Browsing by Author "Chien, Chian-Shiu"

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Ash2l interacts with Oct4-stemness circuitry to promote super-enhancer-driven pluripotency network
(2019-11-04)
Tsai, Ping-Hsing
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Chien, Yueh
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Wang, Mong-Lien
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Hsu, Chih-Hung
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Laurent, Benoit
Pluripotency and cell fates can be modulated through the regulation of super-enhancers; however, the underlying mechanisms are unclear. Here, we showed a novel mechanism in which Ash2l directly binds to super-enhancers of several stemness genes to regulate pluripotency and self-renewal in pluripotent stem cells. Ash2l recruits Oct4/Sox2/Nanog (OSN) to form Ash2l/OSN complex at the super-enhancers of Jarid2, Nanog, Sox2 and Oct4, and further drives enhancer activation, upregulation of stemness genes, and maintains the pluripotent circuitry. Ash2l knockdown abrogates the OSN recruitment to all super-enhancers and further hinders the enhancer activation. In addition, CRISPRi/dCas9-mediated blocking of Ash2l-binding motifs at these super-enhancers also prevents OSN recruitment and enhancer activation, validating that Ash2l directly binds to super-enhancers and initiates the pluripotency network. Transfection of Ash2l with W118A mutation to disrupt Ash2l-Oct4 interaction fails to rescue Ash2l-driven enhancer activation and pluripotent gene upregulation in Ash2l-depleted pluripotent stem cells. Together, our data demonstrated Ash2l formed an enhancer-bound Ash2l/OSN complex that can drive enhancer activation, govern pluripotency network and stemness circuitry.
Cisplatin-selected resistance is associated with increased motility and stem-like properties via activation of STAT3/Snail axis in atypical teratoid/rhabdoid tumor cells
(2015-01-30)
Liu, Wei-Hsiu
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Chen, Ming-Teh
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Wang, Mong-Lien
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Lee, Yi-Yen
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Chiou, Guang-Yuh
Atypical teratoid/rhabdoid tumor (ATRT) is a malignant pediatric brain tumor with great recurrence after complete surgery and chemotherapy. Here, we demonstrate that cisplatin treatment selects not only for resistance but also for a more oncogenic phenotype characterized by high self-renewal and invasive capabilities. These phenomena are likely due to STAT3 upregulatoin which occurred simultaneously with higher expression of Snail, an activator of epithelial-mesenchymal transition (EMT), in ATRT-CisR cells. STAT3 knockdown effectively suppressed Snail expression and blocked motility and invasion in ATRT-CisR cells, while overexpressing Snail reversed these effects. Chromatin immunoprecipitation assay indicated that STAT3 directly bound to Snail promoter. Moreover, STAT3 knockdown effectively suppressed cancer stem-like properties, synergistically enhanced the chemotherapeutic effect, and significantly improved survival rate in ATRT-CisR-transplanted immunocompromised mice. Finally, immunohistochemistrical analysis showed that STAT3 and Snail were coexpressed at high levels in recurrent ATRT tissues. Thus, the STAT3/Snail pathway plays an important role in oncogenic resistance, rendering cells not only drug-resistant but also increasingly oncogenic (invasion, EMT and recurrence). Therefore, the STAT3/Snail could be a target for ATRT treatment.
