Epidermal Cells: Methods and Protocols (Methods in Molecular Biology, 2109) 1071602500, 9781071602508

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Methods in Molecular Biology 2109

Kursad Turksen Editor

Epidermal Cells Methods and Protocols Fourth Edition

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Epidermal Cells Methods and Protocols Fourth Edition

Edited by

Kursad Turksen Ottawa, ON, Canada

Editor Kursad Turksen Ottawa, ON, Canada

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-0250-8 ISBN 978-1-0716-0251-5 (eBook) https://doi.org/10.1007/978-1-0716-0251-5 © Springer Science+Business Media, LLC, part of Springer Nature 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cover Illustration: Artwork created by Kursad Turksen This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Many methods over many years have advanced our understanding of the workings of epidermal cells. Nevertheless, much remains to be deciphered, prompting us to put together this fourth edition of Epidermal Cells. This volume brings together a new set of protocols to arm epidermal cell biologists with tools and approaches to continue the quest to uncover the intricacies and regulatory mechanisms underlying epidermal cell development and function. Once again, the protocols gathered here are faithful to the mission statement of the Methods in Molecular Biology series: They are well established and described in an easy-tofollow, step-by-step fashion so as to be valuable for not only experts but also novices in the epidermal cell field. That goal is achieved because of the generosity of the contributors who have carefully described their protocols in this volume, and I am very grateful for their efforts. My thanks as well go to Dr. John Walker, the Editor-in-Chief of the Methods in Molecular Biology series, for giving me the opportunity to create this volume and for supporting me along the way. I am also grateful to Patrick Marton, the Executive Editor of Methods in Molecular Biology and the Springer Protocols collection, for his continuous support from idea to completion of this volume. A special thank you goes to Anna Rakovsky, Assistant Editor for Methods in Molecular Biology, for continuous support from beginning to end of this project. I would also like to thank David C. Casey, Senior Editor for Methods in Molecular Biology, for his outstanding editorial work during the production of this volume. Finally, I would like to thank Sarumathi Hemachandirane, Anand Ventakachalam, and the rest of the production crew for their work in putting together an outstanding volume. Ottawa, ON, Canada

Kursad Turksen

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The Simplest Protocol for Rapid and Long-Term Culture of Primary Epidermal Keratinocytes from Human and Mouse . . . . . . . . . . . . . . . . . . . . 1 Filipa Pinto, Daisuke Suzuki, and Makoto Senoo Dermal-Epidermal Separation by Heat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Liyan Jian, Yu Cao, and Ying Zou Dermal-Epidermal Separation by Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Liyan Jian, Yu Cao, and Ying Zou Dermal-Epidermal Separation by Chemical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Liyan Jian, Yu Cao, and Ying Zou Isolation and Characterization of Extracellular Vesicles from Keratinocyte Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Sebastian Sjo¨qvist, Aya Imafuku, Dhanu Gupta, and Samir EL Andaloussi Competitive Repopulation Assay of Long-Term Epidermal Stem Cell Regeneration Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Carmen Segrelles, Karla Santos-de-Frutos, Jesu´s M. Paramio, and Corina Lorz Quantification of Melanosome Transfer Using Immunofluorescence Microscopy and Automated Image Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Aishwarya Sridharan, S. Y. John Lim, Graham D. Wright, and Leah A. Vardy Contribution of Immunohistochemistry in Revealing S100A7/JAB1 Colocalization in Psoriatic Epidermal Keratinocyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Mariagrazia Granata, Evangelia Skarmoutsou, Maria Clorinda Mazzarino, Massimo Libra, and Fabio D’Amico Digital Quantification of Epidermal Protein Expression in Paraffin-Embedded Tissue Using Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . 75 Manuel Valdebran, Eric H. Kowalski, Diana Kneiber, Jing Li, Jeffrey Kim, and Kyle T. Amber Keratinocyte Differentiation by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Natalia Sanz-Go mez, Ana Freije, and Alberto Gandarillas In Silico and In Vitro Considerations of Keratinocyte Nuclear Receptor Protein Structural Order for Improving Experimental Analysis. . . . . . . . . . . . . . . . . . . . 93 Rambon Shamilov, Matthew J. Staid, and Brian J. Aneskievich Genetic Modification of Human Primary Keratinocytes by Lentiviral Vectors . . . . . . . 113 Ana Freije, Natalia Sanz-Gomez, and Alberto Gandarillas Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9 . . . . . . . . . 125 Serena Grossi, Gabriele Fenini, Paulina Hennig, Michela Di Filippo, and Hans-Dietmar Beer

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In Vitro Wound Healing Assays to Investigate Epidermal Migration . . . . . . . . . . . . . . . Kegan A. Main, Constantinos M. Mikelis, and Colleen L. Doc¸i Iterative Three-Dimensional Epidermis Bioengineering and Xenografting to Assess Long-Term Regenerative Potential in Human Keratinocyte Precursor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nicolas O. Fortunel, Emmanuelle Bouissou-Cadio, Julien Coutier, and Miche`le T. Martin Generation of a Full-Thickness Human Skin Equivalent on an Immunodeficient Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nicole Diette, Igor Kogut, and Ganna Bilousova Biotin Identification Proteomics in Three-Dimensional Organotypic Human Skin Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Calvin J. Cable, Nihal Kaplan, Spiro Getsios, Paul M. Thomas, and Bethany E. Perez White Information and Statistical Analysis Pipeline for High-Throughput RNA Sequencing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shinji Nakaoka and Keita Matsuyama Cell-Extracellular Matrix Adhesion Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mehmet Varol Methods for Analysis of Keratinocyte Migration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jie Liu and Jiaping Zhang Scratch Wound Healing Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Simona Martinotti and Elia Ranzato Photoprotective Activity Assay Toward Ultraviolet B in Human Keratinocytes . . . . . . Mehmet Varol Photodynamic Therapy Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mehmet Varol A Method to Prepare Claudin-Modulating Recombinant Proteins. . . . . . . . . . . . . . . . . Keisuke Tachibana and Masuo Kondoh Human Fetal Skin Fibroblast Isolation and Expansion for Clinical Application. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parisa Goodarzi, Hamid Reza Aghayan, Moloud Payab, Bagher Larijani, Sepideh Alavi-Moghadam, Masoumeh Sarvari, Hossein Adibi, Fatemeh Khatami, Najmeh Foroughi Heravani, Mahdieh Hadavandkhani, and Babak Arjmand Ciliated Epithelial Cell Differentiation at Air-Liquid Interface Using Commercially Available Culture Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dani Do Hyang Lee, Alina Petris, Robert E. Hynds, and Christopher O’Callaghan Correction to: Isolation and Characterization of Extracellular Vesicles from Keratinocyte Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sebastian Sjo¨qvist, Aya Imafuku, Dhanu Gupta, and Samir EL Andaloussi Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors HOSSEIN ADIBI • Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran HAMID REZA AGHAYAN • Cell Therapy and Regenerative Medicine Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran SEPIDEH ALAVI-MOGHADAM • Metabolomics and Genomics Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran KYLE T. AMBER • Department of Dermatology, University of Illinois at Chicago, Chicago, IL, USA BRIAN J. ANESKIEVICH • Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT, USA BABAK ARJMAND • Cell Therapy and Regenerative Medicine Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran; Metabolomics and Genomics Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran HANS-DIETMAR BEER • Department of Dermatology, University Hospital Zurich, Zurich, Switzerland; Faculty of Medicine, University of Zurich, Zurich, Switzerland GANNA BILOUSOVA • Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA; Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA; Correspondence: Ganna Bilousova, Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA EMMANUELLE BOUISSOU-CADIO • Laboratoire de Ge´nomique et Radiobiologie de la Ke´ ratinopoı¨e`se, CEA/DRF/IBFJ/IRCM, Paris, France; INSERM U967, Paris, France; Universite´ Paris-Diderot, Paris, France; Universite´ Paris-Saclay, Paris, France CALVIN J. CABLE • Department of Dermatology, Northwestern University, Chicago, IL, USA YU CAO • Institute of Precision Medicine, The Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China JULIEN COUTIER • Laboratoire de Ge´nomique et Radiobiologie de la Ke´ratinopoı¨e`se, CEA/ DRF/IBFJ/IRCM, Paris, France; INSERM U967, Paris, France; Universite´ ParisDiderot, Paris, France; Universite´ Paris-Saclay, Paris, France FABIO D’AMICO • Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy MICHELA DI FILIPPO • Department of Dermatology, University Hospital Zurich, Zurich, Switzerland NICOLE DIETTE • Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA; Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA

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Contributors

COLLEEN L. DOC¸I • Department of Biology, College of Arts and Sciences, Marian University Indianapolis, Indianapolis, IN, USA SAMIR EL ANDALOUSSI • Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden GABRIELE FENINI • Department of Dermatology, University Hospital Zurich, Zurich, Switzerland; Faculty of Medicine, University of Zurich, Zurich, Switzerland NICOLAS O. FORTUNEL • Laboratoire de Ge´nomique et Radiobiologie de la Ke´ratinopoı¨e`se, CEA/DRF/IBFJ/IRCM, Paris, France; INSERM U967, Paris, France; Universite´ ParisDiderot, Paris, France; Universite´ Paris-Saclay, Paris, France ANA FREIJE • Cell Cycle, Stem Cell Fate and Cancer Laboratory, Institute for Research Marque´s de Valdecilla (IDIVAL), Santander, Spain ALBERTO GANDARILLAS • Cell Cycle, Stem Cell Fate and Cancer Laboratory, Institute for Research Marque´s de Valdecilla (IDIVAL), Santander, Spain; INSERM, LanguedocRoussillon, Montpellier, France SPIRO GETSIOS • Department of Dermatology, Northwestern University, Chicago, IL, USA PARISA GOODARZI • Brain and Spinal Cord Injury Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran MARIAGRAZIA GRANATA • Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy SERENA GROSSI • Department of Dermatology, University Hospital Zurich, Zurich, Switzerland; Faculty of Medicine, University of Zurich, Zurich, Switzerland DHANU GUPTA • Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden MAHDIEH HADAVANDKHANI • Cell Therapy and Regenerative Medicine Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran PAULINA HENNIG • Department of Dermatology, University Hospital Zurich, Zurich, Switzerland NAJMEH FOROUGHI HERAVANI • Cell Therapy and Regenerative Medicine Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran ROBERT E. HYNDS • UCL Respiratory, University College London, London, UK AYA IMAFUKU • Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan LIYAN JIAN • Institute of Precision Medicine, The Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China S. Y. JOHN LIM • Skin Research Institute of Singapore, A∗STAR, Singapore, Singapore NIHAL KAPLAN • Department of Dermatology, Northwestern University, Chicago, IL, USA FATEMEH KHATAMI • Chronic Diseases Research Center, Endocrinology and Metabolism Population Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran JEFFREY KIM • Department of Pathology, University of California Irvine, Irvine, CA, USA DIANA KNEIBER • Department of Dermatology, University of Illinois at Chicago, Chicago, IL, USA IGOR KOGUT • Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA; Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA

Contributors

xi

MASUO KONDOH • Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan ERIC H. KOWALSKI • Department of Dermatology, University of Illinois at Chicago, Chicago, IL, USA BAGHER LARIJANI • Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran DANI DO HYANG LEE • Respiratory, Critical Care & Anaesthesia, UCL Great Ormond Street Institute of Child Health, London, UK MASSIMO LIBRA • Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy JING LI • Department of Dermatology, University of California Irvine, Irvine, CA, USA JIE LIU • Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Army Military Medical University, Chongqing, China CORINA LORZ • Molecular Oncology Unit, CIEMAT (ed 70A), Madrid, Spain; Molecular Oncology, University Hospital 12 de Octubre, Research Institute 12 de Octubre i+12, Madrid, Spain; Centro de Investigacion Biome´dica en Red de Ca´ncer (CIBERONC), Madrid, Spain KEGAN A. MAIN • Department of Biology, College of Arts and Sciences, Marian University Indianapolis, Indianapolis, IN, USA MICHE`LE T. MARTIN • Laboratoire de Ge´nomique et Radiobiologie de la Ke´ratinopoı¨e`se, CEA/DRF/IBFJ/IRCM, Paris, France; INSERM U967, Paris, France; Universite´ ParisDiderot, Paris, France; Universite´ Paris-Saclay, Paris, France SIMONA MARTINOTTI • DiSIT-Dipartimento di Scienze e Innovazione Tecnologica, University of Piemonte Orientale, Vercelli, Italy KEITA MATSUYAMA • Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan MARIA CLORINDA MAZZARINO • Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy CONSTANTINOS M. MIKELIS • Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, USA SHINJI NAKAOKA • Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan CHRISTOPHER O’CALLAGHAN • Respiratory, Critical Care & Anaesthesia, UCL Great Ormond Street Institute of Child Health, London, UK JESU´S M. PARAMIO • Molecular Oncology Unit, CIEMAT (ed 70A), Madrid, Spain; Molecular Oncology, University Hospital 12 de Octubre, Research Institute 12 de Octubre i +12, Madrid, Spain; Centro de Investigacion Biome´dica en Red de Ca´ncer (CIBERONC), Madrid, Spain MOLOUD PAYAB • Obesity and Eating Habits Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran BETHANY E. PEREZ WHITE • Department of Dermatology, Northwestern University, Chicago, IL, USA; Skin Disease Research Center, Northwestern University, Chicago, IL, USA ALINA PETRIS • Respiratory, Critical Care & Anaesthesia, UCL Great Ormond Street Institute of Child Health, London, UK FILIPA PINTO • Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA ELIA RANZATO • DiSIT-Dipartimento di Scienze e Innovazione Tecnologica, University of Piemonte Orientale, Vercelli, Italy

