• Journal of Embryo Transfer
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• v.28 no.1
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• pp.1-6
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• 2013

A lot of works have been dedicated to clarify the reasons why the establishment of embryonic stem cells (ESCs) from pig is more difficult than that from mouse and human. Several concomitant factors such as culture condition including feeder layer, sensitivity of cell to cell contact, definitive markers of pluripotency for evaluation of the validity and optimal timing of derivation have been suggested as the disturbing factors in the establishment of porcine ESCs Traditionally, attempts to derive stem cells from porcine embryos have depend on protocols established for mouse ESCs using inner cell mass (ICM) for the isolation and culture. And more recently, protocols used for primate ESCs were also applied. However, there is no report for the establishment of porcine ESCs. Indeed, ungulate species including pigs have crucial developmental differences unlike rodents and primates. Here we will review recent studies about issues for establishment of porcine ESCs and discuss the promise and strategies focusing on the timing for derivation and pluripotent state of porcine ESCs.

• Molecules and Cells
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• v.19 no.1
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• pp.46-53
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• 2005

Human embryonic stem (hES) cells, unlike most cells derived from adult or fetal human tissues, represent a potentially unlimited source of various cell types for basic clinical research. To meet the increased demand for characterized hES cell lines, we established and characterized nine new lines obtained from frozen-thawed pronucleus-stage embryos. In addition, we improved the derivation efficiency from inner cell masses (to 47.4%) and optimized culture conditions for undifferentiated hES cells. After these cell lines had been maintained for over a year in vitro, they were characterized comprehensively for expression of markers of undifferentiated hES cells, karyotype, and in vitro/in vivo differentiation capacity. All of the cell lines were pluripotent, and one cell line was trisomic for chromosome 3. Improved culture techniques for hES cells should make them a good source for diverse applications in regenerative medicine, but further investigation is needed of their basic biology.

• Molecules and Cells
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• v.25 no.3
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• pp.358-367
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• 2008

The embryonic germ cell (EGCs) of mice is a kind of pluripotent stem cell that can be generated from pre- and post-migratory primordial germ cells (PGCs). Most previous studies on DNA methylation of EGCs were restricted to 12.5 days post coitum (dpc). This study was designed to establish and characterize murine EGC lines from migrated PGCs as late as 13.5 dpc and to estimate the degrees of methylation of their imprinted genes as well as of the non-imprinted locus, Oct4, using an accurate and quantitative method of measurement. We established five independent EGC lines from post migratory PGCs of 11.5-13.5 dpc from C57BL/6 ${\times}$ DBA/2 F1 hybrid mouse fetuses. All the EGCs exhibited the typical features of pluripotent cells including hypomethylation of the Oct4 regulatory region. We examined the methylation status of three imprinted genes; Igf2, Igf2r and H19 in the five EGC lines using bisulfite genomic sequencing analysis. Igf2r was almost unmethylated in all the EGC lines irrespective of the their sex and stage of isolation; Igf2 and H19 were more methylated than Igf2r, especially in male EGCs. Moreover, EGCs derived at 13.5 dpc exhibited higher levels of DNA methylation than those from earlier stages. These results suggest that in vitro derived EGCs acquire different epigenotypes from their parental in vivo migratory PGCs, and that sex-specific de novo methylation occurs in the Igf2 and H19 genes of EGCs.