Enhanced Antioxidant Capacity of Dental Pulp-Derived iPSC-Differentiated Hepatocytes and Liver Regeneration by Injectable HGF-Releasing Hydrogel in Fulminant Hepatic Failure
(2015-01-01)
Chiang, Chih-Hung
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Wu, Wai-Wah
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Li, Hsin-Yang
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Chien, Yueh
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Sun, Cho-Chin
Acute hepatic failure (AHF) is a severe liver injury leading to sustained damage and complications. Induced pluripotent stem cells (iPSCs) may be an alternative option for the treatment of AHF. In this study, we reprogrammed human dental pulp-derived fibroblasts into iPSCs, which exhibited pluripotency and the capacity to differentiate into tridermal lineages, including hepatocyte-like cells (iPSC-Heps). These iPSC-Heps resembled human embryonic stem cell-derived hepatocyte-like cells in gene signature and hepatic markers/functions. To improve iPSC-Heps engraftment, we next developed an injectable carboxymethyl-hexanoyl chitosan hydrogel (CHC) with sustained hepatocyte growth factor (HGF) release (HGF CHC) and investigated the hepatoprotective activity of HGF CHC-delivered iPSC-Heps in vitro and in an immunocompromised AHF mouse model induced by thioacetamide (TAA). Intrahepatic delivery of HGF CHC iPSC-Heps reduced the TAA-induced hepatic necrotic area and rescued liver function and recipient viability. Compared with PBS-delivered iPSC-Heps, the HGF CHC-delivered iPSC-Heps exhibited higher antioxidant and antiapoptotic activities that reduced hepatic necrotic area. Importantly, these HGF CHC-mediated responses could be abolished by administering anti-HGF neutralizing antibodies. In conclusion, our findings demonstrated that HGF mediated the enhancement of iPSC-Hep antioxidant/antiapoptotic capacities and hepatoprotection and that HGF CHC is as an excellent vehicle for iPSC-Hep engraftment in iPSC-based therapy against AHF.
Enhancing induced pluripotent stem cell toward differentiation into functional cardiomyocytes
(2020-07-01)
Chien, Chian-Shiu
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Wang, Chien-Ying
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Leu, Hsin-Bang
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Chien, Yueh
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Yang, Yi-Ping
Background: Heart diseases, especially myocardial ischemia, remain one of the leading causes of mortality worldwide and usually result in irreparable cardiomyocyte damage and severe heart failure. Recent advances in induced pluripotent stem cell (iPSC) technologies for applied regenerative medicine and stem cell research, especially for iPSC-derived cardiomyocytes have increased the hope for heart repair. However, the driver molecules of myocardial differentiation and the functional reconstruction capacity of iPSC-derived cardiomyocytes are still questionable. Methods: Herein, we established a rapid differentiated platform that is involved in cardiomyogenic differentiation and maturation from iPSCs in vitro. Functional analysis is performed in miR-181a-transfected iPSC-derived cardiomyocyte (iPSC-cardio/miR-181a) under a time-lapse microscope. In addition, we calculated the beating area and frequency of iPSC-cardio/miR-181a cells in the presence of HCN4 shRNA or miR-181a SPONGE. Results: miR-181a enhanced the beating area and maintained the beating frequency of iPSC-derived cardiomyocytes by enhancing HCN4 expression. Conclusion: miR-181a would play a key role on maintaining proper beating function in iPSC-derived cardiomyocytes.
Epigenetic Regulation of the miR142-3p/Interleukin-6 Circuit in Glioblastoma
(2013-12-12)
Chiou, Guang-Yuh
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Chien, Chian-Shiu
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Wang, Mong-Lien
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Chen, Ming-Teh
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Yang, Yi-Ping
Epigenetic regulation plays a critical role in glioblastoma (GBM) tumorigenesis. However, how microRNAs (miRNAs) and cytokines cooperate to regulate GBM tumor progression is still unclear. Here, we show that interleukin-6 (IL-6) inhibits miR142-3p expression and promotes GBM propagation by inducing DNA methyltransferase 1-mediated hypermethylation of the miR142-3p promoter. Interestingly, miR142-3p also suppresses IL-6 secretion by targeting the 3' UTR of IL-6. In addition, miR142-3p also targets the 3' UTR and suppresses the expression of high-mobility group AT-hook 2 (HMGA2), leading to inhibition of Sox2-related sternness. We further show that HMGA2 enhances Sox2 expression by directly binding to the Sox2 promoter. Clinically, GBM patients whose tumors present upregulated IL-6, HMGA2, and Sox2 protein expressions and hypermethylated miR142-3p promoter also demonstrate poor survival outcome. Orthotopic delivery of miR142-3p blocks IL-6/HMGA2/Sox2 expression and suppresses stem-like properties in GBM-xenotransplanted mice. Collectively, we discovered an IL-6/miR142-3p feed-back-loop-dependent regulation of GBM malignancy that could be a potential therapeutic target.
