Browsing by Author "Lu, Huai-En"

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Direct Reprogramming of Human Suspension Cells into Mesodermal Cell Lineages via Combined Magnetic Targeting and Photothermal Stimulation by Magnetic Graphene Oxide Complexes
(2017-08-25)
Chiang, Min-Yu
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Lin, Yi-Zhen
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Chang, Shwu-Jen
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Shyu, Woei-Cherng
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Lu, Huai-En
Suspension cells can provide a source of cells for cellular reprogramming, but they are difficult to transfect by nonviral vectors. An efficient and safe nonviral vector (GO-Fe3O4-PEI complexes) based on iron oxide nanoparticle (Fe3O4)-decorated graphene oxide (GO) complexed with polyethylenimine (PEI) for the first time is developed for delivering three individual episomal plasmids (pCXLE-hOCT3/4-shp53, pCXLE-hSK, and pCXLE-hUL) encoding pluripotent-related factors of Oct3/4, shRNA against p53, Sox2, Klf4, L-Myc, and Lin28 into human peripheral blood mononuclear cells (PBMCs) simultaneously. The combined treatment of magnetic stirring and near-infrared (NIR)-laser irradiation, which can promote contact between the complexes and floating cells and increase the cell membrane permeability, respectively, is used to conduct multiple physical stimulations for suspension PBMCs transfection. The PCR analysis shows that the combinatorial effect of magnetic targeting and photothermal stimulation obviously promoted the transfection efficiency of suspension cells. The transfected cells show positive expression of the pluripotency markers, including Nanog, Oct4, and Sox2, and have potential to differentiate into mesoderm and ectoderm cells. The results demonstrate that the GO-Fe3O4-PEI complex provides a safe, convenient, and efficient tool for reprogramming PBMCs into partially induced pluripotent stem cells, which are able to rapidly transdifferentiate into mesodermal lineages without full reprogramming.
Early Administration of Glutamine Protects Cardiomyocytes from Post-Cardiac Arrest Acidosis
(2016-01-01)
Lin, Yan-Ren
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Li, Chao-Jui
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Syu, Shih-Han
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Wen, Cheng-Hao
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Buddhakosai, Waradee
Postcardiac arrest acidosis can decrease survival. Effective medications without adverse side effects are still not well characterized. We aimed to analyze whether early administration of glutamine could improve survival and protect cardiomyocytes from postcardiac arrest acidosis using animal and cell models. Forty Wistar rats with postcardiac arrest acidosis (blood pH < 7.2) were included. They were divided into study (500 mg/kg L-alanyl-L-glutamine, n = 20) and control (normal saline,n = 20) groups. Each of the rats received resuscitation. The outcomes were compared between the two groups. In addition, cardiomyocytes derived from human induced pluripotent stem cells were exposed to HBSS with different pH levels (7.3 or 6.5) or to culture medium (control). Apoptosis-related markers and beating function were analyzed. We found that the duration of survival was significantly longer in the study group (n < 0.05). In addition, in pH 6.5 or pH 7.3 HBSS buffer, the expression levels of cell stress (p53) and apoptosis (caspase-3, Bcl-xL) markers were significantly lower in cardiomyocytes treated with 50 mM L-glutamine than those without L-glutamine (RT-PCR). L-glutamine also increased the beating function of cardiomyocytes, especially at the lower pH level (6.5). More importantly, glutamine decreased cardiomyocyte apoptosis and increased these cells' beating function at a low pH level.
