused couplets or the number of fused couplets that cleaved between the extract treated or control group. However, there was a significantly decrease in the percentage of fused couplets that developed to the blastocyst stage in the SOE treated group (p<0.05). Histone acetylation status was determined using an antibody to acetylation at lysine 9 on histone 3 (H3K9Ac). The intensity of H3K9Ac staining in 1-cell stage NT embryos was significantly increased when treated with the SOE (p<0.05), similar to that in 1-cell stage IVF embryos. In addition, porcine NT embryos reconstructed by using donor cell exposed to SOE prior to cell fusion significantly decreased developmental competence to the blastocyst stage but increased pluripotent gene expressions (Sox2, Nanog and Oct3/4) when compared with those in normal NT embryos (p<0.05).

Although somatic cell nuclear transfer (SCNT) has successfully been produced cloned animals in several species, the cloning efficiency is extremely low. It is generally believed that the low cloning efficiency is mainly attributed to faulty epigenetic modifications underlying the aberrant reprogramming of donor cell nuclei in recipient cytoplasm after SCNT. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be a key factor in improving the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi), such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increase histone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmental competence and pluripotency-related gene expressions (Nanog, Oct3/4 and Sox2) in porcine cloned blastocysts. The porcine cloned embryos were treated with a 50 nM concentration of TSA or a 100 μm concentration of CBHA during the in vitro early culture (10h) after cell fusion and then were assessed to cleavage rate, development to the blastocyst stage and pluripotency-related gene expressions in NT blastocysts. All data was analyzed by chi-square. Following 4-5 replicates (245, 200 and 222 for NT, TSA and CBHA treated NT embryos respectively) there was no difference between normal NT and CBHA treated NT embryos, whereas TSA treated NT embryos was significantly decreased for cleavage rate (p<0.05). The developmental competence to the blastocyst stage in CBHA treated NT embryos (18.9%) significantly increased than that of normal NT and TSA treated NT embryos (9.4% and 11.5%) (p<0.05). In addition, all of pluripotent transcription factors (Nanog, Oct3/4 and Sox2) were highly expressed in the CBHA treated NT embryos, however, Sox2 and Oct3/4 were expressed in TSA treated NT embryos and Sox2 was only expressed in normal NT embryos (p<0.05). In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increased the developmental competence of porcine NT embryos and pluripotency- related gene expressions (Nanog, Oct3/4 and Sox2) in NT blastocysts.

Somatic cells achieve a pluripotent state by pluripotential reprogramming. During pluripotential reprogramming, somatic cells re-established various features of pluripotent cells, such as the expression of pluripotency markers, inactivation of tissue-specific gene expression, developmental potential to contribute to all three germ layers, and an undifferentiated epigenetic state. Induced pluripotent stem (iPS) cells undergo unlimited self-renewal and have differentiation potential into various types of cells like embryonic stem cells. These iPS cells are potentially a valuable source of immune matched pluripotent stem cells that can be differentiated and used for tissue replacement therapies. Recent technical advance in direct reprogramming of somatic cells lead to a safe, viral- free iPS cell generation. Here we develop new techniques to generate iPS cells. Using titanium oxide (TiO2) nanotubes. we could successfully transfer reprogramming proteins (Oct4, Sox2, Klf4, Nanog and c-Myc) into somatic cells. After two weeks of treatment of protein conjugated nanotubes, somatic cells adopted an ES cell-like morphology and activated Oct4-GFP, which is pluripotency marker, indicating that nanotubes can be used for protein delivery carriers, which induce cellular reprogramming. Next, we induced differentiation of iPS cells into neural stem cells (NSCs) and compared with mouse embryonic stem (ES) cell-derived NSCs. NSCs from ES and iPS cells were morphologically indistinguishable from NSCs from brain tissue and rapidly propagated in the presence EGF and bFGF, and stained positive for NSCs markers Nestin and Sox2. Moreover, these NSCs have capacity of differentiation into multiple cell lineages, such as neurons, astrocytes, and oligodendrocytes. Induction of pluripotency and directed differentiation of iPS cells into a specialized cell type hold considerable promise for regenerative medicine as well as basic research.

Cells that have endogenous multipotent properties can be used as a starting source for the generation of induced pluripotent cells (iPSC). In addition, small molecules associated with epigenetic reprogramming are also widely used to enhance the multi- or pluripotency of such cells. Skinderived precursor cells (SKPs) are multipotent, sphereforming and embryonic neural crest-related precursor cells. These cells can be isolated from a juvenile or adult mammalian dermis. SKPs are also an efficient starting cell source for reprogramming and the generation of iPSCs because of the high expression levels of Sox2 and Klf4 in these cells as well as their endogenous multipotency. In this study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was tested in the generation of iPSCs as a potential enhancer of the reprogramming potential of SKPs. SKPs were isolated from the back skins of 5-6 week old C57BL/6 X DBA/2 F1 mice. After passage 3, the SKPs was treated with 2 mM of VPA and the quantitative real time RT-PCR was performed to quantify the expression of Oct4 and Klf4 (pluripotency specific genes), and Snai2 and Ngfr (neural crest specific genes). The results show that Oct4 and Klf4 expression was decreased by VPA treatment. However, there were no significant changes in neural crest specific gene expression following VPA treatment. Hence, although VPA is one of the most potent of the HDAC inhibitors, it does not enhance the reprogramming of multipotent skin precursor cells in mice.

