Expression of TASK-1 channel in mouse Leydig cells

Min Seok Woo,Eun-Jin Kim,Anjas Happy Prayoga,Yangmi Kim,Dawon Kang 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Leydig cells, crucial for testosterone production, express ion channels like ANO1 that influence hormone secretion. This study investigates the expression and role of the Tandem of P domains in a weak inward rectifying K+ channel-related Acid-Sensitive K+-1 (TASK-1) channel in these cells, exploring its impact on testicular function and steroidogenesis. Methods: TASK-1 expression in Leydig cells was confirmed using immunostaining, while RT-PCR and Western Blot (WB) validated its expression in the TM3 Leydig cell line. The effect of a TASK-1 channel blocker on cell viability was assessed through live/dead staining and MTT assays. Additionally, the blocker’s effect on testosterone secretion was evaluated by measuring testosterone levels. Results: Immunohistochemical analysis revealed a predominant presence of TASK- 1, along with c-Kit and ANO-1, in Leydig cells adjacent to seminiferous tubules and also in Sertoli and spermatogenic cells. Expression levels of TASK-1 mRNA and protein were significantly higher in TM3 Leydig cells compared to TM4 Sertoli cells. In addition, blocking TASK-1 in TM3 cells with ML365 induced cell death but did not affect LHinduced testosterone secretion. Conclusions: These findings suggest that TASK-1 in Leydig cells is crucial for their viability and proliferation, highlighting its potential importance in testicular physiology.

Evaluation of the maintenance of stemness, viability, and differentiation potential of gingiva-derived stem-cell spheroids

Lee, Sung-Il,Ko, Youngkyung,Park, Jun-Beom D.A. Spandidos 2017 Experimental and therapeutic medicine Vol.13 No.5

<P>Gingiva-derived stem cells have been applied for tissue-engineering purposes and may be considered a favorable source of mesenchymal stem cells as harvesting stem cells from the mandible or maxilla may be performed with ease under local anesthesia. The present study was performed to fabricate stem-cell spheroids using concave microwells and to evaluate the maintenance of stemness, viability, and differentiation potential. Gingiva-derived stem cells were isolated, and the stem cells of 4×10<SUP>5</SUP> (group A) or 8×10<SUP>5</SUP> (group B) cells were seeded into polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres and the change of the diameters of the spheroids were evaluated. The viability of spheroids was qualitatively analyzed via Live/Dead kit assay. A cell viability analysis was performed on days 1, 3, 6, and 12 with Cell Counting Kit-8. The maintenance of stemness was evaluated with immunocytochemical staining using SSEA-4, TRA-1-60(R) (positive markers), and SSEA-1 (negative marker). Osteogenic, adipogenic, and chondrogenic differentiation potential was evaluated by incubating spheroids in osteogenic, adipogenic and chondrogenic induction medium, respectively. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A than in group B. The majority of cells in the spheroids emitted green fluorescence, indicating the presence of live cells at day 6. At day 12, the majority of cells in the spheroids emitted green fluorescence, and a small portion of red fluorescence was also noted, which indicated the presence of dead cells. The spheroids were positive for the stem-cell markers SSEA-4 and TRA-1-60(R) and were negative for SSEA-1, suggesting that these spheroids primarily contained undifferentiated human stem cells. Osteogenic, adipogenic, and chondrogenic differentiation was more evident with an increase of incubation time: Mineralized extracellular deposits were observed following Alizarin Red S staining at days 14 and 21; oil globules were increased at day 18 when compared with day 6; and Alcian blue staining was more evident at day 18 when compared with day 6. Within the limits of this study, stem-cell spheroids from gingival cells maintained the stemness, viability, and differentiation potential during the experimental periods. This method may be applied for a promising strategy for stem-cell therapy.</P>

Growth and Migration of BALB/3T3 Fibroblast Cells on Nano-engineered Silica Beads Surface

Kim, Jihee,Chandra, Prakash,Yang, Jiyoon,Rhee, Seog Woo Korean Chemical Society 2013 Bulletin of the Korean Chemical Society Vol.34 No.12

