Publications by authors named "Jennifer H Shin"

49 Publications

Engineering 3D Cortical Spheroids for an Ischemic Stroke Model.

ACS Biomater Sci Eng 2021 08 19;7(8):3845-3860. Epub 2021 Jul 19.

Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, 291, Daehak-ro, Daejeon 34141, Republic of Korea.

Three-dimensional (3D) spheroids composed of brain cells have shown great potential to mimic the pathophysiology of the brain. However, a 3D spheroidal brain-disease model for cerebral ischemia has not been reported. This study investigated an ultralow attachment (ULA) surface-mediated formation of 3D cortical spheroids using primary rat cortical cells to recapitulate the cerebral ischemic responses in stroke by oxygen-glucose deprivation-reoxygenation (OGD-R) treatment. Comparison between two-dimensional (2D) and 3D cell culture models confirmed the better performance of the 3D cortical spheroids as normal brain models. The cortical cells cultured in 3D maintained their healthy physiological morphology of a less activated state and suppressed mRNA expressions of pathological stroke markers, S100B, IL-1β, and MBP, selected based on stroke model. Interestingly, the spheroids formed on the ULA surface exhibited striking aggregation dynamics involving active cell-substrate interactions, whereas those formed on the agarose surface aggregated passively by the convective flow of the media. Accordingly, ULA spheroids manifested a layered arrangement of neurons and astrocytes with higher expressions of integrin β1, integrin α5, N-cadherin, and fibronectin than the agarose spheroids. OGD-R-induced stroke model of the ULA spheroids successfully mimicked the ischemic response as evidenced by the upregulated mRNA expressions of the key markers for stroke, S100B, IL-1β, and MBP. Our study suggested that structurally and functionally distinct cortical spheroids could be generated by simply tuning the cell-substrate binding activities during dynamic spheroidal formation, which should be an essential factor to consider in establishing a brain-disease model.
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http://dx.doi.org/10.1021/acsbiomaterials.1c00406DOI Listing
August 2021

Physical analysis reveals distinct responses of human bronchial epithelial cells to guanidine and isothiazolinone biocides.

Toxicol Appl Pharmacol 2021 08 23;424:115589. Epub 2021 May 23.

School of Environmental Engineering, University of Seoul, 163 Seoulsiripdae-ro, Dongdaemun-gu, Seoul 02504, Republic of Korea. Electronic address:

Changes in the physical state of the cells can serve as important indicators of stress responses because they are closely linked with the changes in the pathophysiological functions of the cells. Physical traits can be conveniently assessed by analyzing the morphological features and the stresses at the cell-matrix and cell-cell adhesions in both single-cell and monolayer model systems in 2D. In this study, we investigated the mechano-stress responses of human bronchial epithelial cells, BEAS-2B, to two functionally distinct groups of biocides identified during the humidifier disinfectant accident, namely, guanidine (PHMG) and isothiazolinone (CMIT/MIT). We analyzed the physical traits, including cell area, nuclear area, and nuclear shape. While the results showed inconsistent average responses to the biocides, the degree of dispersion in the data set, measured by standard deviation, was remarkably higher in CMIT/MIT treated cells for all traits. As mechano-stress endpoints, traction and intercellular stresses were also measured, and the cytoskeletal actin structures were analyzed using immunofluorescence. This study demonstrates the versatility of the real-time imaging-based biomechanical analysis, which will contribute to identifying the temporally sensitive cellular behaviors as well as the emergence of heterogeneity in response to exogenously imposed stress factors. This study will also shed light on a comparative understanding of less studied substance, CMIT/MIT, in relation to a more studied substance, PHMG, which will further contribute to more strategic planning for proper risk management of the ingredients involved in toxicological accidents.
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http://dx.doi.org/10.1016/j.taap.2021.115589DOI Listing
August 2021

Enriching neural stem cell and anti-inflammatory glial phenotypes with electrical stimulation after traumatic brain injury in male rats.

J Neurosci Res 2021 Jul 26;99(7):1864-1884. Epub 2021 Mar 26.

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC, USA.

Traumatic brain injury (TBI) by an external physical impact results in compromised brain function via undesired neuronal death. Following the injury, resident and peripheral immune cells, astrocytes, and neural stem cells (NSCs) cooperatively contribute to the recovery of the neuronal function after TBI. However, excessive pro-inflammatory responses of immune cells, and the disappearance of endogenous NSCs at the injury site during the acute phase of TBI, can exacerbate TBI progression leading to incomplete healing. Therefore, positive outcomes may depend on early interventions to control the injury-associated cellular milieu in the early phase of injury. Here, we explore electrical stimulation (ES) of the injury site in a rodent model (male Sprague-Dawley rats) to investigate its overall effect on the constituent brain cell phenotype and composition during the acute phase of TBI. Our data showed that a brief ES for 1 hr on day 2 of TBI promoted anti-inflammatory phenotypes of microglia as assessed by CD206 expression and increased the population of NSCs and Nestin astrocytes at 7 days post-TBI. Also, ES effectively increased the number of viable neurons when compared to the unstimulated control group. Given the salience of microglia and neural stem cells for healing after TBI, our results strongly support the potential benefit of the therapeutic use of ES during the acute phase of TBI to regulate neuroinflammation and to enhance neuroregeneration.
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http://dx.doi.org/10.1002/jnr.24834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8360147PMC
July 2021

Three-Dimensional Spheroid Culture on Polymer-Coated Surface Potentiate Stem Cell Functions via Enhanced Cell-Extracellular Matrix Interactions.