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Contributors

KARLA SANTOS-DE-FRUTOS • Molecular Oncology Unit, CIEMAT (ed 70A), Madrid, Spain; Molecular Oncology, University Hospital 12 de Octubre, Research Institute 12 de Octubre i +12, Madrid, Spain; Centro de Investigacion Biome´dica en Red de Ca´ncer (CIBERONC), Madrid, Spain NATALIA SANZ-GO´MEZ • Cell Cycle, Stem Cell Fate and Cancer Laboratory, Institute for Research Marque´s de Valdecilla (IDIVAL), Santander, Spain MASOUMEH SARVARI • Cell Therapy and Regenerative Medicine Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran CARMEN SEGRELLES • Molecular Oncology Unit, CIEMAT (ed 70A), Madrid, Spain; Molecular Oncology, University Hospital 12 de Octubre, Research Institute 12 de Octubre i +12, Madrid, Spain; Centro de Investigacion Biome´dica en Red de Ca´ncer (CIBERONC), Madrid, Spain MAKOTO SENOO • Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA RAMBON SHAMILOV • School of Pharmacy, University of Connecticut, Storrs, CT, USA SEBASTIAN SJO¨QVIST • Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden EVANGELIA SKARMOUTSOU • Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy AISHWARYA SRIDHARAN • Skin Research Institute of Singapore, A∗STAR, Singapore, Singapore MATTHEW J. STAID • School of Pharmacy, University of Connecticut, Storrs, CT, USA DAISUKE SUZUKI • Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA KEISUKE TACHIBANA • Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan PAUL M. THOMAS • Department of Molecular Biosciences, Northwestern University, Chicago, IL, USA; Northwestern Proteomics, Northwestern University, Chicago, IL, USA MANUEL VALDEBRAN • Department of Dermatology, University of California Irvine, Irvine, CA, USA LEAH A. VARDY • Skin Research Institute of Singapore, A∗STAR, Singapore, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore, Singapore MEHMET VAROL • Department of Molecular Biology and Genetics, Faculty of Science, Mugla Sitki Kocman University, Mugla, Turkey GRAHAM D. WRIGHT • Skin Research Institute of Singapore, A∗STAR, Singapore, Singapore JIAPING ZHANG • State Key Laboratory of Trauma, Burns and Combined Injury, Plastic Surgery Department, Southwest Hospital, Army Military Medical University, Chongqing, China YING ZOU • Skin & Cosmetic Research Department, Shanghai Dermatology Hospital, Shanghai, China

Methods in Molecular Biology (2020) 2109: 1–22 DOI 10.1007/7651_2019_263 © Springer Science+Business Media New York 2019 Published online: 07 September 2019

The Simplest Protocol for Rapid and Long-Term Culture of Primary Epidermal Keratinocytes from Human and Mouse Filipa Pinto, Daisuke Suzuki, and Makoto Senoo Abstract Although mouse models have been used as an essential tool for studying the physiology and diseases of the skin, propagation of mouse primary epidermal keratinocytes remains challenging. In this chapter, we introduce the simplest, at least to our knowledge, protocol that enables long-term expansion of p63+ mouse epidermal keratinocytes in low Ca2+ media without the need of progenitor cell-purification steps or support by a feeder cell layer. Pharmacological inhibition of TGF-β signaling in crude preparations of mouse epidermis robustly increases proliferative capacity of p63+ epidermal progenitor cells, while preserving their ability to differentiate. Suppression of TGF-β signaling also permits p63+ epidermal keratinocytes to form macroscopically large clones in 3T3-J2 feeder cell co-culture. Suppression of TGF-β signaling also enhances the clonal growth of human keratinocytes in co-culture with a variety of feeder cells. This simple and efficient approach will not only facilitate the use of mouse models by providing p63+ primary epidermal keratinocytes in quantity but also significantly reduce the time needed for preparing the customized skin grafts in Green method. Keywords Mouse models, Primary epidermal keratinocytes, Human keratinocytes, Transcription factor p63, TGF-β signaling, Small molecule inhibitors, Skin transplantation, Feeder cell co-culture

1

Introduction Homeostasis of the epidermis is maintained by tissue-specific stem cells capable of self-renewal, proliferation, and differentiation [1, 2]. Progresses in culture of epidermal stem cells have contributed to the development of therapeutic strategies in regenerative medicine [3], including skin transplantation for patients with severe burn wounds. The Green method, developed for the expansion of autologous keratinocytes from a small skin biopsy, has enabled permanent wound closure with autografts in burn victims [3–6]. In addition, the Green method has been essential in studying skin stem cell biology as it allows the expansion of epithelial stem cells and the assessment of their self-renewal capacity [7–10]. Mouse models have been used in studies of normal and disease conditions of the skin. However, the growth of primary epidermal keratinocytes derived from mice rapidly declines in culture, and the cells become terminally differentiated [11], a limit that restricts the

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use of mouse primary keratinocytes for functional analyses of the skin. As proliferation and differentiation of epithelial cells are tightly coupled through the induction of cyclin-dependent kinase (CDK) inhibitor genes [12–14], suppression of growth arrest and of premature differentiation are both potential approaches to improve the lifespan of mouse primary epidermal keratinocytes [15–18]. Transforming growth factor-β (TGF-β) signaling regulates proliferation and differentiation of many different epithelial progenitor cells [19], including those that are controlled by the transcription factor p63 (e.g., epidermal keratinocytes) [10]. In particular, TGF-β signaling has been shown to suppress the growth of keratinocytes by inhibiting their cell cycle progression [20]. Effects of TGF-β signaling are mediated through a TGF-β receptor complex consisting of the Type I TGF-β receptor (ALK5) and the Type II TGF-β receptor (TGFBRII), both of which possess intrinsic serine/threonine kinase activity [21]. Upon binding of TGF-β ligands, TGFBRII activates ALK5 kinase by phosphorylation which, in turn, activates Smad2/3-mediated transcription of the genes required for the growth arrest of keratinocytes [22]. In co-culture with 3T3-J2 cells, there are three major sources of TGF-β ligands: keratinocytes [23], 3T3-J2 cells [24], and serum present in the co-culture media [25]. The presence of TGF-β ligands in co-culture could restrict the potential growth of epidermal keratinocytes. Indeed, we have shown recently that TGF-β signaling is active in human keratinocytes in co-culture with 3T3-J2 cells as determined by the nuclear localization of Smad2/ 3 [26]. Accordingly, suppression of TGF-β signaling by RepSox, a cell-permeable small molecule inhibitor of ALK5 kinase, increases the proliferation of human epidermal keratinocytes in co-culture with 3T3-J2 cells [26]. In addition, the use of TGF-β signaling inhibitors also enhances significantly the expansion of human epidermal keratinocytes in co-culture with other feeder cell types including human dermal fibroblasts and human preadipocytes, two major cell types utilized as alternatives to 3T3-J2 cells, with a long-term goal of developing customized skin autografts [27, 28]. The Green method utilizes 3T3-J2 cells, a unique mouse mesenchymal cell line, as a feeder layer to support the proliferative potential of epidermal progenitor cells long term [3, 7]. The co-culture of human primary epidermal keratinocytes with 3T3-J2 cells produces cultured epidermal autografts (CEA). Since its inception in the 1980s, the CEA have been used worldwide for patients with severe burn wounds, often saving their lives [3–6]. However, patients with massive burn wounds can spare only limited skin donor sites, and it requires several weeks of amplification of keratinocytes to prepare sufficient numbers of CEA. A methodology to promote the expansion of keratinocytes in Green’s protocol would be beneficial as it can decrease

Rapid and Long-Term Expansion of Primary Keratinocytes

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hospitalization time for patients with massive burn injuries who undergo replacement of skin covering nearly the entire body surface area. Although the use of low calcium (Ca2+) media has extended proliferation of mouse epidermal keratinocytes short term [11], the most effective protocols to propagate these primary cells rely on modified 3T3-J2 feeder co-culture [15, 17] or the use of fluorescence-activated cell sorting (FACS)-purified progenitor populations with triple drug inhibitors [18]. Highly efficient protocols that eliminate the use of undefined factors (e.g., feeder cells), labor-intensive purification steps, and the potentially complex effects of multiple drugs will facilitate the use of primary cells of mice in studies of skin biology. In this chapter, we introduce a newly developed protocol that enables long-term proliferation of p63+ mouse primary epidermal keratinocytes utilizing a TGF-β signaling inhibitor without the need of a feeder layer or progenitor cell-purification steps. Suppression of TGF-β signaling also permits the expanded p63+ mouse epidermal keratinocytes to form macroscopically large clones in 3T3-J2 feeder cell co-culture for clonal evaluation. We anticipate that this simple and efficient approach will facilitate the use of mouse models for studying the physiology and pathogenesis of the epidermis. Furthermore, we present an improved Green method in which expansion of human epidermal keratinocytes is significantly enhanced by the use of this pharmacological inhibition of TGF-β signaling.

2

Materials

2.1

Mouse

2.2

Cells

C57BL/6 mice (Jackson Laboratories or Charles River Laboratories) were used to develop the protocol introduced in this chapter. Animal work described below requires an approval by local Institutional Animal Care and Use Committees (IACUC). 1. Human primary epidermal keratinocytes were purchased from Cellntec (see Note 1). 2. 3T3-J2 cells, derived from mouse embryonic fibroblasts [7], were provided by H. Green at Harvard Medical School. 3. PrimaPure human dermal fibroblasts (hDFs) were purchased from Genlantis. hDFs used in this protocol were derived from the dermis of normal human neonatal foreskin and have been verified to express fibroblast surface proteins (http://www. genlantis.com/human-fibroblasts.html). 4. Human preadipocytes (hPAs) were provided by M. Reilly at the University of Pennsylvania School of Medicine. hPAs used in this protocol were isolated from adipose tissues of patients

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who underwent plastic surgery in the abdominal region. The subjects were free of metabolic or endocrine diseases as assessed by routine clinical examination and laboratory tests. 2.3 Cell Culture Reagents and Media

1. CnT-Prime epithelial culture medium (CnT-PR, Cellntec). 2. Dulbecco’s Modified Eagle’s Medium with 4.5 g/L glucose, 584 mg/L L-glutamine, and 110 mg/mL sodium pyruvate (DMEM, Mediatech). 3. Ham’s F-12 (Mediatech).

medium

(modified)

with

L-glutamine

4. Fetal bovine serum (FBS, Hyclone). 5. Bovine calf serum (CS, Hyclone). 6. Penicillin (10,000 U/mL)—Streptomycin (10,000 μg/mL) (Gibco Invitrogen). 7. Bovine serum albumin (BSA, Fisher Scientific). 8. 0.25% Trypsin-EDTA (Gibco Invitrogen). 9. Phosphate-buffered saline without calcium and magnesium (PBS, Mediatech). 10. Adenine (minimum 99%, Sigma-Aldrich). 11. T3 (3,30 ,5-triiodo-L-thyronine sodium salt) (minimum 99% HPLC, Sigma-Aldrich). 12. Hydrocortisone (>98%, Sigma-Aldrich). 13. Cholera toxin (approx. 95%, Sigma-Aldrich). 14. Insulin from bovine pancreas (Sigma-Aldrich). 15. Human epidermal growth factor (EGF, BD Biosciences). 16. RepSox (ALK5 Inhibitor II, Selleck Chemicals). 17. Basic fibroblast growth factor (bFGF, R&D systems): Dissolve in 1% BSA aqueous solution at 10 μg/mL. Store in 10 μL aliquots at 80  C. 18. Biotin (Sigma-Aldrich): Dissolve in 0.01 N NaOH at 1 mg/ mL and sterilize with a 0.2 μm syringe filter. Store in 0.1 mL aliquots at 80  C. 19. D-Pantothenic acid hemicalcium salt (Sigma-Aldrich): Dissolve in dH2O at 4 mg/mL and sterilize with a 0.2 μm syringe filter. Store in 0.1 mL aliquots at 80  C. 20. 2-[3-(6-Methylpyridine-2-yl)-1H-pyrazol-4-yl]-1,5naphthyridine (RepSox, Selleck Chemicals): Dissolve in DMSO at 25 mM. Store in 50 μL aliquots at 80  C. 2.4 Other Reagents and Chemicals

1. Isoflurane (Piramal Healthcare). 2. Isojin (Meiji Seika Pharma Co., Ltd.). 3. 0.4% Trypan blue solution (Thermo Scientific).