Glutamate Stimulation Dysregulates AMPA Receptors-Induced Signal Transduction Pathway in Leber's Inherited Optic Neuropathy Patient-Specific hiPSC-Derived Retinal Ganglion Cells
(2019-06-01)
Yang, Yi-Ping
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Phan Nguyen Nhi Nguyen
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Lin, Tai-Chi
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Yarmishyn, Aliaksandr A.
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Chen, Wun-Syuan
The mitochondrial genetic disorder, Leber's hereditary optic neuropathy (LHON), is caused by a mutation in MT-ND4 gene, encoding NADH dehydrogenase subunit 4. It leads to the progressive death of retinal ganglion cells (RGCs) and causes visual impairment or even blindness. However, the precise mechanisms of LHON disease penetrance and progression are not completely elucidated. Human-induced pluripotent stem cells (hiPSCs) offer unique opportunities to investigate disease-relevant phenotypes and regulatory mechanisms underlying LHON pathogenesis at the cellular level. In this study, we successfully generated RGCs by differentiation of LHON patient-specific hiPSCs. We modified the protocol of differentiation to obtain a more enriched population of single-cell RGCs for LHON study. Based on assessing morphology, expression of specific markers and electrophysiological activity, we found that LHON-specific hiPSC-derived were more defective in comparison with normal wild-type RGCs. Based on our previous study, whereby by using microarray analysis we identified that the components of glutamatergic synapse signaling pathway were significantly downregulated in LHON-specific RGCs, we focused our study on glutamate-associated alpha -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. We found that the protein expression levels of the subunits of the AMPA receptor, GluR1 and GluR2, and their associated scaffold proteins were decreased in LHON-RGCs. By performing the co-immunoprecipitation assay, we found several differences in the efficiencies of interaction between AMPA subunits and scaffold proteins between normal and LHON-specific RGCs.
Imbalanced Production of Reactive Oxygen Species and Mitochondrial Antioxidant SOD2 in Fabry Disease-Specific Human Induced Pluripotent Stem Cell-Differentiated Vascular Endothelial Cells
(2017-01-01)
Tseng, Wei-Lien
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Chou, Shih-Jie
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Chiang, Huai-Chih
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Wang, Mong-Lien
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Chien, Chian-Shiu
Fabry disease (FD) is an X-linked inherited lysosomal storage disease caused by alpha-galactosidase A (GLA) deficiency. Progressive intracellular accumulation of globotriaosylceramide (Gb3) is considered to be pathogenically responsible for the phenotype variability of FD that causes cardiovascular dysfunction; however, molecular mechanisms underlying the impairment of FD-associated cardiovascular tissues remain unclear. In this study, we reprogrammed human induced pluripotent stem cells (hiPSCs) from peripheral blood cells of patients with FD (FD-iPSCs); subsequently differentiated them into vascular endothelial-likecells (FD-ECs) expressing CD31, VE-cadherin, and vWF; and investigated their ability to form vascular tube-like structures. FD-ECs recapitulated the FD pathophysiological phenotype exhibiting intracellular Gb3 accumulation under a transmission electron microscope. Moreover, compared with healthy control iPSC-derived endothelial cells (NC-ECs), reactive oxygen species (ROS) production considerably increased in FD-ECs. Microarray analysis was performed to explore the possible mechanism underlying Gb3 accumulation-induced ROS production in FD-ECs. Our results revealed that superoxide dismutase 2 (SOD2), a mitochondrial antioxidant, was significantly downregulated in FD-ECs. Compared with NC-ECs, AMPK activity was significantly enhanced in FD-ECs. Furthermore, to investigate the role of Gb3 in these effects, human umbilical vein endothelial cells (HUVECs) were treated with Gb3. After Gb3 treatment, we observed that SOD2 expression was suppressed and AMPK activity was enhanced in a dose-dependent manner. Collectively, our results indicate that excess accumulation of Gb3 suppressed SOD2 expression, increased ROS production, enhanced AMPK activation, and finally caused vascular endothelial dysfunction. Our findings suggest that dysregulated mitochondrial ROS may be a potential target for treating FD.