In this study, the behavior of cells on the modified surface, and the correlation between the modified substrates and the response of cells is described. A close-packed layer of nano-sized silica beads was prepared on a coverslip, and the adhesion, proliferation, and migration of BALB/3T3 fibroblast cells on the silica layer was monitered. The 550 nm silica beads were synthesized by the hydrolysis and condensation reaction of tetraethylorthosilicate in basic solution. The amine groups were introduced onto the surfaces of silica particles by treatment with 3-aminopropyltrimethoxysilane. The close-packed layer of silica beads on the coverslip was obtained by the reaction of the amine-functionalized silica beads and the (3-triethoxysilyl)propylsuccinic anhydride treated coverslip. BALB/3T3 fibroblast cells were loaded on bare glass, APTMS coated glass, and silica bead coated glass with the same initial cell density, and the migration and proliferation of cells on the substrates was investigated. The cells were fixed and stained with antibodies in order to analyze the changes in the actin filaments and nuclei after culture on the different surfaces. The motility of cells on the silica bead coated glass was greater than that of the cells cultured on the control substrate. The growth rate of cells on the silica bead coated glass was slower than that of the control. Because the close-packed layer of silica beads gave an embossed surface, the adhesion of cells was very weak compared to the smooth surfaces. These results indicate that the adhesion of cells on the substrates is very important, and the actin filaments might play key roles in the migration and proliferation of cells. The nuclei of the cells were shrunk on the weakly adhered surfaces, and the S1 stage in which DNA is duplicated in the cell dividing processes might be retarded. As a result, the rate of proliferation of cells was decreased compared to the smooth surface of the control. In conclusion, the results described here are very important in the understanding of the interaction between implanted materials and biosystems.

The Role of Stress Granules in the Neuronal Differentiation of Stem Cells

정신구,온탁범,Chul Ho Jang,Karthikeyan Vijayakumar,조광원 한국분자세포생물학회 2020 Molecules and cells Vol.43 No.10

Cells assemble stress granules (SGs) to protect their RNAs from exposure to harmful chemical reactions induced by environmental stress. These SGs release RNAs, which resume translation once the stress is relieved. During stem cell differentiation, gene expression is altered to allow cells to adopt various functional and morphological features necessary to differentiate. This process induces stress within a cell, and cells that cannot overcome this stress die. Here, we investigated the role of SGs in the progression of stem cell differentiation. SGs aggregated during the neuronal differentiation of human bone marrow-mesenchymal stem cells, and not in cell lines that could not undergo differentiation. SGs were observed between one and three hours post-induction; RNA translation was restrained at the same time. Immediately after disassembly of SGs, the expression of the neuronal marker neurofilament-M (NFM) gradually increased. Assembled SGs that persisted in cells were exposed to salubrinal, which inhibited the dephosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), and in eIF2α/S51D mutant cells. When eIF2α/S51A mutant cells differentiated, SGs were not assembled. In all experiments, the disruption of SGs was accompanied by delayed NF-M expression and the number of neuronally differentiated cells was decreased. Decreased differentiation was accompanied by decreased cell viability, indicating the necessity of SGs for preventing cell death during neuronal differentiation. Collectively, these results demonstrate the essential role of SGs during the neuronal differentiation of stem cells.

Growth and Migration of BALB/3T3 Fibroblast Cells on Nano-engineered Silica Beads Surface

Jihee Kim,Prakash Chandra,Jiyoon Yang,Seog Woo Rhee 대한화학회 2013 Bulletin of the Korean Chemical Society Vol.34 No.12