ACS Biomater Sci Eng 2020 04 9;6(4):2240-2250. Epub 2020 Mar 9.

Department of Chemical and Biomolecular Engineering and KI for Nano Century, Korea Advanced of Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.

The aggregation of mesenchymal stem cells (MSCs) into three-dimensional (3D) spheroids has emerged as a promising therapeutic candidate for the treatment of a variety of diseases. In spite of the numerous 3D culture methods suggested recently for MSC spheroid generation, it is still elusive to fully reflect real stem cell niches; this effort majorly suffers from a lack of cell-extracellular matrix (ECM) interactions within the 3D spheroids. In this study, we develop a simple but versatile method for generating human MSC (hMSC) spheroids by culturing the cells on a functional polymer film surface, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4). Interestingly, the pV4D4-coated surface allows a dynamic cell adhesion to the polymer surface while developing the formation of 3D spheroids. The corresponding mechanotransduction promotes the expression of the endogenous ECM and, in turn, results in a remarkable improvement in self-renewal abilities, pro-angiogenic potency, and multilineage differentiation capabilities. This observation highlights the significance of our method compared to the conventional spheroid-generating methods in terms of recreating the ECM-rich microenvironment. We believe the developed surface can serve as a versatile but reliable method for stem cell-based tissue engineering and regenerative medicine.
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http://dx.doi.org/10.1021/acsbiomaterials.9b01738DOI Listing
April 2020

Remodeling of Adhesion Network within Cancer Spheroids via Cell-Polymer Interaction.

ACS Biomater Sci Eng 2020 10 10;6(10):5632-5644. Epub 2020 Sep 10.

Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea.

3D spheroids are considered as the improved model to mimic the distinct arrangements of the cells . To date, low-attachment surfaces have been most widely used to induce the spontaneous aggregation of cells in suspension by simply tuning the relative strength of the cell-cell adhesion over cell-substrate adhesion. However, aggregating cancer cells into 3D clusters should mean more than just adjoining the cells in the physical proximity. The tumor cell functionality is strongly affected by the adhesion networks between cancer cells and extracellular matrix (ECM). Here, we performed an in-depth analysis of how the nonmetastatic breast cancer cells (MCF7) can be transformed to gain invasive phenotypes through compact aggregation into 3D spheroids on a functional polymer film surface, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4). By comparing the adhesion networks and invasion dynamics between 3D spheroids cultured on the pV4D4 surface with those cultured on conventional ultra-low-attachment (ULA) dishes, we report that only spheroids on the pV4D4 display active and sporadic cell-surface binding activities via dynamic protrusions, which correlates strongly with an increase in integrin β1. Moreover, localized laminin expression at the core of the pV4D4-cultured spheroids confirms the prominence of the intimate integrin-laminin interactions prompted by the exposure to pV4D4. This study suggests that structurally and functionally dissimilar 3D spheroids can be generated from the same type of cells on the surfaces of different physicochemical properties without any chemical treatment or genetic manipulation.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00977DOI Listing
October 2020

Super-Resolution Three-Dimensional Imaging of Actin Filaments in Cultured Cells and the Brain Expansion Microscopy.

ACS Nano 2020 11 23;14(11):14999-15010. Epub 2020 Oct 23.

Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea.

Actin is an essential protein in almost all life forms. It mediates diverse biological functions, ranging from controlling the shape of cells and cell movements to cargo transport and the formation of synaptic connections. Multiple diseases are closely related to the dysfunction of actin or actin-related proteins. Despite the biological importance of actin, super-resolution imaging of it in tissue is still challenging, as it forms very dense networks in almost all cells inside the tissue. In this work, we demonstrate multiplexed super-resolution volumetric imaging of actin in both cultured cells and mouse brain slices expansion microscopy (ExM). By introducing a simple labeling process, which enables the anchoring of an actin probe, phalloidin, to a swellable hydrogel, the multiplexed ExM imaging of actin filaments was achieved. We first showed that this technique could visualize the nanoscale details of actin filament organizations in cultured cells. Then, we applied this technique to mouse brain slices and visualized diverse actin organizations, such as the parallel actin filaments along the long axis of dendrites and dense actin structures in postsynaptic spines. We examined the postsynaptic spines in the mouse brain and showed that the organizations of actin filaments are highly diverse. This technique, which enables the high-throughput 60 nm resolution imaging of actin filaments and other proteins in cultured cells and thick tissue slices, would be a useful tool to study the organization of actin filaments in diverse biological circumstances and how they change under pathological conditions.
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http://dx.doi.org/10.1021/acsnano.0c04915DOI Listing
November 2020

Electrospun Microvasculature for Rapid Vascular Network Restoration.

Tissue Eng Regen Med 2021 02 10;18(1):89-97. Epub 2020 Sep 10.

Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.

Background: Sufficient blood supply through neo-vasculature is a major challenge in cell therapy and tissue engineering in order to support the growth, function, and viability of implanted cells. However, depending on the implant size and cell types, the natural process of angiogenesis may not provide enough blood supply for long term survival of the implants, requiring supplementary strategy to prevent local ischemia. Many researchers have reported the methodologies to form pre-vasculatures that mimic in vivo microvessels for implantation to promote angiogenesis. These approaches successfully showed significant enhancement in long-term survival and regenerative functions of implanted cells, yet there remains room for improvement.