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4. 10% neutral buffered formalin (Fisher Scientific). 5. 1% Rhodamine B: Dissolve Rhodamine B (Sigma-Aldrich) in dH2O at a final concentration of 1% (w/v), filter through Whatman filter papers (Fisher Scientific), and store at room temperature (RT). 6. Antibodies. Rabbit anti-cytokeratin 5 (CK5) (polyclonal, BioLegend). Mouse anti-p63 (4A4, Santa Cruz Biotechnology). Alexa 488-goat anti-mouse IgG (Molecular Probes). Alexa 488-goat anti-rabbit IgG (Molecular Probes). Alexa 594-goat anti-rabbit IgG (Molecular Probes). 7. 1% Triton X-100: Add Triton X-100 (Fisher Scientific) in PBS at a final concentration of 1% (v/v), and store at room temperature. 8. 0.1% Tween-20 (PBS-T): Add Tween-20 (Fisher Scientific) in PBS at a final concentration of 0.1% (v/v) and store at room temperature. 9. Blocking solution: Add FBS in PBS at a final concentration of 10% (v/v). 10. IntraSure kit (BD Biosciences): Used for intracellular staining. 11. Hoechst 33342 (Invitrogen): Dissolve in DMSO at 1 mg/mL as a stock solution. Store in 0.1 mL aliquots at 20  C. To prepare working solution, dilute the stock solution to 1 μg/mL in PBS. 12. Dimethyl sulfoxide (DMSO, Wako Chemicals). 2.5 Instruments and Supplies

1. Humidified CO2 incubators (5% and 10%, Sanyo). 2. CO2 gas cylinders (Airgas). 3. 37  C water bath (Fisher Scientific). 4. Hemocytometer (Hausser Scientific). 5. Inverted microscope (CKX41, Olympus). 6. Fluorescence microscope (BZ-X710, Keyence Corp.). 7. γ-Irradiator with a Cesium-137 source (Gammacell 40, Atomic Energy of Canada). 8. Tissue culture plates (35 mm, 60 mm, 100 mm, Greiner Bio-One). 9. 15 mL and 50 mL polypropylene conical tubes (Greiner Bio-One). 10. 1.5 mL Eppendorf tubes (GeneMate). 11. Cryovials (Denville). 12. Minisart syringe filter unit (0.2 μm, Sartorius). 13. 70 μm cell strainer (Corning).

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14. Parafilm (Bemis Co. Ltd). 15. Whatman filter paper (Sigma-Aldrich). 2.6 Preparation of Cell Culture Media and Freezing Media

Add 100 U/mL penicillin and 100 μg/mL streptomycin to CnT-PR media.

2.6.1 CnT-PR Medium 2.6.2 J2 Medium

Supplement DMEM with 10% CS (v/v), 100 U/mL penicillin, and 100 μg/mL streptomycin [7].

2.6.3 FAD Medium

Mix DMEM and Ham’s F-12 media at a 3:1 ratio (v/v), and supplement with 10% FBS (v/v), 100 U/mL penicillin, and 100 μg/mL streptomycin [7].

2.6.4 Complete FAD (cFAD) Medium

Supplement FAD medium above (Subheading 2.6.3) with 10 ng/ mL EGF, 5 μg/mL insulin, 2  109 M T3, 0.4 μg/mL hydrocortisone, 24 μg/mL adenine, and 1  1010 M cholera toxin [7].

2.6.5 hPA Medium

Mix DMEM and Ham’s F-12 at a 1:1 ratio (v/v), and supplement with 10% FBS (v/v), 10 ng/mL EGF, 1 ng/mL bFGF, 8 μg/mL biotin, 4 μg/mL D-pantothenic acid, 8.7 μM insulin, 100 U/mL penicillin, and 100 μg/mL streptomycin [30].

2.6.6 Freezing Medium

Mix cell culture media above (Subheadings 2.6.1–2.6.5), serum (CS or FBS), and DMSO at a 4:5:1 ratio (v/v/v). To freeze 3T3-J2 cells, use a combination of J2 medium and CS. To freeze epidermal cells, use either CnT-PR or FAD medium along with FBS.

2.7 Preparation of Cell Culture Reagents

1. Adenine stock. Dissolve lyophilized adenine in 0.2 N HCl at a final concentration of 24 mg/mL. Sterilize with a 0.2 μm syringe filter. Store in 0.5 mL aliquots at 20  C. 2. T3 stock. Dissolve lyophilized T3 in PBS at a final concentration of 10 μM. Sterilize with a 0.2 μm syringe filter. Store in 0.5 mL aliquots at 20  C. 3. Hydrocortisone stock. Dissolve lyophilized hydrocortisone in 95% EtOH at a final concentration of 5 mg/mL. Store in 0.2 mL aliquots at 20  C. 4. Cholera toxin stock. Dissolve lyophilized cholera toxin in dH2O at a final concentration of 0.1 mg/mL. Sterilize with a 0.2 μm syringe filter. Store in 0.4 mL aliquots at 80  C. 5. Insulin stock. Dissolve lyophilized insulin in 0.005 N HCl at a final concentration of 0.1 mg/mL. Sterilize with a 0.2 μm syringe filter. Store in 0.5 mL aliquots at 80  C.

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6. EGF stock. Dissolve lyophilized EGF in 1% BSA aqueous solution at a final concentration of 20 μg/mL. Sterilize with a 0.2 μm syringe filter. Store in 0.5 mL aliquots at 80  C. 7. Inhibitor stocks. Dissolve lyophilized inhibitors (RepSox, A83-01, LY364947, SB525334, and SB431542) in DMSO at a final concentration of 25 mM. Store in 25 μL aliquots at 80  C. In the protocol described in this chapter, only RepSox is required.

3

Methods

3.1 Collection of Crude Primary Epidermal Cells from Mouse Skin

1. Euthanize embryos to neonatal mice by decapitation. Postnatal mice should be euthanized by overdosing of isoflurane or CO2 inhalation, followed by cervical dislocation. 2. When mice older than 2 weeks old are used, hair should be clipped prior to isolation of the skin. 3. Harvest 1–2 cm2 back skin per animal as a single sheet of tissue using sterilized surgical scissors. 4. Sterilize the isolated skin by immersing in Isojin solution for 10 s, followed by three washes in PBS. 5. Place the skin on a sterilized 2  2 cm Whatman filter paper with the dermis facing down in a 35 mm tissue culture plate. The dermis is a viscous tissue and adheres tightly to the dried filter paper. 6. Slowly add 2 mL fresh 0.25% trypsin-EDTA in the tissue culture plate above, enough to soak the filter paper, and incubate for 2–4 h at RT in a tissue culture biosafety cabinet. 7. Carefully separate the epidermis from the dermis using sterilized forceps with fine tips. While the epidermis easily peels off as a whitish tissue, the dermis, as a translucent tissue, remains attached to the filter paper. 8. Transfer the isolated epidermis to 1.5 mL Eppendorf tubes. 9. Add 1 mL 0.25% trypsin-EDTA to the tubes, and incubate for 5 min at RT. 10. Add 100 μL FBS (equivalent to 10% the volume of trypsin used) to inactivate the enzymatic reaction, vigorously suspend the cells by pipetting, and spin down the cells at 400  g for 5 min. 11. Resuspend the cells in 1 mL PBS, and filter through a 70 μm cell strainer. 12. Centrifuge the cells at 400  g for 5 min.

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13. Wash the cells two additional times in PBS in order to completely remove FBS, which potentially inhibits epidermal cell growth in the subsequent culture. 14. Resuspend the cells in 100 μL CnT-PR media, and count the cell number using a hemocytometer. 3.2 Enrichment and Expansion of Mouse Primary Epidermal Keratinocytes in Serum-Free Media

1. Plate 1  106 crude primary cells (see Subheading 3.1) in one 35 mm tissue culture plate (or one well in a 6-well plate) that contains 3 mL CnT-PR media. 2. Add RepSox to the culture media at a final concentration of 1 μg/mL (see Note 2). 3. Place the tissue culture plate in a humidified incubator with 5% CO2 at 37  C. Allow the cells to adhere to the plastic plate for 2–3 days (see Note 3). 4. After the incubation, replace media with fresh 3 mL CnT-PR media. Culture media should be changed every 2–3 days thereafter until the cells become subconfluent (see Note 4). 5. When the cells become subconfluent, remove culture media, and wash the cells twice in 3 mL PBS. 6. Add 1 mL of 0.25% trypsin-EDTA to the cells, and incubate the plate in a humidified chamber with 5% CO2 at 37  C for 5 min (see Note 5). 7. Tap the tissue culture plate to dislodge the cells. 8. Dissociate the cells by gentle pipetting, collect them in a 15 mL polypropylene tube, and neutralize the enzymatic reaction by adding 100 μL FBS (equivalent to 10% the volume of trypsin used). 9. Centrifuge the cells at 200  g for 5 min. 10. Remove supernatant, wash the cells twice in 5 mL PBS, and resuspend in 1 mL CnT-PR media. 11. Count cell numbers. Approximately 3–5  105 cells are collected from one subconfluent 35 mm tissue culture plate. 12. Replate the cells at a 1:10 ratio in CnT-PR media containing 1 μg/mL RepSox. These secondary cultures become subconfluent in 5–7 days for subsequent experiments. By passage (P) 1 to P2, virtually all growing cells are cytokeratin (CK)+p63+ epidermal progenitor cells [26] (see Note 6). 13. To prepare frozen stock, resuspend the cell pellet in CnT-PRbased freezing media (see Subheading 2.6.6) at the density of 2  106 cells per mL and aliquot 0.5 mL per cryovial (1  106 cells per vial). Place the cryovials in 80  C overnight, and transfer them to a liquid nitrogen tank for longer storage.

Rapid and Long-Term Expansion of Primary Keratinocytes

3.3 Preparation of Human Epidermal Keratinocyte Stocks

9

Human epidermal keratinocytes used in this protocol were derived from neonatal foreskin and were purchased from Cellntec. The cells have been tested negative for hepatitis B, hepatitis C, and HIV-1 and are free of bacteria, fungi, and mycoplasma contamination. Cryovials containing 5  105 viable cells were supplied on dry ice. The cells can be used immediately or transferred to a liquid nitrogen tank for later use. 1. To grow human epidermal keratinocytes in plastic culture without feeder cells, one cryovial containing 5  105 cells is thawed in a 37  C water bath for 3 min. 2. Spin down the cells at 200  g for 5 min. 3. Remove freezing media, and suspend the cells in 1 mL CnT-PR medium. 4. Plate 5  105 human epidermal keratinocytes in one 100 mm tissue culture plate that contains 10 mL CnT-PR medium. 5. Place the tissue culture plate in a humidified incubator with 5% CO2 at 37  C. 6. After overnight incubation (>16 h), replace media with 10 mL of fresh CnT-PR medium. Culture medium should be changed every 2–3 days thereafter until the cells become subconfluent (see Note 7). 7. When the cells become subconfluent, remove culture media, and wash the cells twice in 5 mL PBS. 8. Add 2 mL of 0.25% trypsin-EDTA to the cells, and incubate the plate in a humidified chamber with 5% CO2 at 37  C for 5 min (see Note 5). 9. Tap the tissue culture plate to dislodge the cells. 10. Dissociate the cells by gentle pipetting, collect them in a 15 mL polypropylene tube, and neutralize the enzymatic reaction by adding 0.2 mL FBS (equivalent to 10% the volume of trypsin used). 11. Centrifuge the cells at 200  g for 5 min. 12. Remove supernatant, wash the cells twice in 5 mL PBS, and resuspend in 1 mL CnT-PR medium. 13. Count cell numbers. Approximately 3–5  106 cells are collected from one subconfluent 100 mm tissue culture plate. 14. Replate human keratinocytes at a 1:15–1:20 ratio at the density of 4  103 cells per cm2 into 100 mm tissue culture plates that contain 10 mL CnT-PR medium. These secondary cultures become subconfluent in 5–7 days. 15. To prepare frozen stock, resuspend the cell pellet in keratinocyte freezing medium (see Subheading 2.6.6) at the density of 2  106 cells per mL, and aliquot 0.5 mL per cryovial (1  106 cells per vial). Place the cryovials in 80  C overnight, and transfer them to a liquid nitrogen tank for longer storage.