In this study, the behavior of cells on the modified surface, and the correlation between the modified substrates and the response of cells is described. A close-packed layer of nano-sized silica beads was prepared on a coverslip, and the adhesion, proliferation, and migration of BALB/3T3 fibroblast cells on the silica layer was monitered. The 550 nm silica beads were synthesized by the hydrolysis and condensation reaction of tetraethylorthosilicate in basic solution. The amine groups were introduced onto the surfaces of silica particles by treatment with 3-aminopropyltrimethoxysilane. The close-packed layer of silica beads on the coverslip was obtained by the reaction of the amine-functionalized silica beads and the (3-triethoxysilyl)propylsuccinic anhydride treated coverslip. BALB/3T3 fibroblast cells were loaded on bare glass, APTMS coated glass, and silica bead coated glass with the same initial cell density, and the migration and proliferation of cells on the substrates was investigated. The cells were fixed and stained with antibodies in order to analyze the changes in the actin filaments and nuclei after culture on the different surfaces. The motility of cells on the silica bead coated glass was greater than that of the cells cultured on the control substrate. The growth rate of cells on the silica bead coated glass was slower than that of the control. Because the close-packed layer of silica beads gave an embossed surface, the adhesion of cells was very weak compared to the smooth surfaces. These results indicate that the adhesion of cells on the substrates is very important, and the actin filaments might play key roles in the migration and proliferation of cells. The nuclei of the cells were shrunk on the weakly adhered surfaces, and the S1 stage in which DNA is duplicated in the cell dividing processes might be retarded. As a result, the rate of proliferation of cells was decreased compared to the smooth surface of the control. In conclusion, the results described here are very important in the understanding of the interaction between implanted materials and biosystems.

Human Nasal Inferior Turbinate-Derived Neural Stem Cells Improve the Niche of Substantia Nigra Par Compacta in a Parkinson’s Disease Model by Modulating Hippo Signaling

최준원,Park Sun Wha,Lee Hyunji,Kim Do Hyun,Kim Sung Won 한국조직공학과 재생의학회 2024 조직공학과 재생의학 Vol.21 No.5

Background: Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases, following Alzheimer’s disease. The onset of PD is characterized by the loss of dopaminergic neurons in the substantia nigra. Stem cell therapy has great potential for the treatment of neurodegenerative diseases, and human nasal turbinate-derived stem cells (hNTSCs) have been found to share some characteristics with mesenchymal stem cells. Although the Hippo signaling pathway was originally thought to regulate cell size in organs, recent studies have shown that it can also control inflammation in neural cells. Methods: Dopaminergic neuron-like cells were differentiated from SH-SY5Y cells (DA-Like cells) and treated with 1-Methyl-4-phenylpyridinium iodide to stimulate Reactive oxidative species (ROS) production. A transwell assay was conducted to validate the effect of hNTSCs on the Hippo pathway. We generated an MPTP-induced PD mouse model and transplanted hNTSCs into the substantia nigra of PD mice via stereotaxic surgery. After five weeks of behavioral testing, the brain samples were validated by immunoblotting and immunostaining to confirm the niche control of hNTSCs. Results: In-vitro experiments showed that hNTSCs significantly increased cell survival and exerted anti-inflammatory effects by controlling ROS-mediated ER stress and hippocampal signaling pathway factors. Similarly, the in-vivo experiments demonstrated an increase in anti-inflammatory effects and cell survival rate. After transplantation of hNTSCs, the PD mouse model showed improved mobility and relief from PD symptoms. Conclusion: hNTSCs improved the survival rate of dopaminergic neurons by manipulating the hippocampal pathway through Yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding motif (TAZ) by reducing inflammatory cytokines. In this study, we found that controlling the niche of hNTSCs had a therapeutic effect on PD lesions. Background: Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases, following Alzheimer’s disease. The onset of PD is characterized by the loss of dopaminergic neurons in the substantia nigra. Stem cell therapy has great potential for the treatment of neurodegenerative diseases, and human nasal turbinate-derived stem cells (hNTSCs) have been found to share some characteristics with mesenchymal stem cells. Although the Hippo signaling pathway was originally thought to regulate cell size in organs, recent studies have shown that it can also control inflammation in neural cells. Methods: Dopaminergic neuron-like cells were differentiated from SH-SY5Y cells (DA-Like cells) and treated with 1-Methyl-4-phenylpyridinium iodide to stimulate Reactive oxidative species (ROS) production. A transwell assay was conducted to validate the effect of hNTSCs on the Hippo pathway. We generated an MPTP-induced PD mouse model and transplanted hNTSCs into the substantia nigra of PD mice via stereotaxic surgery. After five weeks of behavioral testing, the brain samples were validated by immunoblotting and immunostaining to confirm the niche control of hNTSCs. Results: In-vitro experiments showed that hNTSCs significantly increased cell survival and exerted anti-inflammatory effects by controlling ROS-mediated ER stress and hippocampal signaling pathway factors. Similarly, the in-vivo experiments demonstrated an increase in anti-inflammatory effects and cell survival rate. After transplantation of hNTSCs, the PD mouse model showed improved mobility and relief from PD symptoms. Conclusion: hNTSCs improved the survival rate of dopaminergic neurons by manipulating the hippocampal pathway through Yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding motif (TAZ) by reducing inflammatory cytokines. In this study, we found that controlling the niche of hNTSCs had a therapeutic effect on PD lesions.