Methods: This paper suggests a proof-of-concept strategy to utilize novel scaffolds of dimpled/hollow electrospun fibers that enable the formation of highly mature pre-vasculatures with adequate dimensions and fast degrading in the tissue.

Result: Higher surface roughness improved the maturity of endothelial cells mediated by increased cell-scaffold affinity. The degradation of scaffold material for functional restoration of the neo-vasculatures was also expedited by employing the hollow scaffold design based on co-axial electrospinning techniques.

Conclusion: This unique scaffold-based pre-vasculature can hold implanted cells and tissue constructs for a prolonged time while minimizing the cellular loss, manifesting as a gold standard design for transplantable scaffolds.
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http://dx.doi.org/10.1007/s13770-020-00292-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862457PMC
February 2021

Physicochemically Tuned Myofibroblasts for Wound Healing Strategy.

Sci Rep 2019 11 5;9(1):16070. Epub 2019 Nov 5.

Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.

Normal healing of skin wounds involves a complex interplay between many different cellular constituents, including keratinocytes, immune cells, fibroblasts, myofibroblasts, as well as extracellular matrices. Especially, fibroblasts play a critical role in regulating the immune response and matrix reconstruction by secreting many cytokines and matrix proteins. Myofibroblasts, which are differentiated form of fibroblasts, feature high cellular contractility and encourage the synthesis of matrix proteins to promote faster closure of the wounds. We focus on the functional characteristics of these myofibroblasts as the healing strategy for severe wounds where the surplus amount of matrix proteins could be beneficial for better regeneration. In this study, we first employed multiple physicochemical cues, namely topographical alignment, TGF-β1, and electrical field (EF), to induce differentiation of dermal fibroblasts into myofibroblasts, and to further activate the differentiated cells. We then used these cells in a mouse wound model to verify their potential as a transplantable substitute for the severe wound. Our results confirmed that physicochemically stimulated myofibroblasts promoted faster healing of the wound compared to the case with non-stimulated myofibroblasts through elevated matrix reconstruction in the mouse model. Conclusively, we propose the utilization of physicochemically tuned myofibroblasts as a novel strategy for promoting better healing of moderate to severe wounds.
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http://dx.doi.org/10.1038/s41598-019-52523-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831678PMC
November 2019

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration.

J Vis Exp 2019 10 13(152). Epub 2019 Oct 13.

Department of Biomedical Sciences, Korea University;

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.
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http://dx.doi.org/10.3791/60415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202863PMC
October 2019

Reversible Thermal Gradient Device to Control Biased Thermotactic Response of C. elegans.

Anal Sci 2019 Dec 30;35(12):1367-1373. Epub 2019 Aug 30.

Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology (KAIST).

C. elegans exhibits a directional migration toward a remembered temperature setpoint (T) by activating thermo-sensorimotor neurons. While cryophilic thermotaxis is well reproduced, thermophilic thermotaxis requires very stringent temperature regulations - otherwise, worms exhibit random migration in colder side of T. Here, we introduce a thermal stimulus device developed to control worms with different thermotactic behaviors on both colder and warmer sides of the T. On a linear gradient, the worm population displayed a Gaussian distribution near T but in a skewed shape with a peak shifted to the colder side due to their atactic motion in colder temperature than T. By repetitive application of thermal gradient-reversals, we found that their population density became higher near T because the speed at which the worms accumulate toward T was much faster than that of the dispersion by diffusion to the cold side, resulting in forced aggregation of worms at the desired temperature.
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http://dx.doi.org/10.2116/analsci.19P194DOI Listing
December 2019

Effect of Keratinocytes on Myofibroblasts in Hypertrophic Scars.

Aesthetic Plast Surg 2019 10 25;43(5):1371-1380. Epub 2019 Jul 25.

Department of Plastic and Reconstructive Surgery, School of Medicine, Kyungpook National University, 130 Dongdeokro, Jung-gu, Daegu, 41944, Korea.

Myofibroblasts play a central role in matrix formation and wound contraction during wound healing and undergo apoptosis at the end of the healing. Hypertrophic scarring is a pathologic condition in which myofibroblasts persist in the tissue. It has been hypothesized that abnormalities in epidermal-dermal crosstalk underlie this pathology. Therefore, in this study, we investigated whether myofibroblasts are affected by keratinocytes. Transforming growth factor beta-induced myofibroblasts (Imyo) and myofibroblasts from hypertrophic scar tissue (Hmyo) were characterized using microarrays. Keratinocytes were co-cultured with myofibroblasts, and quantitative PCR analysis was performed. We found that numerous extracellular matrix- and smooth muscle cell-associated genes were upregulated in Imyo and Hmyo respectively, and these findings suggest that Hmyo are fully differentiated myofibroblasts and that Imyo are less differentiated than Hmyo. Decreased collagen type 1 gene expression was found in keratinocytes co-cultured with Imyo and Hmyo; further, α-smooth muscle actin expression in Imyo increased in the presence of keratinocytes. These observations indicate that keratinocytes play a role in the development of pathological fibrosis in hypertrophic scar tissue by regulating the behavior of dermal fibroblasts and myofibroblasts. We believe that this study provides the basis for understanding the pathophysiology of hypertrophic scarring and identifying new therapeutic approaches for this dysfunction.No Level Assigned This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors - www.springer.com/00266 .
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http://dx.doi.org/10.1007/s00266-019-01434-1DOI Listing
October 2019

Stretchable ECM Patch Enhances Stem Cell Delivery for Post-MI Cardiovascular Repair.