Impact of Different Initial Epinephrine Treatment Time Points on the Early Postresuscitative Hemodynamic Status of Children With Traumatic Out-of-hospital Cardiac Arrest
(2016-03-01)
Lin, Yan-Ren
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Syue, Yuan-Jhen
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Buddhakosai, Waradee
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Lu, Huai-En
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Chang, Chin-Fu
The postresuscitative hemodynamic status of children with traumatic out-of-hospital cardiac arrest (OHCA) might be impacted by the early administration of epinephrine, but this topic has not been well addressed. The aim of this study was to analyze the early postresuscitative hemodynamics, survival, and neurologic outcome according to different time points of first epinephrine treatment among children with traumatic OHCA.Information on 388 children who presented to the emergency departments of 3 medical centers and who were treated with epinephrine for traumatic OHCA during the study period (2003-2012) was retrospectively collected. The early postresuscitative hemodynamic features (cardiac functions, end-organ perfusion, and consciousness), survival, and neurologic outcome according to different time points of first epinephrine treatment (early: <15, intermediate: 15-30, and late: >30minutes after collapse) were analyzed.Among 165 children who achieved sustained return of spontaneous circulation, 38 children (9.8%) survived to discharge and 12 children (3.1%) had good neurologic outcomes. Early epinephrine increased the postresuscitative heart rate and blood pressure in the first 30minutes, but ultimately impaired end-organ perfusion (decreased urine output and initial creatinine clearance) (all P<0.05). Early epinephrine treatment increased the chance of achieving sustained return of spontaneous circulation, but did not increase the rates of survival and good neurologic outcome.Early epinephrine temporarily increased heart rate and blood pressure in the first 30minutes of the postresuscitative period, but impaired end-organ perfusion. Most importantly, the rates of survival and good neurologic outcome were not significantly increased by early epinephrine administration.
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.
Organ-on-a-Chip: Opportunities for Assessing the Toxicity of Particulate Matter
(2020-05-29)
Yang, Jia-Wei
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Shen, Yu-Chih
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Lin, Ko-Chih
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Cheng, Sheng-Jen
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Chen, Shiue-Luen
Recent developments in epidemiology have confirmed that airborne particulates are directly associated with respiratory pathology and mortality. Although clinical studies have yielded evidence of the effects of many types of fine particulates on human health, it still does not have a complete understanding of how physiological reactions are caused nor to the changes and damages associated with cellular and molecular mechanisms. Currently, most health assessment studies of particulate matter (PM) are conducted through cell culture or animal experiments. The results of such experiments often do not correlate with clinical findings or actual human reactions, and they also cause difficulty when investigating the causes of air pollution and associated human health hazards, the analysis of biomarkers, and the development of future pollution control strategies. Microfluidic-based cell culture technology has considerable potential to expand the capabilities of conventional cell culture by providing high-precision measurement, considerably increasing the potential for the parallelization of cellular assays, ensuring inexpensive automation, and improving the response of the overall cell culture in a more physiologically relevant context. This review paper focuses on integrating the important respiratory health problems caused by air pollution today, as well as the development and application of biomimetic organ-on-a-chip technology. This more precise experimental model is expected to accelerate studies elucidating the effect of PM on the human body and to reveal new opportunities for breakthroughs in disease research and drug development.
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.
The Development of Controllable Magnetic Driven Microphysiological System
(2019-11-07)
Yang, Jia-Wei
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Chen, Yu-Wei
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Ho, Pei-Yi
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Jiang, Liane
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Hsieh, Kuan Yu
Current research has enabled the use of microphysiological systems and creation of models for alveolar and pulmonary diseases. However, bottlenecks remain in terms of medium- and long-term regulation of cell cultures and their functions in microchannel systems, as well as in the enhancement of in vitro representation of alveolar models and reference values of the data. Currently used systems also require on-chip manufacturing of complex units, such as pumps, tubes, and other cumbersome structures for maintaining cells in culture. In addition, system simplification and minimization of all external and human factors major challenges facing the establishment of in vitro alveolar models. In this study, a magnetically driven dynamic alveolus cell-culture system has been developed to use controlled magnetic force to drive a magnetic film on the chip, thereby directing the fluid within it to produce a circulating flow. The system has been confirmed to be conducive with regard to facilitating uniform attachment of human alveolar epithelial cells and long-term culture. The cell structure has been recapitulated, and differentiation functions have been maintained. Subsequently, reactions between silica nanoparticles and human alveolar epithelial cells have been used to validate the effects and advantages of the proposed dynamic chip-based system compared to a static environment. The innovative concept of use of a magnetic drive has been successfully employed in this study to create a simple and controllable yet dynamic alveolus cell-culture system to realize its functions and advantages with regard to in vitro tissue construction.