Interleukin-18 deteriorates Fabry cardiomyopathy and contributes to the development of left ventricular hypertrophy in Fabry patients with GLA IVS4+919 G>A mutation
(2016-12-27)
Chien, Yueh
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Chien, Chian-Shiu
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Chiang, Huai-Chih
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Huang, Wei-Lin
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Chou, Shih-Jie
Rationale: A high incidence of GLA IVS4+919 G>A mutation in patients with Fabry disease of the later-onset cardiac phenotype, has been reported in Taiwan. However, suitable biomarkers or potential therapeutic surrogates for Fabry cardiomyopathy (FC) in such patients under enzyme replacement treatment (ERT) remain unknown. Objective: Using FC patients carrying IVS4+919 G>A mutation, we constructed an induced pluripotent stem cell (iPSC)-based disease model to investigate the pathogenetic biomarkers and potential therapeutic targets in ERT-treated FC. Results and methods: The iPSC-differentiated cardiomyocytes derived from FC-patients (FC-iPSC-CMs) carried IVS4+919 G>A mutation recapitulating FC characteristics, including low a-galactosidase A enzyme activity, cellular hypertrophy, and massive globotriaosylceramide accumulation. Microarray analysis revealed that interleukin-18 (IL-18), a pleiotropic cytokine involved in various myocardial diseases, was the most highly upregulated marker in FC-iPSC-CMs. Meanwhile, IL-18 levels were found to be significantly elevated in the culture media of FC-iPSC-CMs and patients\' sera. Notably, the serum IL-18 levels were highly paralleled with the progression of left ventricular hypertrophy in Fabry patients receiving ERT. Finally, using FC-iPSC-CMs as in vitro FC model, neutralization of IL-18 with specific antibodies combined with ERT synergistically reduced the secretion of IL-18 and the progression of cardiomyocyte hypertrophy in FC-iPSC-CMs. Conclusion: Our data demonstrated that cardiac IL-18 and circulating IL-18 are involved in the pathogenesis of FC and LVH. IL-18 may be a novel marker for evaluating ERT efficacy, and targeting IL-18 might be a potential adjunctive therapy combined with ERT for the treatment of advanced cardiomyopathy in FC patients with IVS4+919 G>A mutation.
Using cationic polyurethane-short branch PEI as microRNA-driven nano-delivery system for stem cell differentiation
(2020-04-01)
Chien, Chian-Shiu
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Wang, Chien-Ying
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Yang, Yi-Ping
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Chou, Shih-Jie
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Ko, Yu-Ling
Background: Non-viral gene delivery, such as using biodegradable polyurethane short-branch polyethylenimine (PU-PEI), has been considered a potentially safer gene delivery system in comparison to conventional virus systems. Methods: The polycationization of DNA complexes protects DNA from nuclease degradation, and these DNA complexes are nanoscale in size to enter the cell through endocytosis. Results: Due to the net positive surface charge of the cell, these polyplexes efficiently bind to the cell through electrostatic interactions with negatively charged membrane components. Cationic PU-PEI has been shown to be non-cytotoxic and has a high transfection efficiency, making it a practical gene delivery material in diseases. Conclusion: We developed a PU-PEI nanomedicine-based platform to efficiently deliver microRNA in promoting differentiation capacity of stem cells, especially on induced pluripotent stem cells.