Adv Healthc Mater 2019 09 15;8(17):e1900593. Epub 2019 Jul 15.

Center for Biomaterials, Korea Institute of Science and Technology (KIST), Seoul, 02792, Republic of Korea.

Current cell-based therapies administered after myocardial infarction (MI) show limited efficacy due to subpar cell retention in a dynamically beating heart. In particular, cardiac patches generally provide a cursory level of cell attachment due to the lack of an adequate microenvironment. From this perspective, decellularized cell-derived ECM (CDM) is attractive in its recapitulation of a natural biophysical environment for cells. Unfortunately, its weak physical property renders it difficult to retain in its original form, limiting its full potential. Here, a novel strategy to peel CDM off from its underlying substrate is proposed. By physically stamping it onto a polyvinyl alcohol hydrogel, the resulting stretchable extracellular matrix (ECM) membrane preserves the natural microenvironment of CDM, thereby conferring a biological interface to a viscoelastic membrane. Its various mechanical and biological properties are characterized and its capacity to improve cardiomyocyte functionality is demonstrated. Finally, evidence of enhanced stem cell delivery using the stretchable ECM membrane is presented, which leads to improved cardiac remodeling in a rat MI model. A new class of material based on natural CDM is envisioned for the enhanced delivery of cells and growth factors that have a known affinity with ECM.
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http://dx.doi.org/10.1002/adhm.201900593DOI Listing
September 2019

Traction microscopy with integrated microfluidics: responses of the multi-cellular island to gradients of HGF.

Lab Chip 2019 04;19(9):1579-1588

Department of Biomedical Sciences, College of Medicine, Korea University, Seoul 02841, Republic of Korea.

Collective cellular migration plays a central role in development, regeneration, and metastasis. In these processes, mechanical interactions between cells are fundamental but measurement of these interactions is often hampered by technical limitations. To overcome some of these limitations, here we describe a system that integrates microfluidics with traction microscopy (TM). Using this system we can measure simultaneously, and in real time, migration speeds, tractions, and intercellular tension throughout an island of confluent Madin-Darby canine kidney (MDCK) cells. The cell island is exposed to hepatocyte growth factor (HGF) at a controlled gradient of concentrations; HGF is known to elicit epithelial-to-mesenchymal transition (EMT) and cell scattering. As expected, the rate of expansion of the cell island was dependent on the concentration of HGF. Higher concentrations of HGF reduced intercellular tensions, as expected during EMT. A novel finding, however, is that the effects of HGF concentration and its gradient were seen within an island. This integrated experimental system thus provides an integrated tool to better understand cellular forces during collective cellular migration under chemical gradients.
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http://dx.doi.org/10.1039/c9lc00173eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161022PMC
April 2019

Electric field-induced migration and intercellular stress alignment in a collective epithelial monolayer.

Mol Biol Cell 2018 09 25;29(19):2292-2302. Epub 2018 Jul 25.

Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea.

During wound healing, cells migrate with electrotactic bias as a collective entity. Unlike the case of the electric field (EF)-induced single-cell migration, the sensitivity of electrotactic response of the monolayer depends primarily on the integrity of the cell-cell junctions. Although there exist biochemical clues on how cells sense the EF, a well-defined physical portrait to illustrate how collective cells respond to directional EF remains elusive. Here, we developed an EF stimulating system integrated with a hydrogel-based traction measurement platform to quantify the EF-induced changes in cellular tractions, from which the complete in-plane intercellular stress tensor can be calculated. We chose immortalized human keratinocytes, HaCaT, as our model cells to investigate the role of EF in epithelial migration during wound healing. Immediately after the onset of EF (0.5 V/cm), the HaCaT monolayer migrated toward anode with ordered directedness and enhanced speed as early as 15 min. Cellular traction and intercellular stresses were gradually aligned perpendicular to the direction of the EF until 50 min. The EF--induced reorientation of physical stresses was then followed by the delayed cell-body reorientation in the direction perpendicular to the EF. Once the intercellular stresses were aligned, the reversal of the EF direction redirected the reversed migration of the cells without any apparent disruption of the intercellular stresses. The results suggest that the dislodging of the physical stress alignment along the adjacent cells should not be necessary for changing the direction of the monolayer migration.
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http://dx.doi.org/10.1091/mbc.E18-01-0077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249807PMC
September 2018

Aging Donor-Derived Human Mesenchymal Stem Cells Exhibit Reduced Reactive Oxygen Species Loads and Increased Differentiation Potential Following Serial Expansion on a PEG-PCL Copolymer Substrate.

Int J Mol Sci 2018 Jan 25;19(2). Epub 2018 Jan 25.

Severance Biomedical Science Institute, College of Medicine, Yonsei University, Seoul 03722, Korea.