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3.4 Preparation of Feeder Cells for Co-culture with Epidermal Keratinocytes

In this protocol, both 3T3-J2 cells and hDFs are expanded in J2 medium under 10% CO2 atmosphere, while hPAs are propagated in hPA medium under 5% CO2 atmosphere. Keep in mind that the conditions of feeder cells, such as the viability, density, and passage number (see Note 8), are critical for successful expansion of human epidermal keratinocytes in feeder cell co-culture.

3.4.1 Preparation of Feeder Cells 3.4.2 Culture, Passage, and Storage of 3T3-J2 Cells and hDFs

1. To expand 3T3-J2 cells and hDFs, one cryovial containing 1  106 cells is taken out of a liquid nitrogen tank and thawed in a 37  C water bath for 3 min. 2. Spin down the cells at 200  g for 5 min. 3. Remove freezing media, and suspend the cells in 1 mL J2 medium. 4. Plate the resuspended cells in two 100 mm tissue culture plates that contain 10 mL J2 medium (5  105 cells per plate). 5. Place tissue culture plates in a humidified incubator with 10% CO2 at 37  C. 6. After overnight incubation (>16 h), replace media with 10 mL of fresh J2 medium. Culture medium should be changed every 2–3 days thereafter until the cells become subconfluent (see Note 9). 7. When the cells become subconfluent, remove culture media and wash the cells twice in 5 mL PBS. 8. Add 2 mL 0.25% trypsin-EDTA to the cells, and incubate the plates in a humidified incubator with 10% CO2 at 37  C for 5 min. 9. Dissociate the cells by gentle pipetting, collect them in a 15 mL polypropylene tube, and neutralize the enzymatic reaction by adding the same volume (2 mL) of J2 medium. 10. Centrifuge the cells at 200  g for 5 min. 11. Remove supernatant and resuspend the cells in 1 mL J2 medium. 12. Count cell numbers. Approximately 3  106 cells are collected from one subconfluent 100 mm tissue culture plate. 13. Replate 3T3-J2 cells or hDFs at a 1:10–1:15 ratio at the density of 3.3  103 cells per cm2 into 100 mm tissue culture plates that contain 10 mL J2 medium. These secondary cultures become subconfluent in 5–7 days.

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11

14. To prepare frozen stock, resuspend the cell pellet in freezing medium (see Subheading 2.6.6) at the density of 2  106 cells per mL, and aliquot 0.5 mL per cryovial (1  106 cells per vial). Place the cryovials in 80  C overnight, and transfer them to a liquid nitrogen tank for longer storage. 3.4.3 Culture, Passage, and Storage of hPAs

1. To expand hPAs, one cryovial containing 1  106 hPAs is taken out of a liquid nitrogen tank and thawed in a 37  C water bath for 3 min. 2. Spin down the cells at 200  g for 5 min. 3. Remove freezing media and suspend the cells in 1 mL hPA medium. 4. Plate 1  106 hPAs in one 100 mm tissue culture plate that contains 10 mL hPA medium. 5. Place the tissue culture plate in a humidified incubator with 5% CO2 at 37  C. 6. After overnight incubation (>16 h), replace media with 10 mL of hPA medium. Culture medium should be changed every 2–3 days, thereafter until the cells become subconfluent. 7. When the cells become subconfluent, remove culture media and wash the cells twice in 5 mL PBS. 8. Add 2 mL 0.25% trypsin-EDTA to the cells, and incubate the plates in a humidified incubator with 5% CO2 at 37  C for 5 min. 9. Dissociate the cells by gentle pipetting, collect them in a 15 mL polypropylene tube, and neutralize the enzymatic reaction by adding the same volume (2 mL) of hPA medium. 10. Centrifuge the cells at 200  g for 5 min. 11. Remove supernatant and resuspend the cells in 1 mL hPA medium. 12. Count cell numbers. 13. Replate hPAs at a 1:10 ratio at the density of 3.3  103 cells per cm2 into 100 mm tissue culture plates that contain 10 mL hPA medium. These secondary cultures become subconfluent in 7–10 days. 14. To prepare frozen stock, resuspend the cell pellet in freezing medium (see Subheading 2.6.6) at the density of 2  106 cells per mL, and aliquot 0.5 mL per cryovial (1  106 cells per vial). Place the cryovials in 80  C overnight and transfer them to a liquid nitrogen tank for longer storage.

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3.5 Promotion of Human and Mouse Primary Epidermal Keratinocyte Expansion in Coculture with Feeder Cells Using a TGF-β Signaling Inhibitor 3.5.1 Preparation of Feeder Cell Layers

Prior to seeding primary epidermal cells, feeder cells will be mitotically arrested by either lethal γ-irradiation or mitomycin C treatment (see Note 10) to prevent propagation of the feeder cells in subsequent passages. In this protocol, we describe the use of lethally γ-irradiated feeder cells on the scale of a 60 mm tissue culture plate as an example. When other sizes of cell culture vessels are used, the number of feeder cells seeded and volume of the cell culture media used should be adjusted proportionally based on the surface area of the culture vessels (see Note 11). 1. Follow the procedures described in Subheading 3.4.2 (also Table 1), and prepare desired numbers of 60 mm tissue culture plates with subconfluent 3T3-J2 cells. 2. When 3T3-J2 cells become subconfluent, seal the culture plates with Parafilm to maintain sterility, and γ-irradiate the cells at a dose of 60 Gy (6000 rads). 3. After irradiation, replace media with 5 mL of fresh J2 media and place the plates in a humidified incubator until use (see Note 12).

3.5.2 Setting Up Coculture

1. Prepare a minimum amount of cFAD media that can be consumed in 1 week as it contains proteins and chemicals that may degrade once diluted in media. 2. Prior to seeding primary epidermal cells, replace J2 media with 5 mL cFAD media, and place the plates in a humidified incubator with 5% CO2 at 37  C for 1–2 h. 3. By following the steps in Subheadings 3.2 and 3.3, prepare epidermal cells in suspension. Use cFAD instead of CnT-PR media to suspend the epidermal cells. 4. Count viable cell numbers with the aid of Trypan blue solution. 5. Adjust the density of epidermal cells to 103~105 viable cells per mL by diluting in cFAD media. 6. Seed the desired number of epidermal cells onto feeder cell layers. When rapid confluency of keratinocytes is planned, 103–104 cells per 60 mm tissue culture plate can be seeded (see Notes 13 and Table 1 Number of feeder cells and the volume of cell culture media Culture vessel

Diameter (mm)

Area (cm2)

Volume of medium (mL)

Number of feeder cells

6-well plate

35

9.6

3

5.0  105 cells

60 mm plate

52

21

5

1.1  106 cells

100 mm plate

84

55

10

3.0  106 cells

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13

14). Seeding at a higher density than 104 cells per 60 mm plate is not recommended as it does not fully allow relatively immature epidermal keratinocytes to expand. When the determination of the proliferative capacity of epidermal cells at a clonal level is required, 102–103 cells per 60 mm plate should be seeded to permit clonal growth of individual epidermal cells (see Note 15). 7. Add 0.1–1 μM RepSox in cFAD media (see Note 16). Note that RepSox treatment significantly boosts the growth of primary epidermal cells with high proliferative potential [26] (see Note 17). 8. cFAD media containing RepSox should be replaced every 2–3 days until epidermal cell clones reach desired clone sizes for evaluation. 3.5.3 Visualization of Epidermal Keratinocyte Clones in Feeder Cell Co-culture by Rhodamine B Staining

1. Wash the 60 mm co-culture plates above (Subheading 3.5.2) twice in 5 mL PBS, and fix them in 10% buffered formalin for 15 min at RT. 2. Remove formalin solution and wash the cells twice in 5 mL PBS. 3. Add 5 mL of 1% Rhodamine B solution to the cells, and stain for more than 15 min at RT. 4. Pour off Rhodamine B solution into a separate container (see Note 18). 5. Gently rinse the cells with tap water. 6. Invert the plates to air-dry. 7. Take photographs of the stained plates (see Fig. 1 and Note 19), and determine the sizes of individual clones as needed using NIH ImageJ or Adobe Photoshop software [26].

3.5.4 Immunofluorescence Staining of p63 and CK5 in Human Epidermal Keratinocyte Clones in Feeder Cell Co-culture

1. Inoculate 1  103 human epidermal keratinocytes (see Subheading 3.3) onto γ-irradiated feeder cells in 60 mm tissue culture plates in 5 mL cFAD medium in the presence or absence of pre-determined doses of RepSox (see Note 16). Change cell culture medium every 2–3 days. 2. At day 14, wash the cells twice in 2 mL PBS, and fix them in 10% buffered formalin for 10 min at room temperature. 3. Remove formalin solution, and add 2 mL of 1% Triton X-100 solution to permeabilize the cells for 5 min at room temperature. 4. Remove Triton X-100 solution, and wash the cells three times in 2 mL PBS-T. 5. Add 2 mL blocking solution (see Subheading 2.4) to the cells, and incubate the plates for 30 min at room temperature. 6. Remove blocking solution, and rinse the cells once in 2 mL PBS-T.

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Fig. 1 Inhibition of TGF-β signaling promotes the expansion of human epidermal keratinocytes in co-culture with feeder cells. Data shown are representative images of Rhodamine B staining of human epidermal keratinocyte clones at day 14 of co-culture with 3T3-J2 cells (upper), hDFs (middle), and hPAs (lower) in the absence (left) or presence (right) of RepSox. Note that while 0.1 μM RepSox was used in co-culture with 3T3-J2 cells, 1.0 μM RepSox was used in co-culture with hDFs and hPAs. Equal numbers of human epidermal keratinocytes (1  103 cells) were plated in each well. Bar ¼ 5 mm

7. Add primary antibodies to the cells, and incubate for 1 h at room temperature. Use a combination of rabbit anti-CK5 polyclonal antibody (1:2000) and mouse anti-p63 monoclonal antibody (1:1000), diluted in 2 mL PBS-T. 8. Remove the primary antibodies, and wash the cells twice in 2 mL PBS-T for 5 min each. 9. Add secondary antibodies diluted in 2 mL PBS-T to the cells, and incubate for 1 h in the dark at room temperature. Use a combination of Alexa 488-goat anti-mouse IgG and Alexa 594-goat anti-rabbit IgG (both 1:1000 dilution). 10. Remove the secondary antibodies, and wash the cells three times in 2 mL PBS-T for 10 min each. 11. Add 2 mL of Hoechst 33342 solution (see Subheading 2.4) to the cells, and incubate for 5 min in the dark at room temperature.

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12. Remove Hoechst 33342, and rinse the cells twice in 2 mL PBS-T for 5 min each. 13. Cover the cells with 2 mL PBS-T, and analyze the signals using a fluorescence microscope as shown in Fig. 2.

Fig. 2 Expression of p63 and CK5 in epidermal keratinocytes co-cultured with 3T3-J2 cells in the absence or presence of RepSox. Human epidermal keratinocytes were grown in co-culture with 3T3-J2 cells in the absence (left) or presence (right) of 0.1 μM RepSox for 14 days. Data shown are representative images of bright field (upper) and immunofluorescence staining with anti-p63 antibodies (middle) and anti-CK5 antibodies and nuclear counterstaining with Hoechst 33342 (lower). Dotted lines indicate the clone borders. Bar ¼ 10 μm

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Notes 1. Isolation of primary human epidermal keratinocytes has been described elsewhere. See, for example, ref. [29]. 2. RepSox treatment robustly increases the CK+ epidermal cell number in a dose-dependent manner (see Fig. 3). Other widely used TGF-β signaling inhibitors also stimulate the growth of CK+ mouse primary epidermal cells in a dose-dependent manner. However, we found that RepSox treatment showed the highest increase in epidermal cell number at all the doses we examined (Fig. 3). In addition, p63+ mouse epidermal progenitor cells grow at a constant rate for at least 60 days in the presence of RepSox (Fig. 4a). Proliferation of mouse epidermal cells is RepSox-dependent, as removal of RepSox from the culture results in a rapid decline in the growth of epidermal cells (Fig. 4a), accompanied by elevated expression of phosphorylated Smad2/3 at both P5 and P20 stages (Fig. 4b, c). 3. Although the majority of the cells are still floating in the culture media 3 days after the inoculation, approximately 10% of the cells adhere to the plates and initiate the growth. 4. Do not over grow mouse epidermal cells as confluent epidermal cells are difficult to dissociate by trypsinization, and they tend to differentiate prematurely in the subsequent passage. The days for primary epidermal cells to become confluent in culture seem to depend on the age of mice (see Fig. 5).

