• Journal of Animal Science and Technology
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• 제66권4호
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• pp.726-739
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• 2024

This study was conducted to investigate whether lysophosphatidic acid (LPA) could improve the development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT-derived embryos were cultured in chemically defined polyvinyl alcohol (PVA)-based porcine zygote medium (PZM)-4 without or with LPA, and the development, cell proliferation potential, apoptosis, and expression levels of pluripotent markers were evaluated. LPA significantly increased the rates of cleavage and blastocyst formation compared to those seen in the LPA un-treatment (control) group. The expression levels of embryonic development-related genes (IGF2R, PCNA and CDH1) were higher (p < 0.05) in the LPA treatment group than in the control group. LPA significantly increased the numbers of total, inner cell mass and EdU (5-ethynyl-2'-deoxyuridine)-positive cells in porcine SCNT blastocysts compared to those seen in the control group. TUNEL assay showed that LPA significantly reduced the apoptosis rate in porcine SCNT-derived embryos; this was confirmed by decreases (p < 0.05) in the expression levels of pro-apoptotic genes, BAX and CASP3, and an increase (p < 0.05) in the expression level of the anti-apoptotic gene, BCL2L1. In addition, LPA significantly increased Oct4 expression at the gene and protein levels. Together, our data suggest that LPA improves the quality and development of porcine SCNT-derived embryos by reducing apoptosis and enhancing cell proliferation and pluripotency.

• 한국수정란이식학회지
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• 제18권3호
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• pp.243-248
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• 2003

1. 생쥐 고환으로부터 얻은 세포를 배양하여 군집을 형성하는 것을 관찰할 수 있었으며, AP, SSEA-1, -3, -4과 Integrin $\alpha$6, $\beta$1 및 Oct4의 발현을 확인하였다. 2. 생쥐 생식줄기세포를 3-5일정도 배양하게 되면, 여러 층으로 이루어진 군집을 이루게 되는데 이는 생쥐 배아줄기세포나 배아생식줄기세포의 형태와 같은 것이었다. 3. 생쥐 생식줄기세포를 체외에서 효과적으로 분리, 배양할 수 있는 조건을 확립하였다.

• 한국발생생물학회지:발생과생식
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• 제14권4호
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• pp.243-251
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• 2010

Estrogen related receptor $\beta$(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.

• 한국발생생물학회지:발생과생식
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• 제12권2호
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• pp.159-168
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• 2008

Nanog is a newly identified member of the homeobox family of DNA binding transcription factors that functions to maintain the undifferentiated state of stem cells. However, molecular mechanisms underlying the function of Nanog remain largely unknown. To elucidate the regulatory roles of Nanog involved in maintenance of P19 embryonal carcinoma (EC) stem cells, we transfected three small interfering RNA (siRNA) duplexes targeted against different regions of the Nanog gene into P19 cells. The Nanog siRNA-100 duplexes effectively decreased the expression of Nanog up to 30.7% compared to other two Nanog siRNAs, the Nanog siRNA-400 (67.9 %) and -793 (53.0%). When examined by RT-PCR and real-time PCR, the expression of markers for pluripotency such as Fgf4, Oct3/4, Rex1, Sox1 and Yes was downregulated at 48 h after transfection with Nanog siRNA-100. Furthermore, expression of the ectodermal markers, Fgf5 and Isl1 was reduced by Nanog knockdown. By contrast, the expression of other markers for pluripotency such as Cripto, Sox2 and Zfp57 was not affected by Nanog knockdown at this time. On the other hand, the expression of Lif/Stat3 pathway molecules and of the endoderm markers including Dab2, Gata4, Gata6 and the germ cell nuclear factor was not changed by Nanog knockdown. The results of this study demonstrated that the knockdown of Nanog expression by RNA interference in P19 cells was sufficient to modulate the expression of pluripotent markers involved in the self-renewal of EC stem cells. These results provide the valuable information on potential downstream targets of Nanog and add to our understanding of the function of Nanog in P19 EC stem cells.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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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.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.100-100
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• 2003