Human mesenchymal stem cells (hMSCs) have been widely studied for therapeutic development in tissue engineering and regenerative medicine. They can be harvested from human donors via tissue biopsies, such as bone marrow aspiration, and cultured to reach clinically relevant cell numbers. However, an unmet issue lies in the fact that the hMSC donors for regenerative therapies are more likely to be of advanced age. Their stem cells are not as potent compared to those of young donors, and continue to lose healthy, stemness-related activities when the hMSCs are serially passaged in tissue culture plates. Here, we have developed a cheap, scalable, and effective copolymer film to culture hMSCs obtained from aged human donors over several passages without loss of reactive oxygen species (ROS) handling or differentiation capacity. Assays of cell morphology, reactive oxygen species load, and differentiation potential demonstrate the effectiveness of copolymer culture on reduction in senescence-related activities of aging donor-derived hMSCs that could hinder the therapeutic potential of autologous stem cell therapies.
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http://dx.doi.org/10.3390/ijms19020359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855581PMC
January 2018

Promotion of Myogenic Maturation by Timely Application of Electric Field Along the Topographical Alignment.

Tissue Eng Part A 2018 05 17;24(9-10):752-760. Epub 2017 Nov 17.

1 Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology , Daejeon, Korea.

Engineered muscular substitutes can restore the impaired muscle functions when integrated properly into the host tissue. To generate functional muscles with sufficient contractility at the site of transplant, the in vitro construction of fully differentiated muscle fibers would be desired. Many previous reports have identified either topographical alignment or electrical stimulation as an effective tool to promote myogenic differentiation. However, optimization of spatial and temporal arrangement of these two physical cues for better differentiation and maturation of skeletal muscles has not been investigated. In this article, we introduce a novel cell culture system that allows simultaneous application of these two independent directional cues at both orthogonal and parallel arrangements. We then show that the parallel arrangement of the aligned topography and the electric field synergistically facilitates better differentiation and maturation of C2C12, generating myotubes with more fused nuclei. Addition of the electric stimulation at the late stage of myogenic differentiation is found to further improve cell fusion to form multinucleate myotubes through a phosphatidylinositol-3-OH-kinase-dependent pathway. As such, we successfully demonstrated that the combined stimulation of topographical and electrical cues could effectively enhance both myogenic differentiation and maturation in a temporal and orientation-dependent manner, providing the basis for therapeutic strategies for regenerative tissue engineering.
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http://dx.doi.org/10.1089/ten.TEA.2017.0055DOI Listing
May 2018

Exposure of keratinocytes to non‑thermal dielectric barrier discharge plasma increases the level of 8‑oxoguanine via inhibition of its repair enzyme.

Mol Med Rep 2017 Nov 11;16(5):6870-6875. Epub 2017 Sep 11.

Department of Biochemistry, School of Medicine and Institute for Nuclear Science and Technology, Jeju National University, Jeju 63243, Republic of Korea.

Oxidative stress enhances cellular DNA oxidation and may cause mutations in DNA bases, including 8‑oxoguanine (8‑oxoG). Our recent study reported that exposure of cells to non‑thermal dielectric barrier discharge (DBD) plasma generates reactive oxygen species and damages DNA. The present study investigated the effect of non‑thermal DBD plasma exposure on the formation of 8‑oxoG in HaCaT human keratinocytes. Cells exposed to DBD plasma exhibited increased level of 8‑oxoG. In addition, mRNA and protein expression levels of 8‑oxoguanine glycosylase 1 (OGG1), an 8‑oxoG repair enzyme, were reduced in plasma‑exposed cells. Furthermore, the expression level of nuclear factor erythroid 2‑related factor 2 (Nrf2), a transcription factor that regulates OGG1 gene expression, was reduced following exposure to DBD plasma. Pretreatment of cells with an antioxidant, N‑acetyl cysteine (NAC), prior to plasma exposure suppressed the formation of 8‑oxoG and restored the expression levels of OGG1 and Nrf2. In addition, phosphorylation of protein kinase B (Akt), which regulates the activation of Nrf2, was reduced following plasma exposure. However, phosphorylation was restored by pretreatment with NAC. These findings suggested that non‑thermal DBD plasma exposure generates 8‑oxoG via inhibition of the Akt‑Nrf2‑OGG1 signaling pathway in HaCaT cells.
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http://dx.doi.org/10.3892/mmr.2017.7454DOI Listing
November 2017

The nesprin-cytoskeleton interface probed directly on single nuclei is a mechanically rich system.

Nucleus 2017 09 22;8(5):534-547. Epub 2017 Jun 22.

b Department of Chemical and Biomolecular Engineering , Vanderbilt University , Nashville , TN , USA.

The cytoskeleton provides structure and plays an important role in cellular function such as migration, resisting compression forces, and transport. The cytoskeleton also reacts to physical cues such as fluid shear stress or extracellular matrix remodeling by reorganizing filament associations, most commonly focal adhesions and cell-cell cadherin junctions. These mechanical stimuli can result in genome-level changes, and the physical connection of the cytoskeleton to the nucleus provides an optimal conduit for signal transduction by interfacing with nuclear envelope proteins, called nesprins, within the LINC (linker of the nucleus to the cytoskeleton) complex. Using single-molecule on single nuclei assays, we report that the interactions between the nucleus and the cytoskeleton, thought to be nesprin-cytoskeleton interactions, are highly sensitive to force magnitude and direction depending on whether cells are historically interfaced with the matrix or with cell aggregates. Application of ∼10-30 pN forces to these nesprin linkages yielded structural transitions, with a base transition size of 5-6 nm, which are speculated to be associated with partial unfoldings of the spectrin domains of the nesprins and/or structural changes of histones within the nucleus.
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http://dx.doi.org/10.1080/19491034.2017.1322237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703230PMC
September 2017

Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer.

