• Reproductive and Developmental Biology
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• 제32권2호
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• pp.135-140
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• 2008

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

• Clinical and Experimental Reproductive Medicine
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• 제39권2호
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• pp.87-93
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• 2012

Objective: Lin28 has been known to control the proliferation and pluripotency of embryonic stem cells. The purpose of this study was to determine the downstream effectors of Lin28 in mouse embryonic stem cells (mESCs) by RNA interference and microarray analysis. Methods: The control siRNA and Lin28 siRNA (Dharmacon) were transfected into mESCs. Total RNA was prepared from each type of transfected mESC and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to confirm the downregulation of Lin28. The RNAs were labeled and hybridized with an Affymetrix Gene-Chip Mouse Genome 430 2.0 array. The data analysis was accomplished by GenPlex 3.0 software. The expression levels of selected genes were confirmed by quantitative real-time RT-PCR. Results: According to the statistical analysis of the cDNA microarray, a total of 500 genes were altered in Lin28-downregulated mESCs (up-regulated, 384; down-regulated, 116). After differentially expressed gene filtering, 31 genes were selected as candidate genes regulated by Lin28 downregulation. Among them, neuropeptide Y5 receptor and oocyte-specific homeobox 5 genes were significantly upregulated in Lin28-downregulated mESCs. We also showed that the families of neuropeptide Y receptor (Npyr) and oocyte-specific homeobox (Obox) genes were upregulated by downregulation of Lin28. Conclusion: Based on the results of this study, we suggest that Lin28 controls the characteristics of mESCs through the regulation of effectors such as the Npyr and Obox families.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8461-8465
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• 2016

Gastric cancer (GC) as the fourth most common cause of malignancies shows high rate of morbidity appropriating the second leading cause of cancer-related death worldwide. Developmental pluripotency associated-2 (DPPA2), cancer-testis antigen (CT100), is commonly expressed only in the human germ line and pluripotent embryonic cells but it is also present in a significant subset of malignant tumors. To investigate whether or not DPPA2 expression is recalled in GC, our aim in this study was to elucidate DPPA2 protein expression in gastric cancer. Fifty five GC tumor and their related margin normal tissues were recruited to evaluate DPPA2 protein expression and its probable associations with different clinicopathological features of the patients. DPPA2 was overexpressed in GC cases compared with normal tissues (P < .005). While DPPA2 expression was detected in all GC samples, its high expression was found in 23 of 55 tumor tissues (41.8%). Interestingly, 50 of 55 normal samples (90.9%) were negative for DPPA2 protein expression and remained 5 samples showed very low expression of DPPA2. DPPA2 protein expression in GC was significantly correlated with lymph node metastasis (p = 0.012). The clinical relevance of DPPA2 in GC illustrated that high level expression of this protein was associated with lymph node metastasis supporting this hypothesis that alteration in DPPA2 was associated with aggressiveness of gastric cancer and may be an early event in progression of the disease. DPPA2 may be introduced as a new marker for invasive and metastatic GCs.

• 한국발생생물학회지:발생과생식
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• 제7권2호
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• pp.61-68
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• 2003

포유동물의 초기 발생과정 중 접합체가 전능성이나 다능성을 가지기 위해서는 전반적인 DNA 메틸화를 포함하는 후성 유전학적 리프로그래밍의 복잡한 과정을 거쳐야만 한다. 본 연구팀에서는 공여핵의 후성 유전학적 리프로그래밍 과정을 조사하기 위하여 소 복제수정란에서 메틸화 양상을 분석하였다. 복제수정란의 비정상적인 메틸화 양상이 다양한 반복염기서열에서 관찰되었지만 single-copy유전자들의 염기서열은 정상적인 메틸화 양상을 보여주었다. 전반적으로 복제수정란의 전반적인 메틸화 상태는 정상수정란과 완전히 다른 양상을 보여주었다. 또한 복제 배반포의 영양외배엽세포에서 특이적으로 높은 메틸화 수준은 현 복제동물에서 빈번히 나타나는 불완전한 태반형성에 작용할 수 있을 것이다. 결론적으로 복제수정란의 비정상적 발생은 공여핵의 불완전한 후성 유전학적 리프로그래밍에 기인할 수 있다는 사실을 제시하게 되었다. 이러한 공여핵의 후성 유전학적 과정의 이해는 복제수정란의 비정상적 발생을 보다 분명히 밝힐 수 있을 것이다.

