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      Cross-talk between human airway epithelial cells and 3T3-J2 feeder cells involves partial activation of human MET by murine HGF

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          Abstract

          There is considerable interest in the ex vivo propagation of primary human basal epithelial stem/progenitor cells with a view to their use in drug development, toxicity testing and regenerative medicine. These cells can be expanded in co-culture with mitotically inactivated 3T3-J2 murine embryonic feeder cells but, similar to other epithelial cell culture systems employing 3T3-J2 cells, the aspects of cross-talk between 3T3-J2 cells and human airway basal cells that are critical for their expansion remain largely unknown. In this study, we investigated secreted growth factors that are produced by 3T3-J2 cells and act upon primary human airway basal cells. We found robust production of hepatocyte growth factor (HGF) from fibroblast feeder cells following mitotic inactivation. Consistent with the limited cross-species reactivity of murine HGF on the human HGF receptor (MET; HGFR), MET inhibition did not affect proliferative responses in human airway basal cells and HGF could not replace feeder cells in this culture system. However, we found that murine HGF is not completely inactive on human airway epithelial cells or cancer cell lines but stimulates the phosphorylation of GRB2-associated-binding protein 2 (GAB2) and signal transducer and activator of transcription 6 (STAT6). Although HGF induces phosphorylation of STAT6 tyrosine 641 (Y641), there is no subsequent STAT6 nuclear translocation or STAT6-driven transcriptional response. Overall, these findings highlight the relevance of cross-species protein interactions between murine feeder cells and human epithelial cells in 3T3-J2 co-culture and demonstrate that STAT6 phosphorylation occurs in response to MET activation in epithelial cells. However, STAT6 nuclear translocation does not occur in response to HGF, precluding the transcriptional activity of STAT6.

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          Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.

          Human diploid epidermis epidermal cells have been successfully grown in serial culture. To initiate colony formation, they require the presence of fibroblasts, but proliferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct density. When trypsinized human skin cells are plated together with the 3T3 cells, the growth of the human fibroblasts is largely suppressed, but epidermal cells grow from single cells into colonies. Each colony consists of keratinocytes ultimately forming a stratified squamous epithelium in which the dividing cells are confined to the lowest layer(s). Hydrocortisone is added to the medium, since in secondary and subsequent subcultures it makes the colony morphology more oderly and distinctive, and maintains proliferation at a slightly greater rate. Under these culture conditions, it is possible to isolate keratinocyte clones free of viable fibroblasts. Like human diploid fibroblasts, human diploid keratinocytes appear to have a finite culture lifetime. For 7 strains studied, the culture lifetime ranged from 20-50 cell generations. The plating efficiency of the epidermal cells taken directly from skin was usually 0.1-1.0%. On subsequent transfer of the cultures initiated from newborns, the plating efficiency rose to 10% or higher, but was most often in the range of 1-5% and dropped sharply toward the end of their culture life. The plating efficiency and culture lifetime were lower for keratinocytes of older persons.
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            Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function.

            Respiratory disease is the third leading cause of death in the industrialized world. Consequently, the trachea, lungs, and cardiopulmonary vasculature have been the focus of extensive investigations. Recent studies have provided new information about the mechanisms driving lung development and differentiation. However, there is still much to learn about the ability of the adult respiratory system to undergo repair and to replace cells lost in response to injury and disease. This Review highlights the multiple stem/progenitor populations in different regions of the adult lung, the plasticity of their behavior in injury models, and molecular pathways that support homeostasis and repair. Copyright © 2014 Elsevier Inc. All rights reserved.
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              An overview of the c-MET signaling pathway.

              c-MET is a receptor tyrosine kinase that, after binding with its ligand, hepatocyte growth factor, activates a wide range of different cellular signaling pathways, including those involved in proliferation, motility, migration and invasion. Although c-MET is important in the control of tissue homeostasis under normal physiological conditions, it has also been found to be aberrantly activated in human cancers via mutation, amplification or protein overexpression. This paper provides an overview of the c-MET signaling pathway, including its role in the development of cancers, and provides a rationale for targeting the pathway as a possible treatment option.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Investigation
                Role: InvestigationRole: Validation
                Role: InvestigationRole: Methodology
                Role: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Supervision
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                17 May 2018
                2018
                : 13
                : 5
                : e0197129
                Affiliations
                [1 ] Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom
                [2 ] CRUK Lung Cancer Centre of Excellence, UCL Cancer Institute, University College London, London, United Kingdom
                [3 ] The Francis Crick Institute, London, United Kingdom
                [4 ] Dipartimento di Scienze Cardio-Toraciche e Respiratorie, Universita’ degli Studi della Campania “L. Vanvitelli”, Naples, Italy
                [5 ] Stem Cell and Regenerative Medicine Section, UCL Institute of Child Health and Great Ormond Street Hospital, London, United Kingdom
                [6 ] Institute of Immunity and Transplantation, University College London, London, United Kingdom
                [7 ] Centre for Cell Therapy and Regenerative Medicine, School of Biomedical Sciences, The University of Western Australia, Perth, Australia
                [8 ] Institute for Respiratory Health, University of Western Australia, Perth, Australia
                University of Alabama at Birmingham, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-2170-8791
                http://orcid.org/0000-0002-6634-5939
                Article
                PONE-D-18-08600
                10.1371/journal.pone.0197129
                5957441
                29771943
                d25c8989-697f-4587-a276-20e01e167983
                © 2018 Hynds 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
                : 21 March 2018
                : 26 April 2018
                Page count
                Figures: 4, Tables: 0, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: WT209199/Z/17
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: WT107963/Z/15/Z
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000781, European Research Council;
                Award ID: 260290
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000781, European Research Council;
                Award ID: 639429
                Award Recipient :
                This work was funded by a UWA-HMGU-UCL Collaborative Seed Funding Award (S.J., C.M.P. and R.E.H.). R.E.H. was supported by a BBSRC-CASE PhD studentship (awarded to A.G.) and is now a Sir Henry Wellcome Postdoctoral Fellow (WT209199/Z/17/Z). R.E.H. also receives support from the CRUK Lung Cancer Centre of Excellence and the Roy Castle Lung Cancer Foundation (2016/07/HYNDS). P.B. is a UCL Excellence Fellow and is supported by the European Research Council (ERC-Stg-2014 639429), the Rosetrees Trust (A1411 and A1179), the GOSH Charity (V6116) and the Cystic Fibrosis Trust (SRC006), and the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London (17DS20). A.G. was supported by an ERC Starting Grant (260290). S.M.J. is a Wellcome Trust Senior Fellow in Clinical Science (WT107963/Z/15/Z) and is supported by the Rosetrees Trust, the Roy Castle Lung Cancer Foundation (2016/17/JANES), the Welton Trust, the Garfield Weston Trust, the UCLH Charitable Foundation, and the UCLH BRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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