• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1309-1316
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• 2023

To exert their beneficial effects, it is essential for the commensal bacteria of probiotic supplements to be sufficiently protected as they pass through the low pH environment of the stomach, and effectively colonize the intestinal epithelium downstream. Here, we investigated the effect of a multilayer coating containing red ginseng dietary fiber, on the acid tolerance, and the adhesion and proliferation capacities of three Lactobacillus strains (Limosilactobacillus reuteri KGC1901, Lacticaseibacillus casei KGC1201, Limosilactobacillus fermentum KGC1601) isolated from Panax ginseng, using HT-29 cells, mucin-coated plates, and human pluripotent stem cell-derived intestinal epithelial cells as in vitro models of human gut physiology. We observed that the multilayer-coated strains displayed improved survival rates after passage through gastric juice, as well as high adhesion and proliferation capacities within the various gut epithelial systems tested, compared to their uncoated counterparts. Our findings demonstrated that the multilayer coat effectively protected commensal microbiota and led to improved adhesion and colonization of intestinal epithelial cells, and consequently to higher probiotic efficacy.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.67-75
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• 2011

The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFR ${\alpha}$-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs ($11.2{\pm}0.8%$) and SSCs ($13.3{\pm}1.1%$). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.

• International Journal of Stem Cells
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• v.16 no.3
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• pp.281-292
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• 2023

Background and Objectives: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) hold great promise as a cellular source of CM for cardiac function restoration in ischemic heart disease. However, the use of animal-derived xenogeneic substances during the biomanufacturing of hiPSC-CM can induce inadvertent immune responses or chronic inflammation, followed by tumorigenicity. In this study, we aimed to reveal the effects of xenogeneic substances on the functional properties and potential immunogenicity of hiPSC-CM during differentiation, demonstrating the quality and safety of hiPSC-based cell therapy. Methods and Results: We successfully generated hiPSC-CM in the presence and absence of xenogeneic substances (xeno-containing (XC) and xeno-free (XF) conditions, respectively), and compared their characteristics, including the contractile functions and glycan profiles. Compared to XC-hiPSC-CM, XF-hiPSC-CM showed early onset of myocyte contractile beating and maturation, with a high expression of cardiac lineage-specific genes (ACTC1, TNNT2, and RYR2) by using MEA and RT-qPCR. We quantified N-glycolylneuraminic acid (Neu5Gc), a xenogeneic sialic acid, in hiPSC-CM using an indirect enzyme-linked immunosorbent assay and liquid chromatography-multiple reaction monitoring-mass spectrometry. Neu5Gc was incorporated into the glycans of hiPSC-CM during xeno-containing differentiation, whereas it was barely detected in XF-hiPSC-CM. Conclusions: To the best of our knowledge, this is the first study to show that the electrophysiological function and glycan profiles of hiPSC-CM can be affected by the presence of xenogeneic substances during their differentiation and maturation. To ensure quality control and safety in hiPSC-based cell therapy, xenogeneic substances should be excluded from the biomanufacturing process.

• International Journal of Oral Biology
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• v.39 no.4
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• pp.177-185
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• 2014

Transfection is a gene delivery tool that is a popular means of manipulating cellular properties, such as induced pluripotent stem cell (iPSC) generation by reprogramming factors (Yamanaka factors). However, the efficiency of transfection needs to be improved. In the present study, three transfection protocols - non-liposomal transfection (NLT), magnetofection and electroporation - were compared by analysis of their transfection efficiencies and cell viabilities using human dental pulp cells (hDPC) and bovine fetal fibroblasts (bFF) as cell sources. Enhanced green fluorescent protein gene was used as the delivery indicator. For magnetofection, Polymag reagent was administrated. NLT, FuGENE-HD and X-treme GENE 9 DNA transfection reagents were used for NLT. For electroporation, the $Neon^{TM}$ and $NEPA21^{TM}$ electroporators were tested. $Neon^{TM}$ electroporation showed highest transfection efficiency when compared with NLT, magnetofection, and $NEPA21^{TM}$ electroporation, with transfection efficiency of about 33% in hDPC and 50% in bFF, based on viable cell population in each cell type. These results suggest that transfection by $Neon^{TM}$ electroporation can be used to deliver foreign genes efficiently in human and bovine somatic cells.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.25-31
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• 2001