• Reproductive and Developmental Biology
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• 제30권4호
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• pp.279-285
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• 2006

Vitrification has been suggested to be an effective method for the cryopreservation of human ES cells. However, the efficiency of vitrification with different vehicles remains a matter of ongoing controversy. The objective of this study was to assess the efficiency of cryopreservation in human ES cells by vitrification using different vehicles. A human ES cell line and a variety of vehicles, including micro-droplet (MD), open-pulled straw (OPS) and electron microscopic grid (EM-grid), were employed in an attempt to assess vitrification efficiency. In order to evaluate the survivability and the undifferentiated state of the post-vitrified human ES cells, we conducted alkaline phosphatase staining and characterization via both RT-PCR and immunofluorescence assays. The survival rates of the post-vitrified human ES cells using MD, OPS and EM-grid were determined to be 61.5%, 66.6% and 53.8%, respectively. There also exist significant differences between slow-freezing and vitrification (p<0.01). However, no significant differences were detected between the vehicle types. Finally, the pluripotency of human ES cells after thawing was verified by teratoma formation. Cryopreservation using vitrification is more effective than slow-freezing, and the efficiency of vehicles proved effective with regard to the preservation of human ES cells.

• Reproductive and Developmental Biology
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• 제30권4호
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• pp.287-291
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• 2006

We have developed a new passaging technique for the expansion of human embryonic stem cells (hESCs) that involves simply pipetting portions of hESCs acquired from colonies, reducing the laborious and time-consuming steps in the expansion of hESCs. Compared to general mechanical methods of passaging, our pipetting method allowed hESCs colonies to be broken into small fragments, which showed significantly higher attachment rates onto feeder cell layers. This technique produced three times the number of hESCs colonies than conventional mechanical methods. In addition, this pipetting method allowed us to distinguish differentiated hESCs from undifferentiated hESCs during hESCs colony pipetting. The hESCs cultured by pipetting method displayed normal human chromosomes for over 60 passages. According to RT-PCR and immunohistochemical analysis, the hESCs successfully maintained their undifferentiated state and pluripotency which was also confirmed by teratoma formation in viva Therefore, the pipetting method described in this study is a useful tool to efficiently and quickly expand hESCs on a large scale without enzyme treatment.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.449-455
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• 2011

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by etopic expression of transcription factors. iPS cells are indistinguishable from ES cells in terms of morphology and stem cell marker expression. Moreover, mouse iPS cells give rise to chimeric mice that are competent for germline transmission. However, mice derived from iPS cells often develop tumors. Furthermore, the low efficiency of iPS cell generation is a big disadvantage for mechanistic studies. Nonviral plasmid.based vectors are free of many of the drawbacks that constrain viral vectors. The histone deacetylase inhibitor valproic acid (VPA) has been shown to improve the efficiency of mouse and human iPS cell generation, and vitamin C (Vc) accelerates gene expression changes and establishment of the fully reprogrammed state. The MEK inhibitor PD0325901 (Stemgent) has been shown to increase the efficiency of the reprogramming of human primary fibroblasts into iPS cells. In this report, we described the generation of mouse iPS cells devoid of exogenous DNA by the simple transient transfection of a nonviral vector carrying 2A-peptide-linked reprogramming factors. We used VPA, Vc, and the MEK inhibitor PD0325901 to increase the reprogramming efficiency. The reprogrammed somatic cells expressed pluripotency markers and formed EBs.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2001년도 제18차 정기총회 및 학술발표 PROCEEDINGS
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• pp.40-46
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• 2001