Fig. 3 Dose-dependent effects of TGF-β signaling inhibitors on cell proliferation of mouse primary epidermal keratinocytes. Newborn mouse-derived, CnT-PRexpanded cytokeratin-positive (CK+) epidermal cells (2  104) were cultivated in CnT-PR media for 10 days in the presence of increasing concentration of TGF-β signaling inhibitors (RepSox, SB525334, LY364947, SB431542, and A83-01). A BMP signaling inhibitor DMH-1 was included as a negative control. Data shown are mean  s.e.m. (n ¼ 3). Adapted from ref. [26]

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Fig. 4 TGF-β signaling inhibition-mediated enhancement of population doubling of p63+ mouse primary epidermal keratinocytes. (a) Population doubling of newborn mouse-derived, RepSox-expanded p63+ epidermal cells grown in CnT-PR media in the presence of 1 μM RepSox for 0, 14, 21, and 60 days. Data shown are representative of three independent experiments with similar results. Arrow indicates continuous cell growth. (b, c) Removal of RepSox associates with increased Smad2/3 phosphorylation. (b) Experimental design. Newborn mouse-derived, RepSox-expanded P5 and P20 epidermal keratinocytes were further grown in continuous presence (upper) or absence (middle and lower) of 1 μM RepSox for 24 h. Culture of P5 cells stimulated with 1 ng/mL TGF-β for 1 h prior to lysis (lower) was used as a positive control. (c) Expression of total and phosphorylated Smad2/3 was determined by Western blot. Lane numbers correspond to those in (b). Antibodies used were rabbit anti-phosphorylated Smad2/3 (D27F4, Cell Signaling Technology), rabbit antiSmad2/3 (D7G7, Cell Signaling Technology), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling Technology). Adapted from ref. [26]

5. Accutase or TrypLE select can be used to more gently dissociate the cells compared with trypsin-EDTA. 6. Inhibition of TGF-β signaling also enriches and expands other p63+ epithelial progenitor cells in primary crude cultures of multiple epithelia, including the cornea, oral and lingual epithelia, salivary gland, esophagus, thymus, and bladder [26]. 7. Do not allow human keratinocytes to become confluent because confluent keratinocytes are difficult to dissociate by enzymatic digestion and they tend to differentiate prematurely in subsequent passages.

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Fig. 5 Age-dependent decline in TGF-β signaling inhibition-mediated cell growth of mouse primary epidermal keratinocytes. (a) Representative images of Rhodamine B staining of crude culture of epidermal cells harvested from newborn (NB), 4-week-old (4w), and 10-week-old (10w) mice and grown in CnT-PR media in the presence (upper) or absence (lower) of 1 μM RepSox for 14 days. One million primary cells were seeded. Data shown are representative of two independent experiments with similar results. Bar ¼ 5 mm. (b) Total number of p63+ epidermal cells. One million primary cells were seeded and adherent cells were counted at day 1 in replicative wells (gray bars) as the majority of the cells remained in suspension. Solid bars and open bars represent total numbers of p63+ epidermal cells at day 14 of culture in the subsequent presence of 1 μM RepSox or 0.1% DMSO, respectively (n ¼ 3). *P < 0.05; **P < 0.01. Adapted from ref. [26]

8. We routinely expand 3T3-J2 cells at passage 4–7 and prepare a large number of frozen stocks before initiating epidermal cell co-culture. We typically use 3T3-J2 cells in co-culture at passage 6–10. 9. Do not allow feeder cells to become confluent as the morphology of the cells is altered and the potential of feeder cells to support keratinocyte growth may decline. 10. Instead of γ-irradiation, mitomycin C treatment can be used to induce growth arrest in feeder cells as follows. (a) Add mitomycin C to 3T3-J2 media (see Subheading 2.6.2) in a 50 mL tube at a final concentration of 10 μg/mL. (b) Aspirate cell culture media from the plates, and add 3 mL of freshly prepared mitomycin C-containing feeder cell media in each 60 mm plate. (c) Place the plates in a humidified incubator with 10% CO2 at 37  C for 2 h. (d) Wash the cells three times in 3 mL PBS. (e) Add 5 mL J2 media, and return the plates to a humidified incubator with 10% CO2 at 37  C until use.

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11. See Table 1 for expected numbers of subconfluent feeder cells at the time of keratinocyte inoculation and appropriate amounts of cell culture media used for feeder cell co-culture. When feeder cells are seeded at the density of 3.3  103 cells per cm2, it will take 5–7 days for 3T3-J2 cells and hDFs to become subconfluent while it will require additional 2–3 days for hPAs to become subconfluent. Higher numbers of feeder cells can be seeded to obtain subconfluent feeder cell layers sooner. In such a circumstance, up to 2  106 viable cells per 100 mm plate can be seeded and the cells can be γ-irradiated in 1–2 days when the feeder cells become subconfluent and adhere tightly to the culture vessels. 12. Although feeder cells treated with γ-irradiation or mitomycin C can remain viable for a few weeks, we typically use them in 5–7 days after the treatment. Feeder cell media should be replaced every 2–3 days until use. 13. Epidermal cells grow faster when they are plated at higher densities, possibly due to an autocrine effect on cell proliferation [31]. 14. Keratinocytes and feeder cells can be distinguished morphologically upon trypsin digestion, while keratinocytes are smaller in size with a smooth periphery, feeder cells are larger in size with an irregular shape. However, flow cytometric analysis with keratinocyte-specific markers is needed for more accurate quantification of keratinocyte numbers. 15. At this plating density, small colonies comprised of 4–10 cells begin to form at day 4–5. By day 7, epidermal clones become macroscopically visible, and they reach 5–15 mm in diameter by day 14 in the presence of 1 μM RepSox (see, e.g., Fig. 6). Cell culture vessels smaller than 6-well plates are not recommended for clonogenic analysis as keratinocyte colonies may fuse before they reach the size suitable for clonal evaluation. 16. Optimum doses of RepSox that promote human epidermal keratinocyte growth in feeder cell co-culture vary from one feeder cell type to another. The optimum dose of RepSox or any other TGF-β signaling inhibitors should be determined for each feeder cell type prior to performing co-culture of keratinocytes (see, e.g., Fig. 7). 17. Both human and mouse epidermal keratinocytes expanded by RepSox treatment in feeder cell co-culture maintain the original high proliferative capacity as determined by serial passages in clonogenic culture with 3T3-J2 cells [26]. In addition,

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Fig. 6 Clonal expansion of mouse primary epidermal keratinocytes in co-culture with 3T3-J2 feeder cells. (a) Rhodamine B staining of newborn mouse-derived, RepSox-expanded P1 epidermal keratinocytes grown in 3T3-J2 co-culture in the presence or absence of 1 μM RepSox for 14 days. Bar ¼ 10 mm. (b) Distribution of epidermal clone sizes at day 14. Data shown are mean  s.e.m. (n ¼ 3). * P < 0.05; **P < 0.01. Adapted from ref. [26]

primary epidermal keratinocytes expanded by RepSox treatment are capable of differentiation in response to Ca2+ stimulation [26]. 18. Rhodamine B solution can be reused multiple times. Used solution should be collected in a separate container and filtered through Whatman filter papers to remove debris before use. 19. Other widely used TGFBRI/ALK5 inhibitors, such as LY364947 and SB525334, also enhance the growth of human epidermal keratinocytes in co-culture with 3T3-J2 cells in a dose-dependent manner [26]. However, the effect of RepSox was superior to any other TGFBRI/ALK5 inhibitors tested [26]. In the case of co-culture with hPAs, we investigated the effects of RepSox at 0.1 and 1 μM and found that the dose-dependent stimulation of human keratinocyte growth was similar to that in co-culture with hDFs.

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Fig. 7 Growth response of human epidermal keratinocytes to RepSox treatment in co-culture with feeder cells. Human epidermal keratinocytes were grown in co-culture with 3T3-J2 cells (upper) and hDFs (lower) in the absence or presence of increasing concentration of TGF-β signaling inhibitor RepSox as indicated. At day 10, the cells were harvested and the cell numbers were counted, followed by flow cytometric analysis of intracellular staining of cytokeratin 5 (CK5). CK5+ epidermal cell numbers were plotted. Data shown are mean  s.e.m. (n ¼ 3)

Acknowledgments This study was supported by an R01AR066755 grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institute of Health to M.S. References 1. Blanpain C, Fuchs E (2014) Stem cell plasticity. Plasticity of epithelial stem cells in tissue regeneration. Science 344:1242281 2. Donati G, Watt FM (2015) Stem cell heterogeneity and plasticity in epithelia. Cell Stem Cell 16:465–476 3. Green H (2008) The birth of therapy with cultured cells. Bioessays 30:897–903

4. Chua AW, Khoo YC, Tan BK, Tan KC, Foo CL, Chong SJ (2016) Skin tissue engineering advances in severe burns: review and therapeutic applications. Burns Trauma 4:3 5. Mcheik JN, Barrault C, Levard G, Morel F, Bernard FX, Lecron JC (2014) Epidermal healing in burns: autologous keratinocyte transplantation as a standard procedure: update

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and perspective. Plast Reconstr Surg Glob Open 2:e218 6. Sun BK, Siprashvili Z, Khavari PA (2014) Advances in skin grafting and treatment of cutaneous wounds. Science 346:941–945 7. Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343 8. Barrandon Y, Green H (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci U S A 84:2302–2306 9. Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y (2001) Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104:233–245 10. Senoo M, Pinto F, Crum CP, McKeon F (2007) p63 is essential for the proliferative potential of stem cells in stratified epithelia. Cell 129:523–536 11. Lichti U, Anders J, Yuspa SH (2008) Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat Protoc 3:799–810 12. Ha L, Ponnamperuma RM, Jay S, Ricci MS, Weinberg WC (2011) Dysregulated ΔNp63α inhibits expression of Ink4a/arf, blocks senescence, and promotes malignant conversion of keratinocytes. PLoS One 6:e21877. https:// doi.org/10.1371/journal.pone.0021877 13. Missero C, Di Cunto F, Kiyokawa H, Koff A, Dotto GP (1996) The absence of p21Cip/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression. Genes Dev 10:3065–3075 14. Paramio JM et al (2001) The ink4a/arf tumor suppressors cooperate with p21cip1/waf in the processes of mouse epidermal differentiation, senescence, and carcinogenesis. J Biol Chem 276:44203–44211 15. Chapman S, McDermott DH, Shen K, Jang MK, McBride AA (2014) The effect of Rho kinase inhibition on long-term keratinocyte proliferation is rapid and conditional. Stem Cell Res Ther 5:60. https://doi.org/10.1186/scrt449 16. King KE et al (2003) ΔNp63α functions as both a positive and a negative transcriptional regulator and blocks in vitro differentiation of murine keratinocytes. Oncogene 22:3635–3644 17. Liu X et al (2012) ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol 180:599–607 18. Mou H et al (2016) Dual SMAD signaling inhibition enables long-term expansion of diverse epithelial basal cells. Cell Stem Cell 19:217–231