• Molecules and Cells
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• v.41 no.2
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• pp.73-82
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• 2018

Cancer preventive activities of green tea and its main constituent, (-)-epigallocatechin gallate (EGCG) have been extensively studied by scientists all over the world. Since 1983, we have studied the cancer chemopreventive effects of EGCG as well as green tea extract and underlying molecular mechanisms. The first part of this review summarizes groundbreaking topics with EGCG and green tea extract: 1) Delayed cancer onset as revealed by a 10-year prospective cohort study, 2) Prevention of colorectal adenoma recurrence by a double-blind randomized clinical phase II trial, 3) Inhibition of metastasis of B16 melanoma cells to the lungs of mice, 4) Increase in the average value of Young's moduli, i.e., cell stiffness, for human lung cancer cell lines and inhibition of cell motility and 5) Synergistic enhancement of anticancer activity against human cancer cell lines with the combination of EGCG and anticancer compounds. In the second part, we became interested in cancer stem cells (CSCs). 1) Cancer stem cells in mouse skin carcinogenesis by way of introduction, after which we discuss two subjects from our review on human CSCs reported by other investigators gathered from a search of PubMed, 2) Expression of stemness markers of human CSCs compared with their parental cells, and 3) EGCG decreases or increases the expression of mRNA and protein in human CSCs. On this point, EGCG inhibited self-renewal and expression of pluripotency-maintaining transcription factors in human CSCs. Human CSCs are thus a target for cancer prevention and treatment with EGCG and green tea catechins.

• Development and Reproduction
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• v.21 no.4
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• pp.425-434
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• 2017

Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1307-1314
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• 2015

Leukemia inhibitory factor (LIF) is a member of the IL-6 cytokine family, having pleiotropic actions such as maintaining stem cell pluripotency and enabling blastocyst implantation. Because the action of LIF is mediated by a ligand-receptor interaction with the LIF receptor (LIF-R), an antagonist for LIF-R has been developed to inhibit LIF-induced signaling. In this study, we present a novel method for the production and purification of an antagonist to human LIF-R (hLA). His-tagged hLA was expressed in E. coli, and simple purification methods without any endopeptidase cleavage were designed. In addition, we determined the optimal temperature conditions for enhancing the production of soluble hLA. Finally, the bioactivity of His-tagged hLA was examined using STAT3 phosphorylation and receptivity of human endometrial ECC-1 cells. Our strategy provides a rapid and efficient method to produce biologically active recombinant hLA.

• Journal of Veterinary Science
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• v.22 no.6
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• pp.86.1-86.13
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• 2021

Background: Conditioned medium is the medium obtained from certain cultured cells and contained secretome from the cells. The secretome, which can be in the form of growth factors, cytokines, exosomes, or other proteins secreted by the cells, can induce the differentiation of cells that still have pluripotent or multipotent properties. Objectives: This study examined the effects of conditioned medium derived from E17 rat brain cells on cells with pluripotent properties. Methods: The conditioned medium used in this study originated from E17 rat brain cells. The CM was used to induce the differentiation of primary colonies of mice blastocysts. Primary colonies were stained with alkaline phosphatase to analyze the pluripotency. The morphological changes in the colonies were examined, and the colonies were stained with GFAP and Neu-N markers on days two and seven after adding the conditioned medium. Results: The conditioned medium could differentiate the primary colony, beginning with the formation of embryoid-body-like structure; round GFAP positive cells were identified. Finally, neuron-like cells testing positive for Neu-N were observed on the seventh day after adding the conditioned medium. Conclusions: Conditioned medium from different species, in this case, E17 rat brain cells, induced and promoted the differentiation of the primary colony from mice blastocysts into neuron-like cells. The addition of CM mediated neurite growth in the differentiation process.

• Molecules and Cells
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• v.42 no.2
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• pp.135-142
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• 2019

OCT4, also known as POU5F1 (POU domain class 5 transcription factor 1), is a transcription factor that acts as a master regulator of pluripotency in embryonic stem cells and is one of the reprogramming factors required for generating induced pluripotent stem cells. The human OCT4 encodes three isoforms, OCT4A, OCT4B, and OCT4B1, which are generated by alternative splicing. Currently, the functions and expression patterns of OCT4B remain largely unknown in malignancies, especially in human glioblastomas. Here, we demonstrated the function of OCT4B in human glioblastomas. Among the isoform of OCT4B, OCT4B-190 ($OCT4B^{19kDa}$) was highly expressed in human glioblastoma stem cells and glioblastoma cells and was mainly detected in the cytoplasm rather than the nucleus. Overexpression of $OCT4B^{19kDa}$ promoted colony formation of glioblastoma cells when grown in soft agar culture conditions. Clinical data analysis revealed that patients with gliomas that expressed OCT4B at high levels had a poorer prognosis than patients with gliomas that expressed OCT4B at low levels. Thus, $OCT4B^{19kDa}$ may play a crucial role in regulating cancer cell survival and adaption in a rigid environment.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.343-348
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• 2018