During early development, a dramatic reduction in methylation levels occurs in mouse (Monk et al., 1987). The process of epigenetic reprogramming in early embryos erases gamete-specific methylation patterns inherited from the parents (Howlett ＆ Reik 1991, Monk et al., 1987, Oswald et al., 2000, Sanford et al., 1984). This genome-wide demethylation process may be a prerequisite for the formation of pluripotent stem cells that are important for the later development (Reik ＆ Surani 1997). During post-implantation development, a wave of de novo methylation takes place; most of the genomic DNA is methylated at defined developmental timepoints, whereas tissue-specific genes undergo demethylation in their tissues of expression (Kafri et al., 1992, Razin ＆ Kafri 1994). Another demethylation-remethylation cycle of epigenetic reprogramming takes place during gametogenesis and is necessary for resetting of genomic imprinting (Solter 1988). The dynamic epigenetic reprogramming events appear to be basic and are probably conserved in eutherian mammals (see below). (omitted)

• Animal Bioscience
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• v.36 no.2_spc
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• pp.339-349
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• 2023

Gene editing (GE) offers a new breeding technique (NBT) of sustainable value to animal agriculture. There are 3 GE working sites covering 5 feasible pathways to generate GE pigs along with the crucial intervals of GE/genotyping, microinjection/electroporation, induced pluripotent stem cells, somatic cell nuclear transfer, cryopreservation, and nonsurgical embryo transfer. The extension of NBT in the new era of pig breeding depends on the synergistic effect of GE and reproductive biotechnologies; the outcome relies not only on scientific due diligence and operational excellence but also on the feasibility of application on farms to improve sustainability.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.60-60
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• 2003

Embryonic stem (ES) cells have property of self-renewal and can differentiate into the cells of all three primary germ layers. Recently, many growth factors, alteration of culture condition and gene modifications have been used to differentiate mouse and human ES cells into specific cell types. This study was performed to evaluate the differentiation protocol for human ES cells to the endodermal lineage cells. Human ES cells (Miz-hESl ) were cultured on STO feeder layer mitotically inactivated with mitemycin C, and embryoid bodies (EBs) were formed by suspension culture. Differentiation protocol of EBs consisted of three steps: stage I, culture of EBs for 6 days with ITSFn medium; stage II, culture of stage I cells for 8 days with N2 medium ; stage III, culture of stage II cells for 22 days with N2 medium. mRNA levels of the endodermal lineage differentiation genes were analyzed by semi- quantitative RT-PCR. The Oct-4 expression, a marker of the pluripotent state, was detected in undifferentiated human ES cells but progressively decreased after EBs formation. Differentiating human ES cells expressed marker genes of endodermal differentiation and pancreatic islet cells. GATA4, a-fetoprotein, Glut-2, and Ngn3 were expressed in all stages. However, albumin and insulin were expressed in only stage III cells. The human ES cells can be differentiated into endodermal lineage cells by multiple step culture system using various supplements. We are developing the more effective protocols for guided differentiation of human ES cells.

