64 EPITHELIAL MESENCHYMAL TRANSITION AND DIFFERENTIATION OF STEROIDOGENIC FACTOR 1 MOUSE EMBRYONIC STEM CELLS INTO THE STEROIDOGENIC CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 162
Author(s):  
H. Y. Kang ◽  
Y.-K. Choi ◽  
J.-U. Hwang ◽  
E.-B. Jeung
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Epithelial Mesenchymal Transition ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Mouse Embryonic Stem Cells ◽  
Mrna Levels ◽  
Forkhead Transcription Factor ◽  
Steroidogenic Factor 1 ◽  
Mesenchymal Transition

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells has been reported to prime the cells for steroidogenesis. In this study, we transfected mouse embryonic stem cells (mESCs) with the mouse SF1 gene (SF1-mESCs) by using the nucleofector (Lonza), and selected SF1-mESCs by G418 250 μg mL–1. The selected cells were differentiated into granulosa-like cells through hanging-drops for 3 days, suspension culture for 1 day, then attachment onto 6-well plates. Expression of steroidogenesis-related genes and gonadal lineage-markers was analysed by real-time PCR. To test the phenotype for granulosa-like cells, transcripts of specific forkhead transcription factor (Foxl2) and follicle stimulating hormone receptor (Fshr) were measured. Also, expression of EMT-related genes, such as E-Cadherin (Cdh1), N-Cadherin (Cdh2), Snai1, Snai2 (Slug), Twist, and Vimentin, was monitored. SF1-mESCs were differentiated into the primitive streak‐mesendoderm and the steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase (Hsd3b1), cytochrome P450-containing enzyme (Cyp)-11a1, and Cyp19a1 were time-dependently changed. Next, the mRNA levels of Foxl2 and Fshr representing granulosa-like cell were increased during differentiation of SF1-mESCs. Especially, the level of oestradiol and Cdh2 was increased at a specific differentiation time. We induced differentiation of mESCs into the functional granulosa-like cells through transfection of the mouse SF1 gene. These cells will be useful for further study and potential application of these cells in steroidogenesis. This research was supported by a grant (15182MFDS460) from the Ministry of Food and Drug Safety in 2015.

336 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS USING SF1 INTO THE STEROID-PRODUCING CELLS

2015 ◽  
Vol 27 (1) ◽  
pp. 256
Author(s):  
H. Y. Kang ◽  
Y.-K. Choi ◽  
E.-B. Jeung
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Selection Process ◽  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Mouse Embryonic Stem Cells ◽  
Forkhead Transcription Factor ◽  
Cmv Promoter ◽  
Mes Cells

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells has been reported to prime the cells for steroidogenesis. In this study, we obtained SF1 cDNA from mRNA of murine pituitary glands and constructed the SF1-expressing vector controlled by the CMV promoter. The SF1 transgenic mouse embryonic stem cells (SF1-mES cells) were established through transfection using the nucleofector (Lonza) and selection process using G418 at 250 μg mL–1. The SF1-mES cells were aggregated in hanging drops for 2 days and were transferred to suspension culture for an additional 1 day in mouse basal differentiation medium. Three-day-old SF1-mESC-derived EB were attached onto 6 well culture plates and differentiated into granulosa-like cells. Differentiated SF1-mES cells were analysed by expression of steroidogenesis-related genes and gonadal lineage-markers to the level of mRNA via real-time PCR method. To test the phenotype for granulosa-like cells, we confirmed transcripts of specific forkhead transcription factor FOXL2 and the follicle-stimulating hormone receptor (FSHR). On the other hand, we monitored some specific genes related to differentiation into testicular tissue. We observed the progress to primitive streak-mesendodermby gene expression analyses. In addition, we observed that differentiated SF1-mES cells express steroidogenic enzymes, such as 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, and CYP19A1. Using the advanced approach, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic and gonadal lineages. We also induced granulosa-like cells. We established the effective protocol to generate ovarian cells. The derivation of these cells explores new avenues for the further study and potential application of these cells in steroidogenesis.

scholarly journals Role of the epithelial–mesenchymal transition and its effects on embryonic stem cells

2014 ◽  
Vol 46 (8) ◽  
pp. e108-e108 ◽  
Author(s):  
Ye-Seul Kim ◽  
Bo-Rim Yi ◽  
Nam-Hyung Kim ◽  
Kyung-Chul Choi
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Epithelial Mesenchymal Transition ◽  
Embryonic Stem ◽  
Mesenchymal Transition

scholarly journals Functional Dissection of pri-miR-290~295 in Dgcr8 Knockout Mouse Embryonic Stem Cells

2019 ◽  
Vol 20 (18) ◽  
pp. 4345
Author(s):  
Ming Shi ◽  
Jing Hao ◽  
Xi-Wen Wang ◽  
Le-Qi Liao ◽  
Huiqing Cao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Mirna Cluster ◽  
Neighboring Gene ◽  
Region Gene ◽  
Mesenchymal Transition

The DiGeorge syndrome critical region gene 8 (Dgcr8) knockout strategy has been widely used to study the function of canonical microRNAs (miRNAs) in vitro and in vivo. However, primary miRNA (pri-miRNA) transcripts are accumulated in Dgcr8 knockout cells due to interrupted processing. Whether abnormally accumulated pri-miRNAs have any function is unknown. Here, using clustered regularly interspaced short palindromic repeats system/CRISPR-associated protein 9 (CRISPR/Cas9), we successfully knocked out the primary microRNA-290~295 (pri-miR-290~295) cluster, the most highly expressed miRNA cluster in mouse embryonic stem cells (ESCs), in Dgcr8 knockout background. We found that the major defects associated with Dgcr8 knockout in mouse ESCs, including higher expression of epithelial-to-mesenchymal transition (EMT) markers, slower proliferation, G1 accumulation, and defects in silencing self-renewal, were not affected by the deletion of pri-miR-290~290 cluster. Interestingly, the transcription of neighboring gene nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 12(Nlrp12) was upregulated upon the deletion of the pri-miR-290~295 cluster. Together, our results suggested that the major defects in Dgcr8 knockout ESCs were not due to the accumulation of pri-miR-290~295, and the deletion of miRNA genes could affect the transcription of neighboring DNA elements.