Pluripotent embryonic stem (ES) cells differentiate spontaneously into beating cardiomyocytes via embryo-like aggregates. We describe the use of mouse embryonic stem (mES03) cells as a reproducible differentiation system for cardiomyocyte. To induce cardiomyocytic differentiation, mES03 cells were dissociated and allowed to aggregate (EB formation) at the presence of 0 75% dimethyl sulfoxide (DMSO) for 4 days and then another 4 days without DMSO (4+/4-). Thus treated EBs were plated onto gelatin-coated dish for differentiation. Spontaneously contracting colonies which appeared in approximately 4-5 days upon differentiation. Expression of cardiac-specific genes were determined by RT-PCR. Rebust expression of myosin light chain (MLC-2V), cardiac myosin heavy chain $\alpha$, cardiac muscle heavy polypeptide 7 $\beta(\beta$-MHC), cardiac transcription factor GATA4 and skeletal muscle-specific ${\alpha}_1$-subunit of the L-type calcium channel (${\alpha}_1 CaCh_{sm}$) were detected as early as 8 days after EB formation, but message of cardiac muscle-specific $\alpha$$_1$-subunit of the L-type calcium channel (${\alpha}_1$CaCh) were revealed at a low level. Strikingly, the expression of atrial natriuretic factor (ANF) was not detected. When spontaneous contracting cell masses were examined their electrophysiological features by patch-clamp technique, it showed ventricle-like action potential 17 days after the EB formation. This study indicates that mES03 cell-derived cardiomyocytes displayed biochemical and electrophysiological properties of cardiomyocytes and DMSO enhanced development of cardiomyocytes in 4+/4- method.

• Reproductive and Developmental Biology
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• 제38권2호
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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.

• 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.

• Asian Pacific Journal of Cancer Prevention
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• 제16권9호
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• pp.3793-3797
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• 2015

Background: Chronic myeloid leukemia (CML) is a myeloproliferative disorder of pluripotent stem cells, caused by reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11), known as the Philadelphia chromosome. Materials and Methods: A total of 51 CML patients were recruited in this study. Complete blood counts of all CML patients were performed to find out their total leukocytes, hemoglobin and platelets. FISH was performed for the detection of BCR-ABL fusion and cryptogenic tests using bone marrow samples were performed for the conformation of Ph (9;22)(q34;q11) and variant translocation mechanisms. Results: In cytogenetic analysis we observed that out of 51 CML patients 40 (88.9%) were Ph positive and 4 (8.88%) had Ph negative chromosomes. Mean values of WBC 134.5 $10^3/{\mu}l$, hemoglobin 10.44 mg/dl, and platelets 288.6 $10^3/{\mu}l$ were observed in this study. Conclusions: In this study, Ph positive translocation between chromosome (9:22)(q34;q11) were observed in 40 (88.9%) CML patients.

• Molecules and Cells
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• 제41권2호
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• pp.110-118
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• 2018

The objective of this study was to induce the production of isthmic organizer (IsO)-like cells capable of secreting fibroblast growth factor (FGF) 8 and WNT1 from human embryonic stem cells (ESCs). The precise modulation of canonical Wnt signaling was achieved in the presence of the small molecule CHIR99021 ($0.6{\mu}M$) during the neural induction of human ESCs, resulting in the differentiation of these cells into IsO-like cells having a midbrain-hindbrain border (MHB) fate in a manner that recapitulated their developmental course in vivo. Resultant cells showed upregulated expression levels of FGF8 and WNT1. The addition of exogenous FGF8 further increased WNT1 expression by 2.6 fold. Gene ontology following microarray analysis confirmed that IsO-like cells enriched the expression of MHB-related genes by 40 fold compared to control cells. Lysates and conditioned media of IsO-like cells contained functional FGF8 and WNT1 proteins that could induce MHB-related genes in differentiating ESCs. The method for generating functional IsO-like cells described in this study could be used to study human central nervous system development and congenital malformations of the midbrain and hindbrain.