Browsing by Author "Hwang, Shiaw-Min"

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Disparate mesenchyme-lineage tendencies in mesenchymal stem cells from human bone marrow and umbilical cord blood
(2006-03-01)
Chang, Yu-Jen
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Shih, Daniel Tzu-Bi
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Tseng, Ching-Ping
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Hsieh, Tzu-Bou
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Lee, Don-Ching
Bone marrow and umbilical cord blood are reported to be the main sources of mesenchymal stem cells (MSCs), which have been proposed for many clinical applications. This study evaluated and quantitated the differentiation potential of bone marrow-derived MSCs (bmMSCs) and cord blood-derived MSCs (cbMSCs) by in vitro induction. Results indicated that cbMSCs had a significantly stronger osteogenic potential but lower capacity for adipogenic differentiation than bmMSCs. Leptin, an important regulator of mesenchymal differentiation, has a significantly stronger effect of promoting osteogenesis and inhibiting adipogenesis in bmMSCs than in cbMSCs. Moreover, Cbfa1 mRNA expression in bmMSCs and cbMSCs was affected to different degrees by leptin during osteogenesis. In contrast, leptin reduced PPAR gamma 2 mRNA expression to the same level during adipogenesis in both types of MSCs. These results demonstrate the disparate capacities of MSCs from bone marrow and cord blood and suggest that they be used differently in experimental and therapeutic studies. In addition, the disparate differentiation tendencies of MSCs from different sources should be considered in further applications.
High Growth Rate Downregulates fumA mRNA Transcription but Is Dramatically Compensated by Its mRNA Stability in Escherichia coli
(2012-05-01)
Lin, Hsiao-Hsien
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Lin, Ching-Hsueh
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Hwang, Shiaw-Min
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Tseng, Ching-Ping
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生物科技學系
Little is known about the association among the transcription, post-transcription, and protein production of the fumA gene. This study demonstrates that increasing growth rate (k) from 0.24/h to 0.96/h causes a marked eightfold reduction in fumA transcription as assessed using the beta-galactosidase activity from fumA promoter fused with a lacZ reporter. It was further confirmed using Northern blot analysis. Most interestingly, the FumA protein levels remained unchanged over the growth rate, as indicated by Western blot analysis. Therefore, whether the reduced fumA mRNA expression under the high growth rate can be overcome by increasing the stability of the fumA mRNA was tested. The half-life of fumA mRNA was established to significantly increase by fivefold when the growth rate was increased to 0.96/h. This finding suggests that the cells could turn down the expression of fumA mRNA because of increased stability of its mRNA under the high growth rate. This notion indicates that mRNA stability plays an essential role in maintaining a critical cellular level of a given protein when the mRNA transcript is downregulated by a metabolic event.
Highly efficient drug delivery systems based on functional supramolecular polymers: In vitro evaluation
(2016-03-15)
Cheng, Chih-Chia
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Chang, Feng-Chih
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Kao, Wan-Yi
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Hwang, Shiaw-Min
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Liao, Li-Chuan
The novel concept of modifying and enhancing the properties of existing functional micelles through self-complementary interactions has significant potential. In this study, a practical approach to living polymerization of functionalized thermoresponsive monomers enabled the incorporation of self constituted multiple hydrogen bonded groups into micelles that have potential as supramolecular drug-delivery systems. Phase transitions and morphological studies in aqueous solution showed that the microstructure can be controlled to achieve well-defined vesicle-like micelles with respect to the strength of the hydrogen bond segment. Thus, the resulting micelles have a very low critical micellization concentration and very high loading capacity (16.1%), making the loading process extremely stable and efficient. Incorporation of the anticancer drug doxorubicin (DOX) affected the micellization process in aqueous solution and enabled fine-tuning of drug loading and precise control of drug release rate with excellent sensitivity. Release studies in vitro showed that DOX-loaded micelles exerted dose dependent cytotoxicity against human liver carcinoma (HepG2) cells at the physiological temperature of 37 degrees C. In addition, DOX-loaded micelles were efficiently endocytosed by the cancer cells, which may enable the micelles to serve as suitable vehicles for effective delivery of anticancer drugs to primary tumors and metastatic disease. This newly developed material may provide a potential route towards next-generation drug delivery vehicles. Statement of Significance A breakthrough innovation in water-based thermo-responsive polymers has enabled significant progress in developing smart stimuli-responsive nanocarriers by generating novel "supramolecular polymeric micelles" via self-complementary hydrogen-bonding interactions. These newly developed micelles exhibit extremely high micellar stability and drug loading capacity (up to 16%), excellent thermo-responsive behavior and precise control of drug release rate due to hydrogen-bond-induced physical cross-linking. In addition, doxorubicin-loaded micelles were efficiently endocytosed by the cancer cells, which allows them to serve as suitable vehicles for effective delivery of anticancer drugs to primary tumors and metastatic disease. Thus, this work provides a potential route for the development of next generation multifunctional nanocarriers that have improved safety and to increase the therapeutic efficacy of anticancer therapy. (C) 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Inhibitory Effect of Excessive Glucose on Its Biochemical Pathway and the Growth of Chinese Hamster Ovary (CHO) Cells
(2015-01-02)
Lee, Tsung-Yih
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Lin, Hsiao-Hsien
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Chen, Chien-Lung
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Hwang, Shiaw-Min
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Tseng, Ching-Ping
In this study, we found the specific growth rate of CHO cells being reduced when glucose increased from 1 to 10g/L. Under high glucose, we show a 41% increase in lactate dehydrogenase activity consistent with a 70% rate increase in lactate production. The pyruvate kinase activity was significantly increased. However, the activities of citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase involved in the TCA cycle were attenuated. We propose that pyruvate produced from glycolysis is utilized in a lesser extent over the TCA cycle under high-glucose conditions. Thus, the pyruvate is largely converted to lactate, which is toxic to the cells.