• Reproductive and Developmental Biology
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• v.39 no.3
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• pp.89-95
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• 2015

The addition of growth factors and cytokines to in vitro culture (IVC) media could affect embryo development and the quality of the resulting blastocysts. The present study was performed to investigate the effect of porcine induced pluripotent stem cell (piPSC)-culture conditioned medium (CM) on the in vitro maturation (IVM) and development of parthenogentic embryos (parthenotes) in pigs. Cumulus-oocyte complexes (COCs) or activated oocytes were cultured in IVM or IVC medium supplemented with 0 (control), 25, or 50% of stem cell medium (SM) or CM, respectively. The maturation rate of CM-25% group was significantly improved when compared with control group (p<0.05), but that was not different among SM or CM groups. Blastocyst formation rate was significantly higher in CM-25% group (29.2%) than that of control (20.7%), SM-50% (19.6%) and CM-50% (23.66%, p<0.05). Cell number and the apoptotic cell index in blastocysts was significantly lower in SM-25% than in CM-25% group (p<0.05). The embryo quality related genes, OCT4, KLF4, TERT and ZFP42, were significantly increased in CM-25% group compared with control (p<0.05). In conclusion, the addition of 25% of CM to IVM and IVC medium positively influences not only the developmental potential also quality of parthenotes in pig.

• Reproductive and Developmental Biology
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• v.38 no.2
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• pp.79-84
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• 2014

MicroRNAs (miRNAs) are approximately 22 nucleotides of small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The miRNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, metabolism, imprinting, and differentiation. Recently, a paper has shown that expression of the miRNA-302/367 cluster expressed abundantly in mouse and human embryonic stem cells (ESCs) can directly reprogram mouse and human somatic cells to induced pluripotent stem cells (iPSCs) efficiently in the absence of any of the four factors, Oct4, Sox2, c-Myc, and Klf4. To apply this efficient method to porcine, we analyzed porcine genomic sequence containing predicted porcine miRNA-302/367 cluster through ENSEMBL database, generated a non-replicative episomal vector system including miRNA-302/367 cluster originated from porcine embryonic fibroblasts (PEF), and tried to make porcine iPSCs by transfection of the miRNA-302/367 cluster. Colonies expressing EGFP and forming compact shape were found, but they were not established as iPSC lines. Our data in this study show that pig miRNA-302/367 cluster could not satisfy requirement of PEF reprogramming conditions for pluripotency. To make pig iPSC lines by miRNA, further studies on the role of miRNAs in pluripotency and new trials of transfection with conventional reprogramming factors are needed.

• International Journal of Stem Cells
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• v.16 no.3
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• pp.326-341
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• 2023

Background and Objectives: Osteoarthritis (OA) is a degenerative disease that leads to the progressive destruction of articular cartilage. Current clinical therapeutic strategies are moderately effective at relieving OA-associated pain but cannot induce chondrocyte differentiation or achieve cartilage regeneration. We investigated the ability of wedelolactone, a biologically active natural product that occurs in Eclipta alba (false daisy), to promote chondrogenic differentiation. Methods and Results: Real-time reverse transcription-polymerase chain reaction, immunohistochemical staining, and immunofluorescence staining assays were used to evaluate the effects of wedelolactone on the chondrogenic differentiation of mesenchymal stem cells (MSCs). RNA sequencing, microRNA (miRNA) sequencing, and isobaric tags for relative and absolute quantitation analyses were performed to explore the mechanism by which wedelolactone promotes the chondrogenic differentiation of MSCs. We found that wedelolactone facilitates the chondrogenic differentiation of human induced pluripotent stem cell-derived MSCs and rat bone-marrow MSCs. Moreover, the forkhead box O (FOXO) signaling pathway was upregulated by wedelolactone during chondrogenic differentiation, and a FOXO1 inhibitor attenuated the effect of wedelolactone on chondrocyte differentiation. We determined that wedelolactone reduces enhancer of zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation of the promoter region of FOXO1 to upregulate its transcription. Additionally, we found that wedelolactone represses miR-1271-5p expression, and that miR-1271-5p post-transcriptionally suppresses the expression of FOXO1 that is dependent on the binding of miR-1271-5p to the FOXO1 3'-untranscribed region. Conclusions: These results indicate that wedelolactone suppresses the activity of EZH2 to facilitate the chondrogenic differentiation of MSCs by activating the FOXO1 signaling pathway. Wedelolactone may therefore improve cartilage regeneration in diseases characterized by inflammatory tissue destruction, such as OA.