The use of pluripotent stem cells has tremendous advantages for various purposes but these cell lines with proven germ-line transmission have been completely established only in the mouse. Embryonic germ (EG) cell lines are also pluripotent and undifferentiated stem cells established from primordial germ cells (PGCs). This study was conducted to establish and characterize the chicken EG cells derived from gonadal primordial germ cells. We isolated gonadal PGCs from 5.5-day-old (stage 28) White leghorn (WL) embryos and established chicken EG cells lines with EG culture medium supplemented with human stem cell factor (hSCF), murine leukemia inhibitory factor (mLIF), bovine basic fibroblast growth factor (bFGF), human interleukin-11 (hIL-11), and human insulin-like growth factor-I (hIGF-I). These cells grew continuously for 4 months (10 passages) on a feeder layer of mitotically active chicken embryonic fibroblasts. These cells were characterized by screening with the Periodic acid-Shiff＇s reaction, anti-SSEA-1 antibody, and a proliferation assay after several passages. As the results, the chicken EG cells maintained characteristics of undifferentiated stem cells as well as that of gonadal PGCs. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types when re-seeded onto culture dish. The chicken EG cells were injected into blastodermal layer at stage X and dorsal aorta of recipient embryo at stage 14 (incubation of 53hrs) and produced chimeric chickens with various differentiated tissues derived from the EG cells. The germline chimeras were also successfully induced by using EG cells. Thus, Chicken EG cells will be useful for the production of transgenic chickena and for studies of germ cell differentiation and genomic imprinting.

• Toxicological Research
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• 제23권3호
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• pp.271-278
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• 2007

Several recent studies demonstrated the potential of bioengineering using stem cells in regenerative medicine. Adult mesenchymal stem cells (MSCs) have the pluripotency to differentiate into cells of mesodermal origin, i.e., bone, cartilage, adipose, and muscle cells; they, therefore, have many potential clinical applications. On the other hand, stem cells possess a self-renewal capability similar to cancer cells. For safety evaluation of MSCs, in this study, we tested tumorigenecity of canine adipose derived mesenchymal stem cells (cAD-MSCs) using Balb/c-nu mice. In this study, there were no changes in mortality, clinical signs, body weights and biochemical parameters of all animals treated. In addition, there were no significant changes between control and treated groups in autopsy findings. These results indicate that cAD-MSC has no tumorigenic potential under the condition in this study.

• 한국수정란이식학회지
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• 제27권3호
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• pp.189-192
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• 2012

Although embryonic stem cells (ESCs) or ES-like cells are reported from many mammalian species other than the mouse, the culture system for murine ESCs may not be suitable to the other species. Previously many other research groups have modified either human or mouse ESC culture systems for bovine ESC culture. In this study, we compared three different culture mediums consisting of DMEM, ${\alpha}$-MEM or KnockOut$^{TM}$-DMEM (KO), which are modified from human or mouse ESC culture system, for the generation of bovine ESCs. In this study, some pre-requisite events which are important for establishment and long-term propagation of ESCs such as inner cell mass (ICM) attachment on feeder cells, primary colony formation and sustainability after passaging. Once the ICM clumps attached on feeder cells, this was designated as passage 0. In regards to the rate of ICM attachment, ${\alpha}$-MEM was superior to the other systems. For primary colony formation, there was no difference between DMEM and ${\alpha}$-MEM whereas KO showed lower formation rate than the other groups. For passaging, the colonies were split into 2~4 pieces and passed every 5~6 days. From passage 1 to passage 3, DMEM system seemed to be appropriate for maintaining putative bovine ESCs. On the other hand, ${\alpha}$-MEM tended to be more suitable after passage 6. Although ${\alpha}$-MEM support to maintain a ES-like cell progenies to passage 15, all three culture systems which are modified from human or mouse ESC culture media failed to retain the propagation and long-term culture of putative bovine ESCs. Our findings imply that more optimized alternative culture system is required for establishing bovine ESC lines.