Isolation of Mesenchymal Stem Cells with Neurogenic Potential from the Mesoderm of the Amniotic Membrane
(2010)
Chang, Yu-Jen
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Hwang, Shiaw-Min
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Tseng, Ching-Ping
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Cheng, Fu-Chou
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Huang, Shih-Hung
The amniotic membrane has been clinically applied as a therapeutic material in wound covering and corneal surface reconstruction. Recently, mesenchymal stem cells (MSCs) have been isolated from the placenta, specifically from the amniotic membrane. However, the localization of MSCs in the amniotic membrane has not been determined. In this study, term placenta was collected, and we performed immunohistochemical staining techniques to identify and localize MSCs in the mesoderm of the amniotic membrane in situ with MSC antibodies, including CD90 and CD105. We further directly cultured and characterized MSCs from the amniotic membrane mesoderm (AMSCs). The AMSCs were easily isolated and represented a homogenous fibroblastic morphology at early passages. In addition to MSC surface markers, AMSCs expressed Sox2, Oct-4 and Nanog. AMSCs could be induced into osteocytes, adipocytes and chondrocytes in vitro and show immunosuppressive effects on T-cell proliferation. Under appropriate conditions, AMSCs could differentiate into neuronal-like cells, which were identified by neuronal-specific markers and their ability to secrete dopamine. This study reveals that AMSCs provide a promising source for stem cell studies and also extend the clinical potential of the amniotic membrane in the field of regenerative medicine. Copyright (C) 2010 S. Karger AG, Basel
Modeling neurogenesis impairment in down syndrome with induced pluripotent stem cells from Trisomy 21 amniotic fluid cells
(2013-02-15)
Lu, Huai-En
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Yang, Yao-Chen
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Chen, Sheng-Mei
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Su, Hong-Lin
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Huang, Pai-Cheng
Down syndrome (DS), or Trisomy 21 (121) syndrome, one of the most common chromosomal abnormalities, is caused by an extra duplication of chromosome 21. In studies of neuron development, experimental models based on human cells are considered to be the most desired and accurate for basic research. The generation of diseased induced pluripotetn stem (iPS) cell is a critical step in understanding the developmental stages of complex neuronal diseases. Here, we generated human DS iPS cell lines from second trimester amniotic fluid (AF) cells with 121 by co-expressing Yamanaka factors through lentiviral delivery and subsequently differentiated them into neuronal progenitor cells (NPCs) for further analyses. 121 AF-iPS cells were characterized for the expression of pluripotent markers and for their ability to differentiate into all three germ layers by forming embryoid bodies in vitro and teratomas in vivo. The 121 AF-iPS cells maintained their unique pattern of chromosomal karyotypes: three pairs of chromosome 21. The level of amyloid precursor protein was significantly increased in NPCs derived from 121 AF-iPS cells compared with NPCs from normal AF-iPS cells. The expression levels of miR-155 and miR-802 in 121 AF-iPS-NPCs were highly elevated in the presence of low expression of MeCP2. We observed that 121 iPS-NPCs generated fewer neurons compared with controls. 121 iPS-NPCs exhibit developmental defects during neurogenesis. Our findings suggest that 121 AF-iPS cells serve as a good source to further elucidate the impairment neurogenesis of DS and the onset of Alzheimer's disease. (C) 2012 Elsevier Inc. All rights reserved.
Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system
(2011-08-01)
Lu, Huai-En
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Tsai, Ming-Song
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Yang, Yao-Chen
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Yuan, Chen-Ching
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Wang, Tzu-Hao
Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28%AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84%AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG,TRA-1-60,TRA-1-81,SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening. (C) 2011 Elsevier Inc. All rights reserved.
Submicron-grooved culture surface extends myotube length by forming parallel and elongated motif
(2013-08-01)
Yuan, Chen-Ching
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Ma, Kuang-Jen
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Li, Kuei-Chi
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Chien, Hsi-Hsin
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Lu, Huai-En
During skeletal muscle development, correct cellular orientation is vital to generate desired longitudinal contraction for functional muscle fibres. In this reported study, submicron-imprint lithography was used to generate submicron-grooved surfaces on polystyrene plates to induce striated myotubes in vitro. Mouse muscle myoblast cells cultured on a submicron-grooved surface migrated faster in a directionally uniform fashion; in comparison, cells cultured on a flat surface grew and migrated slower in indiscriminate directions. Subsequent maturation of the myoblast cells formed along the submicron-groove surface resulted in a tandem of parallel myotubes that were both longer and greater in circumference than in the case of the flat surface. In a functional test, the co-culture submicron-groove-grown myotubes with neurotransmitter secreting cells further demonstrated contraction abilities, suggesting submicron-groove-guided growth served to enhance myotube formation while retaining striated motifs and physiological functionality for muscle tissue engineering.