19. Watabe T, Miyazono K (2009) Roles of TGF-β family signaling in stem cell renewal and differentiation. Cell Res 19:103–115 20. Shipley GD, Pittelkow MR, Wille JJ Jr, Scott RE, Moses HL (1986) Reversible inhibition of normal human prokeratinocyte proliferation by type beta transforming growth factor-growth inhibitor in serum-free medium. Cancer Res 46:2068–2071 21. Schmierer B, Hill CS (2007) TGF-β-SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol 8:970–982 22. Ikushima H, Miyazono K (2010) TGF-β signalling: a complex web in cancer progression. Nat Rev Cancer 10:415–424 23. Ghahary A, Marcoux Y, Karimi-Busheri F, Tredget EE (2001) Keratinocyte differentiation inversely regulates the expression of involucrin and transforming growth factor β1. J Cell Biochem 83:239–248 24. Suzuki D, Senoo M (2015) Dact1 regulates the ability of 3T3-J2 cells to support proliferation of human epidermal keratinocytes. J Invest Dermatol 135:2894–2897 25. Robertson IB, Rifkin DB (2013) Unchaining the beast; insights from structural and evolutionary studies on TGF-β secretion, sequestration, and activation. Cytokine Growth Factor Rev 24:355–372 26. Suzuki D, Pinto F, Senoo M (2017) Inhibition of TGF-β signaling supports high proliferative potential of diverse p63+ mouse epithelial progenitor cells in vitro. Sci Rep 7:6089. https:// doi.org/10.1038/s41598-017-06470-y 27. Bullock AJ, Higham MC, MacNeil S (2006) Use of human fibroblasts in the development of a xenobiotic-free culture and delivery system for human keratinocytes. Tissue Eng 12:245–255 28. Sugiyama H, Maeda K, Yamato M, Hayashi R, Soma T, Hayashida Y, Yang J, Shirakabe M, Matsuyama A, Kikuchi A, Sawa Y, Okano T, Tano Y, Nishida K (2008) Human adipose tissue-derived mesenchymal stem cells as a novel feeder layer for epithelial cells. J Tissue Eng Regen Med 2:445–449 29. Rasmussen C, Thomas-Virnig C, AllenHoffmann BL (2013) Classical human epidermal keratinocyte cell culture. Methods Mol Biol 945:161–175 30. Skurk T, Ecklebe S, Hauner H (2007) A novel technique to propagate primary human preadipocytes without loss of differentiation capacity. Obesity 15:2925–2931 31. Lim X, Tan SH, Koh WL, Chau RM, Yan KS, Kuo CJ, van Amerongen R, Klein AM, Nusse R (2013) Interfollicular epidermal stem cells selfrenew via autocrine Wnt signaling. Science 342:1226–1230

Methods in Molecular Biology (2020) 2109: 23–25 DOI 10.1007/7651_2019_270 © Springer Science+Business Media New York 2019 Published online: 03 December 2019

Dermal-Epidermal Separation by Heat Liyan Jian, Yu Cao, and Ying Zou Abstract The skin contains three primary layers: epidermis, dermis, and hypodermis. Separation of epidermal components from the dermis (dermal-epidermal separation) is an important basic investigation technique for pharmacology, toxicology, and biology. There are different systems of epidermal separation, including typical methods of chemical, enzyme, heat, etc. Each approach has advantages versus disadvantages, and thus the appropriate method should be chosen for a given research question. Here we described the method of heat separation. Keywords Epidermis, Dermis, Separation, Heat

1

Introduction Dermal-epidermal separation is a basic technique for experiment and research in dermatology. The methods have been developed to conduct separation using various approaches, such as chemicals, enzymes, and heat, each with its own advantages and problems. Therefore, care should be taken in aligning the separated cell products with specific purpose of subsequent studies. Since chemical reagents could disturb cellular electrolyte equilibrium and enzymes might disrupt important components on cell surface [1] (see Note 1), heat separation has been extensively used for its simplicity and efficacy when the combination of time and temperature is optimized. Dermal-epidermal separation by heat was introduced in 1942 by Baumberger during their early studies of epidermal tissue [2]. Since then their method has been modified for different research purposes such as detecting autoantigens [3] (see Note 2), studying cyclic AMP levels [4], phospholipid analysis [5], and cellular signal pathway analysis [6], because of the simple procedure and the short manipulation time it requires. The epidermal-dermal junction consists of four parts, i.e., the basal cell plasma membrane, the electron-lucent area, the basal lamina, and the subbasal lamina fibrous components, including anchoring fibrils, dermal microfibril bundles, and collagen fibers [7]. As an increase in temperature could lead to a softening of collagen fibers, carefully heating the skin tissue may be used to

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separate epidermis from dermis [2]. It was reported that heating at 60  C for 45 s could easily separate the epidermis from the dermis by peeling with a scalpel blade or forceps [8].

2

Materials 1. Fresh human skin biopsy was obtained from surgical specimens. 2. Water bath of 60  C and ice-water mix (both prepared from deionized water with a resistivity of 18 MΩ cm at 25  C). 3. Prepare aseptic forceps and Dulbecco’s Phosphate-Buffered Saline (DPBS) solution without calcium and magnesium ions (Thermo Fisher Scientific).

3

Methods 1. Remove subcutaneous tissue from skin samples carefully with aseptic scalpel. Cut into pieces according to the requirement of experiment. 2. Preheat DPBS in 60  C water bath as H-DPBS and in ice-water mix as C-DPBS. 3. Immerse the skin pieces in the H-DPBS for 45–60 s. 4. Transfer the skin section from the H-DPBS and place in C-PBS for immediate cooling. 5. Get the skin section out of C-PBS. Remove excess PBS. 6. Separate epidermis layer from dermis by peeling with aseptic forceps. 7. Put the epidermal and dermal preparations in individual dishes with DPBS for next-step usage.

4

Notes 1. For an ideal dermal-epidermal separation, the combination of heat temperatures and treatment durations should be tested to obtain the optimal condition ensuring a complete separation and intact tissue structures. 2. Peeling epidermis with forceps should be done gently and slowly to keep the epidermis intact.

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Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC) grant No. 81602757, the Science and Technology Commission of Shanghai Municipality grant No. 16411961800, and the Science Foundation of Shanghai Hospital Development Center grant No. 16CR3096B. References 1. Zou Y, Maibach HI (2018) Dermal-epidermal separation methods: research implications. Arch Dermatol Res 310(1):1–9 2. Baumberger JP, Suntzeff V, Cowdry EV (1942) Methods for separation of epidermis from dermis and some physiologic and chemical properties of isolated epidermis. J Natl Cancer Inst 2:413–416 3. Ohata Y, Hashimoto T, Nishikawa T (1995) Comparative study of autoantigens for various bullous skin diseases by immunoblotting using different dermo-epidermal separation techniques. Clin Exp Dermatol 20:454–458 4. Wadskov S, Sondergaard J (1978) Determination of cyclic AMP in heat-separated human epidermal tissue. Acta Derm Venereol 58:191–195

5. Tsambaos D, Mahrle G (1979) The phospholipid pattern in the involved and the uninvolved psoriatic epidermis. Arch Dermatol Res 266:177–180 6. Kassis V, Sondergaard J (1982) Heat-separation of normal human skin for epidermal and dermal prostaglandin analysis. Arch Dermatol Res 273:301–306 7. Briggaman RA, Wheeler CE Jr (1975) The epidermal-dermal junction. J Invest Dermatol 65:71–84 8. Fort JJ, Mitra AK (1994) Effects of epidermal/ dermal separation methods and ester chain configuration on the bioconversion of a homologous series of methotrexate dialkyl esters in dermal and epidermal homogenates of hairless mouse skin. Int J Pharm 102:241–247

Methods in Molecular Biology (2020) 2109: 27–30 DOI 10.1007/7651_2019_267 © Springer Science+Business Media New York 2019 Published online: 03 December 2019

Dermal-Epidermal Separation by Enzyme Liyan Jian, Yu Cao, and Ying Zou Abstract The skin contains three primary layers: epidermis, dermis, and hypodermis. Separation of epidermal components from dermis (dermal-epidermal separation) is an important basic investigation technique for pharmacology, toxicology, and biology. There are different systems of epidermal separation, including typical methods of chemical, enzyme, heat, etc. Each approach has advantages versus disadvantages, and thus the appropriate method should be chosen for a given research question. Here we described the method of enzyme separation. Keywords Epidermis, Dermis, Separation, Enzymes, Dispase

1

Introduction Epidermal-dermal junction (EDJ) is majorly maintained by groups of cell surface protein complexes including collagens, laminins, and fibronectin [1] via molecular interactions such as salt bridge, hydrophobic interaction, hydrogen bond, etc. The breakdown of cell surface protein complexes is the critical step in dermal-epidermal separation and methods have been developed to disrupt the molecular interactions abovementioned, e.g., heat method and neutral salt method. In addition to the disassembly of the quaternary structure of macromolecular complexes between cells, digestion on the protein components within the complexes represents an alternative approach in releasing cells from the adhesive matrix. Proteases are enzymes capable of cleavage of peptide chain and are powerful reagents in protein science and cell biology when the engineering, fragmentation, or cleanup of proteins is desired. Various proteases have been tested in dermal-epidermal separation including trypsin, pancreatin, elastase, pronase, collagenase, and Dispase [2–5] (see Notes 1 and 2). Trypsin treatment might represent the most widely used method for detaching cells of interest from tissues, culture vessels, or agglomeration with minimal influence on cell integrity and function. However, the low efficacy of trypsin digestion on desmosome, the protein junction between epidermal cells, and the vulnerability of trypsin to serum deactivation could result in incomplete separation and low yield [6] (see Notes 1–3). Dispase is a

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Fig. 1 Action site of dispase separation on epidermal-dermal junction

neutral protease found in Bacillus polymyxa capable of digesting lamina densa by cleaving the extracellular matrix components such as type IV collagen and fibronectin [7]. Dispase digestion activity is insensitive to serum and exerts little effect on anchoring fibrils and epidermal cells, making it a good tool in dermal-epidermal separation. Figure 1 shows the action site of dispase separation on epidermal-dermal junction. By combining the enzymatic activities from trypsin and Dispase was developed an efficient method in preparing individual and relatively homogenous cell groups from primary skin layers.

2

Materials 1. Fresh human skin was obtained from surgical specimens. 2. Dispase II and trypsin were purchased from Thermo Fisher Scientific in the forms of lyophilized powder and solution, respectively. 3. To prepare Dispase stock solution, the powder of Dispase (Roche) is reconstituted in phosphate-buffered saline (PBS) at a final concentration of 10 mg/ml (or about 6–10 U/ml∗) without calcium and magnesium ions. Dilute the Dispase stock solution to a final concentration of 2 U/ml with Ca2+/Mg2+free PBS as Dispase digestion solution.

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Methods All the procedure should be carried out aseptically. 1. Cut the skin biopsy into pieces measuring 3–5 mm in diameter and rinse with Ca2+, Mg2+-free PBS. Soak the skin samples in Dispase digestion solution at 4  C for 24 h. 2. Peel the epidermis from the dermis with fine forceps and gently wash the separated sheets with Ca2+/Mg2+-free PBS twice. 3. To further obtain epidermal cell culture, incubate the epidermal sheets in 0.25% trypsin solution at room temperature for 12–15 min. Transfer the sheets into Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 20% fetal bovine serum and gently shake to dissociate the epidermal sheet. 4. Perform cell count on the epidermal cell suspension and place epidermal cells into 25-cm2 culture flask at a density of 2–4  104 cells/cm2.

4

Notes 1. One unit of Dispase is equivalent to 181 unit of protease (PU), or about 600 Japanese unit of Dispase (JPU). 2. Peeling epidermis with aseptic forceps should be gently and slowly to keep epidermis intact. 3. All the solutions should be prepared with ultrapure water, enzyme preparation, and analytical grade chemicals followed by filter sterilization.