Nuclear receptor regulation of stemness and stem cell differentiation

David J. Mangelsdorf,정양식 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.8

Stem cells include a diverse number of toti-, pluri-, and multi-potent cells that play important roles in cellular genesis and differentiation, tissue development, and organogenesis. Genetic regulation involving various transcription factors results in the self-renewal and differentiation properties of stem cells. The nuclear receptor (NR) superfamily is composed of 48 ligand-activated transcription factors involved in diverse physiological functions such as metabolism, development, and reproduction. Increasing evidence shows that certain NRs function in regulating stemness or differentiation of embryonic stem (ES) cells and tissue-specific adult stem cells. Here, we review the role of the NR superfamily in various aspects of stem cell biology, including their regulation of stemness, forward- and trans-differentiation events; reprogramming of terminally differentiated cells; and interspecies differences. These studies provide insights into the therapeutic potential of the NR superfamily in stem cell therapy and in treating stem cell-associated diseases (e.g., cancer stem cell). Stem cells include a diverse number of toti-, pluri-, and multi-potent cells that play important roles in cellular genesis and differentiation, tissue development, and organogenesis. Genetic regulation involving various transcription factors results in the self-renewal and differentiation properties of stem cells. The nuclear receptor (NR) superfamily is composed of 48 ligand-activated transcription factors involved in diverse physiological functions such as metabolism, development, and reproduction. Increasing evidence shows that certain NRs function in regulating stemness or differentiation of embryonic stem (ES) cells and tissue-specific adult stem cells. Here, we review the role of the NR superfamily in various aspects of stem cell biology, including their regulation of stemness, forward- and trans-differentiation events; reprogramming of terminally differentiated cells; and interspecies differences. These studies provide insights into the therapeutic potential of the NR superfamily in stem cell therapy and in treating stem cell-associated diseases (e.g., cancer stem cell).

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming

NO, Jin-Gu,CHOI, Mi-Kyung,KWON, Dae-Jin,YOO, Jae Gyu,YANG, Byoung-Chul,PARK, Jin-Ki,KIM, Dong-Hoon 家畜繁殖硏究所 2015 Journal of Reproduction and Development Vol.61 No.2

<P> Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot<SUP>TM</SUP> reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of <I>Jmjd2b</I>, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, <I>Bax</I> and <I>p53</I>, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, <I>Bax</I> and <I>p53</I> gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.</P>

Reaction of Mast Cells and Goblet Cells in the Small Intestine of C57BL/6 and C3H/HeN Mice Infected with Echinostoma hortense

Park Kyeong-Yeol,Lee Kyu-Jae,Kim In-Sik,Yang Eun-Ju,Lim Su-jung,Lim Byung-Hyuk,Ryang Yong-Suk The Korean Society for Biomedical Laboratory Scien 2005 Journal of biomedical laboratory sciences Vol.11 No.3