Although the efforts to establish fish embryonic stem cells (ESCs) have been made for a long time, derivation of authentic ESCs that possess pluripotency is still difficult suggesting a need for the stepwise optimization of the methods to establish fish ESCs. Primary culture of the blastomeres from the embryos at blastula stage is a critical step for establishing continuous ESC lines. Here, we evaluated the effects of temperatures and basal media on primary culture of blastula embryo-derived blastomeres in marine medaka (Oryzias dancena). The blastomeres were isolated from the blastula embryos and cultured in various conditions designed by the combination of 4 temperatures including $28^{\circ}C$, $31^{\circ}C$, $34^{\circ}C$, and $37^{\circ}C$ and 2 basal media including Dulbecco's modified eagle's medium (DMEM) and Leibovitz's L-15 medium (L15). With the exception of a case cultured in L15 at $31^{\circ}C$, the rate of primary cell adherence reached 100% when the blastomeres were cultured over $31^{\circ}C$. The period for primary adherence was significantly shorter in the groups cultured in $34^{\circ}C$ and $37^{\circ}C$ than in the ones in $28^{\circ}C$ and $31^{\circ}C$. The proportion of subculture was significantly high in the group cultured in DMEM at $31^{\circ}C$ compared to the other groups. Collectively, we demonstrated that the culture in DMEM at $31^{\circ}C$ was effective to primary culture of the blastomeres derived from blastula embryos.

• Biomaterials Research
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• v.22 no.4
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• pp.337-345
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• 2018

Background: Human mesenchymal stem cells (hMSCs) are, due to their pluripotency, useful sources of cells for stem cell therapy and tissue regeneration. The phenotypes of hMSCs are strongly influenced by their microenvironment, in particular the extracellular matrix (ECM), the composition and structure of which are important in regulating stem cell fate. In reciprocal manner, the properties of ECM are remodeled by the hMSCs, but the mechanism involved in ECM remodeling by hMSCs under topographical stimulus is unclear. In this study, we therefore examined the effect of nanotopography on the expression of ECM proteins by hMSCs by analyzing the quantity and structure of the ECM on a nanogrooved surface. Methods: To develop the nanoengineered, hMSC-derived ECM, we fabricated the nanogrooves on a coverglass using a UV-curable polyurethane acrylate (PUA). Then, hMSCs were cultivated on the nanogrooves, and the cells at the full confluency were decellularized. To analyze the effect of nanotopography on the hMSCs, the hMSCs were re-seeded on the nanoengineered, hMSC-derived ECM. Results: hMSCs cultured within the nano-engineered hMSC-derived ECM sheet showed a different pattern of expression of ECM proteins from those cultured on ECM-free, nanogrooved surface. Moreover, hMSCs on the nano-engineered ECM sheet had a shorter vinculin length and were less well-aligned than those on the other surface. In addition, the expression pattern of ECM-related genes by hMSCs on the nanoengineered ECM sheet was altered. Interestingly, the expression of genes for osteogenesis-related ECM proteins was downregulated, while that of genes for chondrogenesis-related ECM proteins was upregulated, on the nanoengineered ECM sheet. Conclusions: The nanoengineered ECM influenced the phenotypic features of hMSCs, and that hMSCs can remodel their ECM microenvironment in the presence of a nanostructured ECM to guide differentiation into a specific lineage.