To identify genes implicated in the control of pluripotency as well as characteristics of stem cells, we analyzed expression profiles of genes derived from mouse morulas, blastocysts, embryonic stem cells, mesenchymal stem cells, and uterus tissue using cDNA microarray. Comparative analyses of their expression profiles identified putative clones that expressed specifically in specific samples or not in a specific sample. The expression pattern of these condidate clones was analyzed using RT-PCR and non-radioactive in situ hybridization. Functional annotation of these clones on pluripotency and stem cell plasticity is in ongoing. These studies may further our understanding on the nature of the stem cells and molecular mechanisms underlying many facets of mammalian development and differentiation.

The formation of definitive endoderm (DE) is a fundamental step for the development of the gastrointestinal tract, respiratory tract and endocrine organs. We present a CRISPR-based pooled screening approach to identify genes which contribute to DE induction from hiPSCs in vitro. CRISPR-based pooled genetic screens in mammalian cell culture enable researchers to identify genes required for a cellular phenotype of interest in an unbiased way. To enable a CRISPR-based forward genetic screen for identifying regulatory genes required for DE differentiation from hiPSCs, we performed pooled screens using a human genome-scale CRISPR knockout library. In addition, we performed a transcriptional activation screen using a lentiviral CRISPRa library to identify the downstream targets of the TGF/nodal/activin signaling pathway, which is a key signaling pathway for DE specification. We identified several signaling pathways including TGFβ, Erk, JNK, and CREB pathways are involved with DE differentiation. We suggest that CRISPR-based pooled genetic screens are a useful tool to identify key signaling pathways and genes required for in vitro differentiation processes and are served as a platform to improve differentiation protocols.

Estrogen related receptor β(Esrrb)는 오르판 수용체 중 하나로 전분화능 관련유전자인 Oct4와 Nanog의 발현을 조절함으로써 줄기세포의 미분화를 유지시키고, 지속적인 자기 복제를 가능케 하는 유전자로 알려져 있다. 또한 Feng 등 (2009)은 체세포에 Oct4, Sox2와 함께 Esrrb 유전자를 함께 도입하면, 유전자가 변형된 체세포가 배아 줄기세포와 유사한 유도만능줄기세포로 리프로그래밍(reprograming)되어 진다는 결과를 보고한 바 있다. 본 연구에서는 인간 ESRRB 단백질을 양수유래줄기세포 내로 직접도입하는 방법을 개발하고, 이를 통해 전분화능 관련유전자의 기능 조절을 확인하고자 하였다. 클로닝 된 인간 short-form ESRRB를 세포투과 펩타이드(cell-penetrating peptide, CPP)의 일종인 R7(아르기닌 7개)에 접합(Fusion)하였고, 합성단백질 (R7-ESRRB-His6)의 형태로 배양중인 인간 양수 유래 줄기세포에 처리하여 세포내로 도입하였다. R7-ESRRB-His6 단백질은 5시간 내에 세포막을 통과하였고, 24시간 내에 핵 내로 이동하였다. 또한 핵 내로 이동한 ESRRB 단백질은 OCT4와 NANOG 유전자의 발현을 증가시켰을 뿐만 아니라, 또 다른 전분화능 관련유전자인 SOX2의 발현도 함께 증가시킨다는 것을 확인하였다. 이상의 결과는 세포투과 펩타이드와 유전자의 접합을 통해 생산된 R7-ESRRB-His6 합성단백질이 양수유래줄기세포내로 원활하게 도입되는 것을 확인하였고, 유전자의 변형 없이 전분화능 관련유전자의 기능을 조절할 수 있는 방법임을 확인하였다.

To identify genes implicated in the control of pluripotency as well as characteristics of stem cells, we analyzed expression profiles of genes derived from mouse morulas, blastocysts, embryonic stem cells, mesenchymal stem cells, and uterus tissue cDNA microarray. Comparative analyses of their expression profiles identified putative clones that expressed specifically in specific samples or not in a specific sample. The expression pattern of these candidate clones was analyzed using RT-PCR and non-radioactive in situ hybridization. Functional annotation of these clones on pluripotency and stem cells and molecular mechanisms underlying many facets of mammalian development and differentiation.