Mast cells and goblet cells have been known to protect the host against parasites. In this study, we examined the response of the mast cells and goblet cells over a period of 6 weeks in the duodenum, jejunum and ileum of C3H/HeN and C57BL/6 mice infected with Echinostoma hortense (E. hortense). In addition, we investigated whether the worm recovery rate of uninfected mice (the control group) or E. hortense-infected mice (the experimental group) was associated with the number of mast cells and goblet cells. The worm recovery rate was higher in the C3H/HeN mice than in the C57BL/6 mice. The number of goblet cells significantly increased in the experimental group of the C3H/HeN and C57BL/6 mice compared with the control group of both strains (P<0.005). Worm recovery peaked 3 weeks after the infection of the C57BL/6 mice and at 2 weeks after the infection of the C3H/HeN mice, and it was higher in the duodenum than in the jejunum and ileum. However, the infected site in the intestine had no relation with worm expulsion. In the C3H/HeN and C57BL/6 mice, the number of goblet cells in the experimental group was significantly higher than that in the control group (P<0.005). The number reached a peak 2 weeks after the infection and it even increased in duodenum, jejunum and ileum. The increased number of goblet cells was retained 6 weeks after infection. The number of goblet cells was higher in the C3H/HeN mice than in the C57BL/6 mice (P<0.01). These results indicate that goblet cells are related with the worm expulsion. Furthermore, immunohistostaining of the antral intestinal walls for lectin showed the significant increase of the number of goblet cells in the experimental group (P<0.001). The high infection rate in the duodenum was found during the early infection. An increased infection rate in the jejunum and ileum was found 3 weeks after infection and the infection rate was higher in the C3H/HeN mice than in the C57BL/6 mice. Taken together, the present study indicates that goblet cells, rather than mast cells, may play critical roles in parasite expulsion.

실험적 간암 발생 과정에 출현하는 난원형 세포의 면역 조직화학적 연구

황택근,이중달,원치규 한양대학교 의과대학 1992 한양의대 학술지 Vol.12 No.1

Oval cells have been observed during chemical carcinogenesis in the livers of animals fed 2-acetylaminofluorene (2-AAF), a potent chemical carcinogen. Biological behavior of these cells remained poorly understood with regard to gepatic carcinogenesis. The author carried out the histological, enzyme histochemical and immunohistochemical studies of oval cells in the livers from 80 rats fed 2-AAF for five months. Throughout the experimental period oval cells along with hepatocytes and bile ductular cells were subjected to immunostain for monoclonal antibodies. The antibodies included those to carcinoembryonic antigen(CEA), alpha-fetoprotein(AFP), low molecular cytokeratin(CAM 5.2), prealbumin, and endothelial cells. Alkaline phosplatase(ALP) reaction to oval cells was also evaluated. The followings were results obtained: Oval cells appeared about the portal areas at the 10th day of the experiment. The cells penetrated the hepatic lobule as they proliferated. The proliferation of oval cells was most prominent at the 6th week and then the cells were gradually decreased in number until the late stage of the experiment. There were foci of transformation of oval cells to cholangiolar cells and foci of cholangiofibrosis made up of proliferated bile ductules. PAS reaction was negative on oval cells, where as hepatocytes were reactive for PAS. ALP reaction was positive on both oval cells and ductular epithelial cells. The hepatocytes were non-reactive for ALP. Both oval cells and bile ductular cells showed positive staining for AFP and CAM 5.2, and negative for CEA and prealbumin. Hepatocytes showed negative staining for AFP, CAM 5.2, CEA and prealbumin. However, an impressive finding that AFP- and CAM-positive hepatocytes were scattered through the hepatic lobules, was observed early during hepatocarcinogenesis. The results indicated that oval cells had no evidence of the conversion to hepatocytes in 2-AAF induced hepatocarcinogenesis, and that hepatocellular carcinoma arose not from oval cells. Oval cells showed immonohistochemical characteristics similar to those of the bile ductular epithelium.