Sci Rep 2017 04 4;8:45844. Epub 2017 Apr 4.

Department of Biomedical Sciences, College of Medicine, Korea University, Seoul 02841, Korea.

Hepatocyte growth factor (HGF) induces cell migration and scattering by mechanisms that are thought to tip a local balance of competing physical forces; cell-to-cell and cell-to-substrate forces. In this local process, HGF is known to attenuate local cadherin-dependent adhesion forces for cell-cell junction development and enhance local integrin-dependent contractile forces for pulling neighboring cells apart. Here we use an expanding island of confluent Madin-Darby canine kidney (MDCK) cells as a model system to quantify the collective cell migration. In the absence of HGF, cell trajectories are highly tortuous whereas in the presence of HGF, they become far less so, resembling free expansion of a gas. At the level of cell-to-cell junctions, HGF attenuates the linkage of stress fibers to cell-to-cell junctions with concomitant decrease in intercellular stress. At the level of cell-to-substrate junctions, HGF augments the linkage of stress fibers to cell-to-substrate junctions with no apparent effect on traction. Together, HGF induces both structural changes in the actin-bound junctional protein complex and physical forces spanning multicellular clusters, which further promotes the expansion of confluent cellular layer.
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http://dx.doi.org/10.1038/srep45844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379206PMC
April 2017

Islet-like organoids derived from human pluripotent stem cells efficiently function in the glucose responsiveness in vitro and in vivo.

Sci Rep 2016 10 12;6:35145. Epub 2016 Oct 12.

Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea.

Insulin secretion is elaborately modulated in pancreatic ß cells within islets of three-dimensional (3D) structures. Using human pluripotent stem cells (hPSCs) to develop islet-like structures with insulin-producing ß cells for the treatment of diabetes is challenging. Here, we report that pancreatic islet-like clusters derived from hESCs are functionally capable of glucose-responsive insulin secretion as well as therapeutic effects. Pancreatic hormone-expressing endocrine cells (ECs) were differentiated from hESCs using a step-wise protocol. The hESC-derived ECs expressed pancreatic endocrine hormones, such as insulin, somatostatin, and pancreatic polypeptide. Notably, dissociated ECs autonomously aggregated to form islet-like, 3D structures of consistent sizes (100-150 μm in diameter). These EC clusters (ECCs) enhanced insulin secretion in response to glucose stimulus and potassium channel inhibition in vitro. Furthermore, ß cell-deficient mice transplanted with ECCs survived for more than 40 d while retaining a normal blood glucose level to some extent. The expression of pancreatic endocrine hormones was observed in tissues transplanted with ECCs. In addition, ECCs could be generated from human induced pluripotent stem cells. These results suggest that hPSC-derived, islet-like clusters may be alternative therapeutic cell sources for treating diabetes.
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http://dx.doi.org/10.1038/srep35145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059670PMC
October 2016

Non-thermal gas plasma-induced endoplasmic reticulum stress mediates apoptosis in human colon cancer cells.

Oncol Rep 2016 Oct 24;36(4):2268-74. Epub 2016 Aug 24.

School of Medicine and Institute for Nuclear Science and Technology, Jeju National University, Jeju 63243, Republic of Korea.

Colorectal cancer is a common type of tumor among both men and women worldwide. Conventional remedies such as chemotherapies pose the risk of side‑effects, and in many cases cancer cells develop chemoresistance to these treatments. Non‑thermal gas plasma (NTGP) was recently identified as a potential tool for cancer treatment. In this study, we investigated the potential use of NTGP to control SNUC5 human colon carcinoma cells. We hypothesized that NTGP would generate reactive oxygen species (ROS) in these cells, resulting in induction of endoplasmic reticulum (ER) stress. ROS generation, expression of ER stress‑related proteins and mitochondrial calcium levels were analyzed. Our results confirmed that plasma‑generated ROS induce apoptosis in SNUC5 cells. Furthermore, we found that plasma exposure resulted in mitochondrial calcium accumulation and expression of unfolded protein response (UPR) proteins such as glucose‑related protein 78 (GRP78), protein kinase R (PKR)‑like ER kinase (PERK), and inositol‑requiring enzyme 1 (IRE1). Elevated expression of spliced X‑box binding protein 1 (XBP1) and CCAAT/enhancer‑binding protein homologous protein (CHOP) further confirmed that ROS generated by NTGP induces apoptosis through the ER stress signaling pathway.
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http://dx.doi.org/10.3892/or.2016.5038DOI Listing
October 2016

Effects of minimal exposures to atmospheric pressure plasma on the activity of Salmonella Typhimurium: Deactivation of bacterial motility and suppression of host-cell invasion.

Arch Biochem Biophys 2016 09 23;605:67-75. Epub 2016 Jun 23.

Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea. Electronic address:

Atmospheric pressure plasma (APP) has been shown effective in sterilization by reducing the number of viable microbes during surface cleaning, food processing, or human tissue treatment. For safe conduct, the majority of previous research focused on complete abolition of microbes, which may require severe treatments. Our aim is to investigate the minimal treatment conditions necessary for effective inactivation of bacteria in such a manner that the APP treated bacteria would not be able to harm the host cells. For this, we ought to identify the objective criteria to make the bacteria dysfunctional. We choose the motile properties and the host-cell invasion capability as two measures to quantify the pathogenic state of bacteria. In this paper, we investigated how the APP treatment in a minimal dosage affects the activity of Salmonella Typhimurium. At 100 W and 15 kHz for 20 s, the APP treatment effectively suppressed active "run and tumble" type motility and induced formation of abnormally long structures. With 20 s exposure, the bacterial cells failed to cause pyroptosis in the host cells with >90% survival after 12 h of co-incubation. Our results suggest novel measures to evaluate the functional pathogenic state for identifying safe APP treatment conditions.
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http://dx.doi.org/10.1016/j.abb.2016.06.012DOI Listing
September 2016

Efficient nematode swimming in a shear thinning colloidal suspension.