323 ESTROGEN-INDUCED EPITHELIAL-MESENCHYMAL TRANSITION AND EXPRESSION OF PROAPOPTOTIC MARKERS IN PORCINE STEM CELLS

2015 ◽  
Vol 27 (1) ◽  
pp. 250
Author(s):  
Y.-S. Kim ◽  
S.-H. Hyun ◽  
C.-K. Lee ◽  
K.-C. Choi
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Epithelial Mesenchymal Transition ◽  
Steroid Hormone ◽  
Embryonic Stem ◽  
Transgenic Pig ◽  
Rt Pcr ◽  
Mesenchymal Transition ◽  
Apoptotic Markers ◽  
E Cadherin

In transgenic pig production for generating animal models of human diseases, apoptosis of early implantation embryo disturbs the transgenic pig production. In general, epithelial-mesenchymal transition (EMT) is considered important in embryo development and apoptosis. In addition, it was reported that 17β-oestradiol (E2), among hormones that participate in early implantation of embryo, could induce EMT and neural differentiation in mouse embryonic stem cells. Therefore, in this study, we examined the effects of the steroid hormone, E2, in the changes of EMT and apoptotic markers in porcine embryonic stem cells (pESC) and porcine induced pluripotent stem cells (piPSC). During the study, we cultured pESC and piPSC in pESC media containing basic fibroblast growth factor (b-FGF) and leukemia inhibitory factor (LIF) and performed RT-PCR and an alkaline phophatase (AP) test to measure pluripotent and undifferentiation markers of these porcine stem cells. The RT-PCR results showed that OCT4, NANOG, and SOX2 were expressed in these pESC and piPSC, indicating their pluripotency as stem cells. Also, these porcine stem cells showed positive AP activity, demonstrating undifferentiation. Additionally, we treated pESC and piPSC with E2 to examine effects of steroid hormone on the changes of EMT and apoptotic markers (i.e. bcl-2, bax, E-cad, and vimentin). The E2 treatment increased the expression of vimentin and bcl-2, while decreased the expression of E-cadherin and bax. By using immunocytochemistry (ICC), we examined the protein expression of EMT markers, which are vimentin and E-cadherin at the translational level, and found that expression of vimentin protein was increased while E-cadherin protein level was reduced at periphery of the colonies in pESC and piPSC. In conclusion, these results indicate that E2 can promote EMT process and reverse apoptosis in these pESC and iPSC. In a future study, we will further examine the effects of progesterone on the expressions of EMT and apoptotic markers in pESC and piPSC. Consequently, this study will contribute to elucidate the underlying mechanisms of EMT and apoptosis controlled by steroid hormones in porcine stem cells.

62 EFFECT OF PROGESTERONE ON CALCIUM REGULATION DURING DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO CARDIOMYOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 161
Author(s):  
Y.-K. Choi ◽  
H. Y. Kang ◽  
J.-U. Hwang ◽  
T. D. Nam ◽  
E.-B. Jeung
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Calcium Regulation ◽  
Mouse Embryonic Stem Cells ◽  
Cardiac Differentiation ◽  
Mrna Levels ◽  
Cellular Functions ◽  
Skeletal Muscle Contraction ◽  
Cardiac Lineage

During spontaneous cardiac differentiation of mouse embryonic stem cells (mESCs, cell line E14), the effect of progesterone on calcium regulation was investigated. Calcium (Ca2+) release from sarcoplasmic reticulum (SR) regulates various cellular functions including the smooth or skeletal muscle contraction. The cardiac L-type Ca2+ channel plays a key role in excitation-contraction coupling of cardiomyocytes and contraction-related gene expression. The mESCs formed mouse embryonic bodies (mEBs) by hanging-drop for 4 days, and mEBs were suspended for 2 days in differentiation medium; DMEM/F:12, 15% charcoal-dextran-treated FBS, β-mercaptoethanol, minimal essential medium NEAA, and penicillin/streptomycin. Then, mEBs were attached onto 6-well culture plates and differentiated into cardiomyocytes. We analysed mRNA expressions for the cardiac lineage markers and calcium-regulating genes. Percentage of beating mEBs was time-dependently increased during differentiation. Differentiated mEBs showed the highest beating ratio (92.11 ± 2.98%) after attachment for 12 days. Beating ratio was decreased to 64.86 ± 4.25% in progesterone-treated mEBs. The mRNA levels of cardiac markers such as Tbx20, Isl1, Foxh1, cTn1, and RyR2 were increased, and troponin protein was observed in beating mEBs via immunocytochemistry. Expression of calcium/contraction regulating genes including Trpv2, Ryr2, CaM2, and Mlck3 was down-regulated by progesterone treatment. These results indicate that progesterone has influences on cardiac differentiation and contraction of cardiomyocytes through regulating intercellular calcium ion. This research was supported by a grant (15182MFDS460) from the Ministry of Food and Drug Safety in 2015.

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