Acknowledgments This work was supported by National Natural Science Foundation of China (NSFC) grant No. 81602757, Science and Technology Commission of Shanghai Municipality grant No. 16411961800, and the Science Foundation of Shanghai Hospital Development Center grant No. 16CR3096B. References 1. Briggaman RA (1982) Biochemical composition of the epidermal-dermal junction and other basement membrane. J Invest Dermatol 78:1–6

2. Zou Y, Maibach HI (2018) Dermal-epidermal separation methods: research implications. Arch Dermatol Res 310:1–9

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3. Einbinder JM, Walzer RA, Mandl I (1966) Epidermal-dermal separation with proteolytic enzymes. J Invest Dermatol 46:492–504 4. Becker SW Jr, Fitzpatrick TB, Montgomery H (1952) Human melanogenesis; cytology and histology of pigment cells (melanodendrocytes). AMA Arch Derm Syphilol 65:511–523 5. Walzer C, Benathan M, Frenk E (1989) Thermolysin treatment: a new method for dermo-

epidermal separation. J Invest Dermatol 92:78–81 6. Takahashi H, Sano K, Yoshizato K et al (1985) Comparative studies on methods of isolating rat epidermal cells. Ann Plast Surg 14:258–266 7. Stenn KS, Link R, Moellmann G et al (1989) Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase. J Invest Dermatol 93:287–290

Methods in Molecular Biology (2020) 2109: 31–33 DOI 10.1007/7651_2019_266 © Springer Science+Business Media New York 2019 Published online: 03 December 2019

Dermal-Epidermal Separation by Chemical Liyan Jian, Yu Cao, and Ying Zou Abstract The skin contains three primary layers: epidermis, dermis, and hypodermis. Separation of epidermal components from dermis (dermal-epidermal separation) is an important basic investigation technique for pharmacology, toxicology, and biology. There are different systems of epidermal separation, including typical methods of chemical, enzyme, heat, etc. Each approach has advantages versus disadvantages, and thus the appropriate method should be chosen for a given research question. Here we described the method of chemical separation. Keywords Epidermis, Dermis, Separation, Chemical, Sodium thiocyanate

1

Introduction Dermal-epidermal separation represents the starting point for a variety of procedures and experiments in dermatology. Different approaches have been tested in separating epidermis and dermis in a rapid but mild way, including heating skin samples, treatment with chemicals, and enzymatic digestion. Comparing to the heat separation with potential thermal damages and the enzymatic separation causing damages on proteins at the cell surface, chemical separation is of higher adaptive capacity for its variability from adjusting factors such as salt selections, ion strengths, and pHs. Dermal-epidermal separation could be conducted with chemicals including acids, alkali, and neutral salts, while the neutral salts showed the highest efficacy in separation [1] (see Note 1). Other merits of neutral salts method include an accurate separation interface at epidermaldermal junction and the integrity of resultant cells [2]. Various neural salts have been tested in dermal-epidermal separation, such as sodium-EDTA (ethylenediaminetetraacetic acid), sodium thiocyanate, ammonium thiocyanate, sodium iodide, and sodium bromide [3, 4]. Among them, sodium thiocyanate showed the fastest reaction on epidermal-dermal junction and was thus extensively utilized [1]. Pilot study found that treatment of skin samples with a 2 N solution of sodium thiocyanate could result in rapid epidermal swelling and glazing, in turn inducing the separation [4]. Following the sodium thiocyanate separation treatment immuno-

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Fig. 1 Action site of sodium thiocyanate separation on epidermal-dermal junction

fluorescence and electron microscopy demonstrated a complete cleavage at the level of the lamina, and the basement membrane was shown remained on dermis [2]. Figure 1 shows the action site of sodium thiocyanate separation on epidermal-dermal junction.

2

Materials 1. Fresh human skin was obtained from surgical specimens. 2. Sodium thiocyanate solution: dissolve analytical grade sodium thiocyanate in ultrapure water at a final concentration of 2 M. The pH was adjusted to 6.8 with hydrochloric acid. Sodium thiocyanate solution should be filter sterilized and kept at 4  C prior to use. 3. Dulbecco’s phosphate-buffered saline (DPBS) solution without calcium and magnesium ions was purchased from Thermo Fisher Scientific.

3

Methods 1. Remove the subcutaneous tissue from skin samples by an aseptic scalpel. Cut into pieces of 2–4 mm cube.

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2. Immerse skin samples in cold sodium thiocyanate solution for 5 min. 3. Peel epidermis from dermis with an aseptic forceps. 4. Wash the separated epidermal and dermal tissues in DPBS at ambient temperature and the samples should be ready for further studies.

4

Note 1. Peeling epidermis with aseptic forceps should be gently and slowly to keep epidermis intact.

Acknowledgments This work was supported by National Natural Science Foundation of China (NSFC) grant No. 81602757, Science and Technology Commission of Shanghai Municipality grant No. 16411961800, and the Science Foundation of Shanghai Hospital Development Center grant No. 16CR3096B. References 1. Zou Y, Maibach HI (2018) Dermal-epidermal separation methods: research implications. Arch Dermatol Res 310:1–9 2. Diaz LA, Heaphy MR, Calvanico NJ et al (1977) Separation of epidermis from dermis with sodium thiocyanate. J Invest Dermatol 68:36–38

3. Trost A, Bauer JW, Lanschu¨tzer C et al (2007) Rapid, high-quality and epidermal-specific isolation of RNA from human skin. Exp Dermatol 16:185–190 4. Felsher Z (1947) Studies on the adherence of the epidermis to the corium. J Invest Dermatol 8:35–47

Methods in Molecular Biology (2020) 2109: 35–44 DOI 10.1007/7651_2019_264 © Springer Science+Business Media New York 2019 Published online: 21 September 2019

Isolation and Characterization of Extracellular Vesicles from Keratinocyte Cultures Sebastian Sjo¨qvist, Aya Imafuku, Dhanu Gupta, and Samir EL Andaloussi Abstract Extracellular vesicles (EVs), including exosomes, are nano-sized membrane-bound particles which are released by cells. They have been found in all examined body fluids and can be isolated from conditioned cell culture media. These vesicles have gained increasing attention due to their importance in cellular cross talk, in both health and disease. For example, keratinocyte-derived EVs have been described to modulate melanin production in epidermis. Similar EVs were also shown to have an important role in skin immunology, by stimulating dendritic cells. In this chapter, we will describe how to isolate EVs from keratinocyte cultures and how to perform characterization by Western blot, nanoparticle tracking analysis, and transmission electron microscopy. Keywords Extracellular characterization

1

vesicles,

Exosomes,

Keratinocytes,

Epidermis,

EV

isolation,

EV

Introduction It is likely that all cells secrete extracellular vesicles (EVs) including exosomes. These vesicles are enveloped by a lipid bilayer and contain a plethora of bioactive molecules such as mRNA, microRNA, noncoding RNA, surface receptors, and bioactive lipids [1, 2]. Extracellular vesicles have been isolated from the majority of body fluids, including cerebrospinal fluid [3], serum [4], saliva [5], and urine [6]. The role of EVs in skin physiology remains largely unknown, but Lo Cicero et al. demonstrated their importance for the cross talk between keratinocytes and melanocytes regarding skin pigmentation [7]. Kotzerke et al. showed that keratinocyte-derived EVs also are of importance for cutaneous immunity. The EVs stimulated dendritic cells to increase expression of CD40 and their production of cytokines such as interleukins 6, 10, and 12 [8].

The original version of this chapter was revised. An erratum to this chapter can be found at https://doi.org/10. 1007/7651_2019_274

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We recently published our findings based on oral keratinocytederived EVs. Since the oral mucosa has exceptional healing capacity, we hypothesized that EVs from oral keratinocytes could have a similar effect. We found that fibroblasts stimulated with EVs drastically increased their production of pro-regenerative factors such as FGF and HGF. The EVs also accelerated full-thickness wound healing, even in a xenogeneic setting, which could be related to their modulation of fibroblasts and epithelial cell growth [9, 10]. In this chapter, we will describe how to isolate EVs from conditioned culture media from keratinocytes. Several methods to isolate EVs exist, including ultracentrifugation, density gradient, and precipitation, each with advantages and disadvantages [11]. Here we describe the use of ultrafiltration followed by sizeexclusion chromatography, a method that has been demonstrated to be efficient and yield pure EV isolates [12, 13]. Further, we will describe the characterization of isolated extracellular vesicles, following the recommendations of the international society of extracellular vesicles (ISEV) minimal requirement [14]. These requirements include identification of positive and negative protein markers (e.g., by Western blot), morphological evaluation (e.g., by transmission electron microscopy), and size distribution (e.g., by nanoparticle tracking analysis). Although we here explain how to isolate and characterize EVs from conditioned media, the protocols could be adopted to isolate EVs from other sources, such as wound exudates.

2 2.1

Materials Cell Culture

1. Epithelial cells, for example, HaCaT cell line. 2. Suitable cell culture media, for example, Dulbecco’s Eagle’s Minimal Essential Medium supplemented with 10% fetal bovine serum and 1% antibiotics. 3. Serum-free or vesicle-depleted media (see Note 1). 4. Physiological phosphate-buffered saline (PBS). 5. Cell detachment reagent, for example, trypsin EDTA. 6. 0.4% trypan blue. 7. Cell culture plastic, for example, 100 mm tissue culture plastic dishes (see Note 2).

2.2 Extracellular Vesicle Isolation Using Size-Exclusion Chromatography

1. 100 and 10 kDa ultrafiltration spin filters. 2. Commercially available size-exclusion chromatography columns, here we use qEV from Izon. 3. PBS. 4. 20% ethanol (diluted in PBS).

Isolation and Characterization of Extracellular Vesicles from Keratinocyte. . .

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5. 0.22 μm syringe filter. 6. 10 mL sterile syringe. 2.3 Nanoparticle Tracking Analysis

1. NanoSight NS500 (Malvern). 2. 1
phosphate-buffered saline (PBS). 3. 0.22 μm sterile syringe filters. 4. 50 mL sterile syringe.

2.4 Transmission Electron Microscopy

1. 2% uranyl acetate. All the steps are performed in a fume hood, with proper protection as uranyl acetate is radioactive. Prepare 4% uranyl acetate solution in distilled water by mixing 4 g of uranyl acetate powder (Sigma-Aldrich) and 100 mL of warm double distilled water. Cool down the solution to room temperature and store at 4  C in dark until further use. For negative staining prepare a small stock of 2% uranyl acetate by adding 200 μL of 4% uranyl acetate into a tube followed by 200 μL of double distilled water. 2. Formvar-Carbon type B coated electron microscopy grids (300 Mesh Ted Pella Inc.). 3. ddH2O. 4. Whatman filter paper. 5. Tweezers. 6. Tecnai 10 electron microscope.

2.5

Western Blot

1. Radioimmunoprecipitation assay buffer (RIPA) with 1% protease/phosphatase inhibitor cocktail. 2. Sonicator. 3. Protein quantification kit, for example, BCA. 4. Loading buffer, for example, Laemmli buffer. 5. Protein gels, for example, 4–12% Bis-Tris polyacrylamide. 6. Protein ladder, for example, “Precision Plus All Blue.” 7. MES buffer. 8. Protein electrophoresis system. 9. Nitrocellulose membrane. 10. Dry blotter, for example, iBlot 2 Dry Blotting System. 11. TBS-T (BioRad). 12. Blocking buffer: 5% dry milk. 13. Primary antibody dilution buffer: 1
TBST-T, 5% BSA. 14. Detection reagent such as ECL Prime WB Detection Kit. 15. Biomolecular Imager such as LAS4000.

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Methods

3.1 Expansion of Cells and Production of Conditioned Media

Cell culture conditions vary depending on cell source. Here we explain general routines, but details such as seeding densities, media replacement intervals, etc. should be considered for each cell type. In general it is recommended to use pre-warmed media when working with cells. 1. Thaw and seed the cells at the recommended cell density, for example, 5000 cells per cm2. 2. In general, it is good to replace the media the following day, in order to reduce the amount of residual freezing media which might be harmful for the cells. Avoiding complete media replacement (i.e., leaving some of the used media) can improve cellular proliferation. 3. Observe the cells daily and note morphology and any signs of contamination. 4. Once the cells have reached about 80–90% confluency, wash the cells with PBS, detach them using trypsin, and passage into a suitable number of culture dishes (see Note 3). 5. Once an adequate amount of dishes have been seeded (15
100 mm dishes are a good start for one batch of EVs), wait until the cells reach about 80% confluency, then wash with PBS, and replace the media with either serum-free or EV-depleted media. 6. After an appropriate incubation time (time varies with cell type, but 24–48 h have yielded good results in our experience), collect the media and save for next step. 7. Observe the cells for any morphological changes and contamination. Count the number of cells and viability using trypan blue staining (see Note 4).

3.2 Concentration of Conditioned Media and EV Isolation Using Size-Exclusion Chromatography

1. The conditioned media has to be cleared from cells, cellular debris, and other contaminants. This can be achieved by centrifugation at 300
g for 10 min at 4  C, transferring the supernatant to new tubes and centrifuge at 3000
g for 10 min at 4  C, again transferring the supernatant to new tubes (see Note 5). 2. The cleared conditioned media has to be concentrated before performing the size-exclusion chromatography. This can be achieved by ultrafiltration using 100 kDa filters such as Amicon spin filters, which are spun at a speed depending on the centrifuge, but 5000
g at 4  C has been working well for us. Continue concentrating the media until the volume is within the limits of the size-exclusion column, for example, 500 μL (see Note 6).