Soft Matter 2016 Feb 21;12(6):1892-7. Epub 2015 Dec 21.

School of Mechanical, Aerospace and Systems Engineering, Division of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea.

The swimming behavior of a nematode Caenorhabditis elegans (C. elegans) is investigated in a non-Newtonian shear thinning colloidal suspension. At the onset value (ϕ∼ 8%), the suspension begins to exhibit shear thinning behavior, and the average swimming speed of worms jumps by approximately 12% more than that measured in a Newtonian solution exhibiting no shear dependent viscosity. In the shear thinning regime, we observe a gradual yet significant improvement in swimming efficiency with an increase in ϕ while the swimming speed remains nearly constant. We postulate that this enhanced swimming can be explained by the temporal change in the stroke form of the nematode that is uniquely observed in a shear thinning colloidal suspension: the nematode features a fast and large stroke in its head to overcome the temporally high drag imposed by the viscous medium, whose effective viscosity (ηs) is shown to drop drastically, inversely proportional to the strength of its stroke. Our results suggest new insights into how nematodes efficiently maneuver through the complex fluid environment in their natural habitat.
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http://dx.doi.org/10.1039/c5sm01824bDOI Listing
February 2016

Focal Adhesion Assembly Induces Phenotypic Changes and Dedifferentiation in Chondrocytes.

J Cell Physiol 2016 Aug 4;231(8):1822-31. Epub 2016 Feb 4.

Division of Mechanical Engineering, School of Mechanical, Aerospace, and Systems Engineering, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea.

The expansion of autologous chondrocytes in vitro is used to generate sufficient populations for cell-based therapies. However, during monolayer culture, chondrocytes lose inherent characteristics and shift to fibroblast-like cells as passage number increase. Here, we investigated passage-dependent changes in cellular physiology, including cellular morphology, motility, and gene and protein expression, as well as the role of focal adhesion and cytoskeletal regulation in the dedifferentiation process. We found that the gene and protein expression levels of both the focal adhesion complex and small Rho GTPases are upregulated with increasing passage number and are closely linked to chondrocyte dedifferentiation. The inhibition of focal adhesion kinase (FAK) but not small Rho GTPases induced the loss of fibroblastic traits and the recovery of collagen type II, aggrecan, and SOX9 expression levels in dedifferentiated chondrocytes. Based on these findings, we propose a strategy to suppress chondrogenic dedifferentiation by inhibiting the identified FAK or Src pathways while maintaining the expansion capability of chondrocytes in a 2D environment. These results highlight a potential therapeutic target for the treatment of skeletal diseases and the generation of cartilage in tissue-engineering approaches. J. Cell. Physiol. 231: 1822-1831, 2016. © 2015 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcp.25290DOI Listing
August 2016

Inhibition of Rho-Associated Protein Kinase Increases the Angiogenic Potential of Mesenchymal Stem Cell Aggregates via Paracrine Effects.

Tissue Eng Part A 2016 Feb 21;22(3-4):233-43. Epub 2016 Jan 21.

1 Department of Biomedical Engineering, College of Medicine, Korea University , Seoul, Korea.

The aggregation of multiple cells, such as mesenchymal condensation, is an important biological process in skeletal muscle development, osteogenesis, and adipogenesis. Due to limited in vivo study model systems, a simple and effective in vitro three-dimensional (3D) aggregation system is required to study the mechanisms of multicellular aggregation and its applications. We first generated controlled mesenchymal stem cell (MSC) aggregates using a bioprinting technique to monitor their aggregation and sprouting. We induced the angiogenic potential of the MSCs through chemical inhibition of the Rho/Rho-associated protein kinase (ROCK) pathway, which led to hairy sprouting in the aggregates. The angiogenic potential of this 3D construct was then tested by subcutaneously implanting the Matrigel with 3D MSC aggregates in a rat. Treatment of 3D MSCs with the ROCK inhibitor, Y27632, increased their angiogenic activity in vivo. The gene expressions and histological staining indicated that angiogenesis and neovascularization were mainly regulated by the paracrine factors secreted from human 3D MSC constructs. Our results demonstrate the enhancement of the angiogenic potential of the MSC constructs through the secretion of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) by the inhibition of the Rho/ROCK pathway.
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http://dx.doi.org/10.1089/ten.TEA.2015.0289DOI Listing
February 2016

Non-thermal dielectric-barrier discharge plasma damages human keratinocytes by inducing oxidative stress.

Int J Mol Med 2016 Jan 9;37(1):29-38. Epub 2015 Nov 9.

School of Medicine and Institute for Nuclear Science and Technology, Jeju National University, Jeju 63243, Republic of Korea.

