Browsing by Author "Yang, Yi-Ping"

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Carboxylated nanodiamond-mediated CRISPR-Cas9 delivery of human retinoschisis mutation into human iPSCs and mouse retina
(2020-01-01)
Yang, Tien-Chun
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Chang, Chia-Yu
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Yarmishyn, Aliaksandr A.
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Mao, Yen-Shiang
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Yang, Yi-Ping
Nanodiamonds (NDs) are considered to be relatively safe carbon nanomaterials used for the transmission of DNA, proteins and drugs. The feasibility of utilizing the NDs to deliver CRISPR-Cas9 system for gene editing has not been clearly studied. Therefore, in this study, we aimed to use NDs as the carriers of CRISPR-Cas9 components designed to introduce the mutation in RS1 gene associated with Xlinked retinoschisis (XLRS). ND particles with a diameter of 3 nm were functionalized by carboxylation of the surface and covalently conjugated with fluorescent mCherry protein. Two linear DNA constructs were attached to the conjugated mCherry: one encoded Cas9 endonuclease and GFP reporter, another encoded sgRNA and contained insert of HDR template designed to introduce RS1 c.625C>T mutation. Such nanoparticles were successfully delivered and internalized by human iPSCs and mouse retinas, the efficiency of internalization was significantly improved by mixing with BSA. The delivery of ND particles led to introduction of RS1 c.625C>T mutation in both human iPSCs and mouse retinas. Rs1 gene editing in mouse retinas resulted in several pathological features typical for XLRS, such as aberrant photoreceptor structure. To conclude, our ND-based CRISPR-Cas9 delivery system can be utilized as a tool for creating in vitro and in vivo disease models of XLRS. Statement of significance X-linked retinoschisis (XLRS) is a prevalent hereditary retinal disease, which is caused by mutations in RS1 gene, whose product is important for structural organization of the retina. The recent development of genome editing techniques such as CRISPR-Cas9 significantly improved the prospects for better understanding the pathology and development of treatment for this disease. Firstly, gene editing can allow development of appropriate in vitro and in vivo disease models; secondly, CRISPR-Cas9 can be applied for gene therapy by removing the disease-causative mutation in vivo. The major prerequisite for these approaches is to develop safe and efficient CRISPR-Cas9 delivery system. In this study, we tested specifically modified nanodiamonds for such a delivery system. We were able to introduce Rs1 mutation into the mouse retina and, importantly, observed several XLRS-specific effects. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd.
Development of a Graphene Oxide-Incorporated Polydimethylsiloxane Membrane with Hexagonal Micropillars
(2018-09-01)
Lin, Yi-Ying
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Chien, Yueh
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Chuang, Jen-Hua
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Chang, Chia-Ching
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Yang, Yi-Ping
Several efforts have been made on the development of bioscaffolds including the polydimethylsiloxane (PDMS) elastomer for supporting cell growth into stable sheets. However, PDMS has several disadvantages, such as intrinsic surface hydrophobicity and mechanical strength. Herein, we generated a novel PDMS-based biomimetic membrane by sequential modifications of the PMDS elastomer with graphene oxide (GO) and addition of a hexagonal micropillar structure at the bottom of the biomembrane. GO was initially homogenously mixed with pure PDMS and then was further coated onto the upper surface of the resultant PDMS. The elastic modulus and hydrophilicity were significantly improved by such modifications. In addition, the development of hexagonal micropillars with smaller diameters largely improved the ion permeability and increased the motion resistance. We further cultured retinal pigment epithelial (RPE) cells on the surface of this modified PDMS biomembrane and assayed its biocompatibility. Remarkably, the GO incorporation and coating exhibited beneficial effect on the cell growth and the new formation of tight junctions in RPE cells. Taken together, this GO-modified PDMS scaffold with polyhexagonal micropillars may be utilized as an ideal cell sheet and adaptor for cell cultivation and can be used in vivo for the transplantation of cells such as RPE cells.
Elongation of Axon Extension for Human iPSC-Derived Retinal Ganglion Cells by a Nano-Imprinted Scaffold
(2017-09-01)
Yang, Tien-Chun
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Chuang, Jen-Hua
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Buddhakosai, Waradee
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Wu, Wen-Ju
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Lee, Chen-Ju
Optic neuropathies, such as glaucoma and Leber's hereditary optic neuropathy (LHON) lead to retinal ganglion cell (RGC) loss and therefore motivate the application of transplantation technique into disease therapy. However, it is a challenge to direct the transplanted optic nerve axons to the correct location of the retina. The use of appropriate scaffold can promote the proper axon growth. Recently, biocompatible materials have been integrated into the medical field, such as tissue engineering and reconstruction of damaged tissues or organs. We, herein, utilized nano-imprinting to create a scaffold mimicking the in vitro tissue microarchitecture, and guiding the axonal growth and orientation of the RGCs. We observed that the robust, long, and organized axons of human induced pluripotent stem cell (iPSC)-derived RGCs projected axially along the scaffold grooves. The RGCs grown on the scaffold expressed the specific neuronal biomarkers indicating their proper functionality. Thus, based on our in vitro culture system, this device can be useful for the neurophysiological analysis and transplantation for ophthalmic neuropathy treatment.
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.
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.