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      Application of co-culture technology of epithelial type cells and mesenchymal type cells using nanopatterned structures

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          Abstract

          Various nanopatterning techniques have been developed to improve cell proliferation and differentiation efficiency. As we previously reported, nanopillars and pores are able to sustain human pluripotent stem cells and differentiate pancreatic cells. From this, the nanoscale patterns would be effective environment for the co-culturing of epithelial and mesenchymal cell types. Interestingly, the nanopatterning selectively reduced the proliferative rate of mesenchymal cells while increasing the expression of adhesion protein in epithelial type cells. Additionally, co-cultured cells on the nanopatterning were not negatively affected in terms of cell function metabolic ability or cell survival. This is in contrast to conventional co-culturing methods such as ultraviolet or chemical treatments. The nanopatterning appears to be an effective environment for mesenchymal co-cultures with typically low proliferative rates cells such as astrocytes, neurons, melanocytes, and fibroblasts without using potentially damaging treatments.

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          Most cited references29

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          The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder.

          A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.
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            Stem cells and their niches.

            A constellation of intrinsic and extrinsic cellular mechanisms regulates the balance of self-renewal and differentiation in all stem cells. Stem cells, their progeny, and elements of their microenvironment make up an anatomical structure that coordinates normal homeostatic production of functional mature cells. Here we discuss the stem cell niche concept, highlight recent progress, and identify important unanswered questions. We focus on three mammalian stem cell systems where large numbers of mature cells must be continuously produced throughout adult life: intestinal epithelium, epidermal structures, and bone marrow.
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              Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.

              The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Young's moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs. (c) 2009 Elsevier Ltd. All rights reserved.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysis
                Role: Data curationRole: Formal analysis
                Role: Data curationRole: Writing – original draft
                Role: Data curationRole: Writing – original draft
                Role: Formal analysisRole: Methodology
                Role: Formal analysisRole: Methodology
                Role: ResourcesRole: Supervision
                Role: Formal analysisRole: Methodology
                Role: ConceptualizationRole: Supervision
                Role: ConceptualizationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: SupervisionRole: Writing – original draft
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 May 2020
                2020
                : 15
                : 5
                : e0232899
                Affiliations
                [1 ] Research Institute, T&R Biofab Co. Ltd, Siheung, Republic of Korea
                [2 ] Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, Republic of Korea
                [3 ] Department of Mechanical Engineering, Pohang University of Science and Technology, (POSTECH) Pohang, Pohang, Republic of Korea
                [4 ] Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
                [5 ] R&D Unit, Amorepacific Corporation, Yongin-si, Gyeonggi-do, Republic of Korea
                [6 ] Department of Polymer Science and Engineering Chungnam National University, Daejeon, Republic of Korea
                [7 ] Research Group for Biomimetic Advanced Technology, Korea Institute of Toxicology, Daejeon, Republic of Korea
                [8 ] Department of Human and Environmental Toxicology, University of Science and Technology, Daejeon, Republic of Korea
                [9 ] Department of Medical Science, School of Medicine, Konkuk University, Seoul, Republic of Korea
                Georgetown University, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist. T&R Biofab Co. Ltd and Amorepacific Corporation do not alter our adherence to PLOS ONE policies on sharing data and materials.

                ‡ These authors also contributed equally to this work.

                Author information
                http://orcid.org/0000-0002-0593-6284
                http://orcid.org/0000-0002-7906-495X
                Article
                PONE-D-19-35930
                10.1371/journal.pone.0232899
                7213697
                32392240
                1d1ff6e5-c198-48e1-b8f1-6ad563da3419
                © 2020 Jung et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 30 December 2019
                : 23 April 2020
                Page count
                Figures: 5, Tables: 0, Pages: 17
                Funding
                This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation funded by the MSIP (NRF-2016M3A9B4919616, NRF-2019M3A9H1103331) and by a grant (20000325) from the Technology Innovation Program funded from the Ministry of Trade, Industry and Energy (MOTIE), and National Research Foundation grant (MSIT) (No. 2017R1A2A1A05001090) funded by the Republic of Korea government. T&R Biofab Co. Ltd and Amorepacific Corporation did not provide financial support, and did not have any roles in this study. The authors belonging to T&R Biofab and Amorepacific companies recently moved to the companies, and no funds have been contributed by the companies.
                Categories
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                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Connective Tissue Cells
                Fibroblasts
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                Biological Tissue
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