The aim of this study was to identify the mechanisms through which dielectric-barrier discharge plasma damages human keratinocytes (HaCaT cells) through the induction of oxidative stress. For this purpose, the cells were exposed to surface dielectric-barrier discharge plasma in 70% oxygen and 30% argon. We noted that cell viability was decreased following exposure of the cells to plasma in a time-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The levels of intracellular reactive oxygen species (ROS) were determined using 2',7'-dichlorodihydrofluorescein diacetate and dihydroethidium was used to monitor superoxide anion production. Plasma induced the generation of ROS, including superoxide anions, hydrogen peroxide and hydroxyl radicals. N-acetyl cysteine, which is an antioxidant, prevented the decrease in cell viability caused by exposure to plasma. ROS generated by exposure to plasma resulted in damage to various cellular components, including lipid membrane peroxidation, DNA breaks and protein carbonylation, which was detected by measuring the levels of 8-isoprostane and diphenyl-1-pyrenylphosphine assay, comet assay and protein carbonyl formation. These results suggest that plasma exerts cytotoxic effects by causing oxidative stress-induced damage to cellular components.
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http://dx.doi.org/10.3892/ijmm.2015.2405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687437PMC
January 2016

Role of atmospheric pressure plasma (APP) in wound healing: APP-induced antifibrotic process in human dermal fibroblasts.

Exp Dermatol 2016 Feb 23;25(2):159-61. Epub 2015 Nov 23.

Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Korea.

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http://dx.doi.org/10.1111/exd.12865DOI Listing
February 2016

Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage.

Biomol Ther (Seoul) 2015 Jul 1;23(4):357-66. Epub 2015 Jul 1.

School of Medicine, Jeju National University, Jeju 690-756.

Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death.
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http://dx.doi.org/10.4062/biomolther.2015.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489831PMC
July 2015

Human endothelial colony forming cells from adult peripheral blood have enhanced sprouting angiogenic potential through up-regulating VEGFR2 signaling.

Int J Cardiol 2015 Oct 15;197:33-43. Epub 2015 Jun 15.

Department of Cardiology, Cardiovascular Center, Korea University Anam Hospital, Seoul, Republic of Korea. Electronic address:

Background: Endothelial colony forming cells (ECFCs), a subtype of endothelial progenitor cells, have been studied as a promising cellular source for therapeutic angiogenesis. Although ECFCs are very similar to mature endothelial cells, details regarding the role of ECFCs during angiogenesis are not known. We compared the cellular and angiogenic properties of ECFCs and mature endothelial cells (HUVECs).

Methods: HUVECs were used as control. Quantitative RT-PCR, western blotting, immunofluorescence staining, flow cytometric analyses and angiogenic cytokine array were performed. 3D-microfluidic angiogenesis assay system was adopted for in vitro angiogenic potential. In vivo angiogenic potential was assessed by Matrigel plug assay.

Results: ECFCs had higher expression of activated endothelial tip cell markers (Dll4, CXCR4, CD34, and VCAM1) and arterial genes (DLL4 and CX40), but lower expression of venous and lymphatic genes (COUP-TFII and PROX1). In 3D-microfluidic angiogenesis assay system, ECFCs induced robust sprouting vascular structures. Co-cultivation of both ECFCs and HUVECs gave rise to lumen-formed hybrid vascular structures, with the resulting ECFCs predominantly localized to the tip portion. This finding suggests that the ECFC has a role as a sprouting endothelial tip cell. Interestingly, VEGF-A phosphorylated VEGFR2 and its downstream signaling molecules more strongly in ECFCs than in HUVECs. Even small amount of VEGF-A successfully induced the sprouting angiogenesis of ECFCs. Finally, co-administration of ECFCs and human dermal fibroblasts successfully induced lumen-formed maturated neovessels in vivo.

Conclusion: ECFCs derived from adult peripheral blood had enhanced sprouting angiogenic potential in vitro and in vivo through up-regulation of the VEGFR2 signaling pathway.
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http://dx.doi.org/10.1016/j.ijcard.2015.06.013DOI Listing
October 2015

Photo-protective effect of americanin B against ultraviolet B-induced damage in cultured human keratinocytes.

Environ Toxicol Pharmacol 2014 Nov 3;38(3):891-900. Epub 2014 Sep 3.

School of Medicine and Institute for Nuclear Science and Technology, Jeju National University, Jeju 690-756, Republic of Korea. Electronic address:

Excessive ultraviolet (UV) radiation, a constituent of sunlight, can induce multiple types of skin damage. We recently demonstrated that americanin B, a lignin compound, protected cells against hydrogen peroxide (H2O2)-induced damage by exerting antioxidant effects and inhibiting apoptosis. In this study, we investigated the ability of americanin B to protect against cell injury induced by UVB (280-320nm), the most harmful UV wavelengths, in human HaCaT keratinocytes. Americanin B absorbed UVB, eliminated UVB-induced intracellular reactive oxygen species (ROS), and decreased the extent of UVB-induced oxidative modification of lipids, proteins, and DNA. In addition, americanin B inhibited UVB-induced apoptosis, as indicated by reductions in apoptotic body formation and DNA fragmentation. Furthermore, americanin B reversed the depolarization of the mitochondrial membrane induced by UVB exposure. These protective activities were associated with down-regulation of apoptosis-promoting proteins, Bax, caspase-9, and caspase-3 and up-regulation of an apoptosis inhibitor, Bcl-2. These results suggest that americanin B can protect human keratinocytes against UVB-induced cell damage.
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http://dx.doi.org/10.1016/j.etap.2014.08.017DOI Listing
November 2014
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