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3. Equilibrate the column by rinsing with 10 mL filtered PBS (see Note 7). Note the time for 5 mL buffer to pass through the column. When working with size-exclusion chromatography columns, avoid introducing bubbles (see Note 8). 4. Once all PBS have passed through the top filter, add your sample. Start collecting the elute fractions; the size of each fraction varies with the column, but 500 μL is common. The first fractions are “void volume,” which don’t contain any part of your sample. The volume varies with columns, but 3 mL (¼6 fractions) is the volume for Izon columns. These fractions can generally be discarded (see Note 9). 5. After the void volume, the EV fractions elute. Collect the fractions according to the instructions of your column. When running a new set of samples, it is a good idea to keep the fractions separated (in contrast to pooling them) and analyze them separately. After running several of the same sample type, you can learn which fractions include the EVs and pool them during the collection. 6. Some columns can be reused. If so, you have to flush them thoroughly with PBS, and at this step, you should again check the time for 5 mL flow through. A different flow rate could indicate blockage or damage to the column. Next, flush with freshly filtered ethanol solution, and keep the column refrigerated until next use. 7. In general, the EVs need to be concentrated, but this depends on your downstream application. 10 kDa spin filters are useful for concentration of EVs. A target volume of less than 80 μL has been sufficient in our experience. 3.3 Nanoparticle Tracking Analysis (NTA)

NTA is a widely used method for quantifying nanoparticle concentration and size in liquid suspension. Nanoparticles in a liquid suspension are visualized upon illumination through a laser to determine the concentration, and the Brownian motion of the particles is used to determine the size. 1. Dilute the EV samples (1:100) and (1:1000) on a final volume of 1 mL using 0.22 μm filtered PBS. 2. Prime the NTA NS500 with 0.22 μm filtered PBS 0.01 M. Make sure the gasket is clean. 3. Once the fluid is primed, load 0.22 μm filtered PBS first. Make sure the solvent is free from aggregates (see Note 10). 4. Flush the machine five times with 0.22 μm filtered PBS. Load the diluted EV sample. Start first with the highest dilution. If the concentration is below 5
108, measure the lower dilution sample (see Note 11).

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5. Adjust the camera level between 10 and 13 according to the sample, so that light scattering from nearby nanoparticles does not affect the visualization of other nanoparticles (see Note 12). 6. Capture five 60 s videos at ten frames per second at 23.5  C (see Note 13). 7. Run the batch process with a screen gain set at 10 and detection threshold adjusted to 7 or 8, depending on the sample. Keep the minimum track length to automatic. 8. Obtained results show particle size distribution and concentration in the sample. 3.4 Transmission Electron Microscopy

1. Glow and discharge the 300-mesh Formvar-Carbon type B coated electron microscopy grids to increase the hydrophilicity. 2. Cut the Whatman filter paper in wedges. 3. Dilute the EVs sample to a final concentration of 1
1012/mL in 0.22 μm filtered PBS. 4. Hold the grid from the edge using tweezers with a shiny side of the grid facing upwards. 5. Pipette 5 μL of EV sample on the shiny side of the grid and incubate for 30 s. 6. Gently blot dry the grid with the tip of Whatman filter paper wedge pre-soaked in ddH2O. 7. Wash the grid with 5 μL of ddH2O and blot dry with pre-soaked Whatman filter paper. 8. Pipette 5 μL of 2% uranyl acetate solution on top of the grid and incubate for 30 s for negative staining of the sample (see Note 14). 9. Blot dry the grid with the tip of Whatman filter paper wedge. 10. Let the grid to air dry. 11. The grid is then imaged using a Tecnai 10 electron microscope at 16,500
, 26,500
, and 60,000
.

3.5

Western Blot

Western blot can be performed to identify protein expression on your EV samples. It is generally recommended to show at least two positive, “EV-markers” and at least one negative marker. Common for positive markers are tetraspanins (e.g., CD63, CD81, or CD82), cytosolic proteins such as Alix, or flotillin-1, while endoplasmin precursor GRP94 can be used as a negative marker. The ISEV 2018 position statement has a more extensive list of suggested protein groups and proteins [14]. 1. Cell lysates serves as an important control for the Western blot analysis. To prepare cell lysate, count 4
106 cells and

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centrifuge 300
g for 5 min. Discard the supernatant and resuspend the pellet in PBS and centrifuge again using the same settings. Discard the supernatant and resuspend the pellet in 1 mL RIPA buffer. Sonicate the suspension for 5 min (15 s on/15 s off intervals, high setting), and centrifuge at 8000
g for 10 min at 4  C. Aliquot the supernatant and store in 80  C. 2. Determine the protein concentration of your cell lysate and EV samples, using, for example, bicinchoninic acid assay. 3. Dilute 3 μg of EV and cell lysate samples 1:1 with Laemmli buffer, put on a heat block at 95  C for 5 min, and immediately cool the samples on ice. 4. Mount the dock in your electrophoresis system and fill with MES buffer. 5. Load the samples and ladder into the gel according to the recommended loading volumes. 6. Perform electrophoresis; we used the following settings successfully: 200 V, 125 mA, and 25 min. 7. Transfer the proteins to a nitrocellulose membrane using iBlot system. 8. Block the membrane using the 5% milk solution. 9. Rinse the membrane in TBS-T buffer. 10. Dilute your primary antibody at an appropriate dilution in the primary antibody buffer. Add to the membrane and incubate overnight in 4  C on a rocking platform. 11. The following day, wash the membrane three times in TBS-T for 10 min each, on a rocking platform in room temperature (see Note 15). 12. Add the appropriate secondary antibody diluted in blocking buffer. 13. Incubate for 1 h in room temperature with gentle rocking. 14. Wash the membrane three times in TBS-T for 10 min each, on a rocking platform in room temperature. 15. Prepare the detection reagent according to manufacturer’s instructions. 16. Add the detection reagent to the filter, wait for 5 min, and drain the reagent. 17. Visualize in a biomolecular imager such as LAS 4000.

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Notes 1. If you are using vesicle-depleted media, it is a good idea to analyze it for any residual particles by NTA. 2. For most small-scale experiments, regular tissue culture dishes work well. Three-dimensional or multilayered culture systems can be more efficient but also require more extensive quality control of the cells as monitoring them is more difficult and media perfusion, etc. can affect the phenotype. 3. Note that confluency percentage suitable for passaging, detachment reagent, and number of culture dishes to which to expand the cells are all cell-specific variables; please check with your cell type. Further, when passaging the cells, it is recommended to count the cells using, for example, trypan blue staining. This way you can identify if the cells have a lower viability than expected or if the cell number differs from expected. 4. Do not reuse the same cells for EV production or other downstream analysis as the serum-starvation/EV-depleted media exposure might affect the cells. 5. It is generally advised to do as many steps as possible in a laminar airflow bench to reduce the risk of contamination of your samples. 6. In case you have a large volume of media, the spin filters can sometimes be reused (please check the manufacturer’s recommendation), if you plan to reuse the filters, make sure that they never get dry. For large volumes of media, tangential flow filtration can be useful [15]. 7. We usually keep the PBS in the syringe with a fitted syringe filter and add the PBS to the column freshly filtered. 8. It can be helpful to add the buffer to the wall of the column instead of straight onto the filter, to prevent bubbles from forming. For larger volumes of media, column chromatogra¨ kta by GE LifeScience can be useful. phy systems such as A 9. If you are reusing the column, it might be useful to make sure that the void volume is clear of particles or other material that could contaminate your EV samples. 10. Avoid using solvents in EVs preparation which includes albumin, as they impose risk of noise in the TEM image accusation and NTA quantification. 11. Make sure to clean the sample feeding tube between samples, to avoid cross-contamination. This can be done by submerging the tube in PBS and drying it with a clean, lint-free tissue. 12. While performing NTA for quantification, adjust the focus until you get a sharp image of small nanoparticles in

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the background. To get accurate quantification with NTA, perform the calibration of NTA at regular intervals with polymer nanoparticles of defined size and concentration. Avoid using solvents in EVs preparation which includes albumin, as they impose risk of noise in the image accusation and NTA quantification. 13. The NTA is very sensitive to vibration. Make sure that you have no other hardware that could cause vibrations (e.g., centrifuge) on the same table. 14. Please make sure to wear suitable protective gear and follow local guidelines regarding the handling of uranyl acetate as it is very toxic. 15. If you experience high background, it could be useful to experiment with the settings of the rocking platform. For example, a vigorous setting for the washing step, but a milder one for antibody incubation steps, has improved the results in our experience.

Acknowledgments This work was supported by the Swedish Society of Medicine. SELA is supported by the Swedish Research Council (VR-Med) and the Swedish foundation of Medical Research (SSF-IRC). References ´ , Lo¨tvall J et al 1. Wiklander OPB, Brennan MA (2019) Advances in therapeutic applications of extracellular vesicles. Sci Transl Med 11: eaav8521 2. Andaloussi SEL, M€ager I, Breakefield XO et al (2013) Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 12:347–357 3. Otake K, Kamiguchi H, Hirozane Y (2019) Identification of biomarkers for amyotrophic lateral sclerosis by comprehensive analysis of exosomal mRNAs in human cerebrospinal fluid. BMC Med Genet 12:7 4. Caradec J, Kharmate G, Hosseini-Beheshti E et al (2014) Reproducibility and efficiency of serum-derived exosome extraction methods. Clin Biochem 47:1286–1292 5. L€asser C, Alikhani VS, Ekstro¨m K et al (2011) Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med 9(9) 6. Zhou H, Yuen PST, Pisitkun T et al (2006) Collection, storage, preservation, and

normalization of human urinary exosomes for biomarker discovery. Kidney Int 69 (8):1471–1476 7. Lo CA, Delevoye C, Gilles-Marsens F et al (2015) Exosomes released by keratinocytes modulate melanocyte pigmentation. Nat Commun 6:7506 8. Kotzerke K, Mempel M, Aung T et al (2013) Immunostimulatory activity of murine keratinocyte-derived exosomes. Exp Dermatol 22:650–655 9. Sjoqvist S, Kasai Y, Shimura D et al (2019) Oral keratinocyte-derived exosomes regulate proliferation of fibroblasts and epithelial cells. Biochem Biophys Res Commun 514:706–712 10. Sjo¨qvist S, Ishikawa T, Shimura D et al (2019) Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing. J Extracell Vesicles 8:1565264 11. Lobb RJ, Becker M, Wen SW et al (2015) Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles 1:1–11

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12. Nordin JZ, Lee Y, Vader P et al (2015) Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine 11:1–5 13. Stranska R, Gysbrechts L, Wouters J et al (2018) Comparison of membrane affinitybased method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma. J Transl Med 16:1

14. The´ry C, Witwer KW, Aikawa E et al (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7(1):1535750 15. Corso G, M€ager I, Lee Y et al (2017) Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography. Sci Rep 7:11561

Methods in Molecular Biology (2020) 2109: 45–53 DOI 10.1007/7651_2019_234 © Springer Science+Business Media New York 2019 Published online: 14 May 2019

Competitive Repopulation Assay of Long-Term Epidermal Stem Cell Regeneration Potential Carmen Segrelles, Karla Santos-de-Frutos, Jesu´s M. Paramio, and Corina Lorz Abstract Epidermal stem cells are responsible for normal tissue homeostasis and contribute to tissue regeneration during injury. Several assays measuring stem cell frequency and function can be used to assess epidermal stem cell potential. However, the ultimate assay that accounts for stemness is the capacity to sustain in vivo long-term tissue regeneration and maintenance. We can use this type of analysis to interrogate whether a specific genetic alteration (e.g., activation or inactivation of any gene thought to be involved in stem cell quiescence or proliferation) confers increased or decreased stem cell potential. Keywords Epidermal stem cells, Keratinocyte transplantation, Skin regeneration, Silicone chambers

1

Introduction Epidermal stem cells sustain normal tissue homeostasis, contribute to regenerate the epidermis during the process of wound healing, and participate in the formation of the epidermal appendages (hair follicles and sebaceous glands). Several methods have been developed to assess epidermal stem cell frequency and function. Murine epidermal stem cells can be detected in vivo using specific biomarkers (cytokeratin K15, CD34, CD71, integrin alpha-6, Lgr5) or labelling strategies based on their quiescent properties (detection of labelling retaining cells) [1, 2]. Their abundance can be quantified through directly counting positive cells in the tissue using immunohistochemistry or immunofluorescence [1, 2] or by harvesting whole epidermal cells [3] followed by flow cytometry analysis of epidermal stem cell marker surface expression [1, 2]. Epidermal stem cells can be isolated using fluorescent activated cell sorting (FACS) [4] or magnetic cell sorting (MACS) [5] and used to

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analyze stem cell potential [4] or molecular profiling (i.e., gene expression [2]). Additionally, epidermal stem cell potential can be assessed using unsorted epidermal keratinocyte populations in in vitro clonogenic assays [3] or in in vivo regeneration assays [6–8]. Long-term in vivo regeneration assays in which epidermal keratinocytes are harvested from the skin of neonatal mice [6–8] or from the dorsal skin of adult mice [1], mixed with dermal fibroblasts and transplanted into silicon chambers implanted onto the back skin of immunodeficient mice, constitute the most rigorous test of epidermal stem cell potential. In this context, after 6–7 weeks, only cells with long-term repopulating ability (true stem cells) will persist in the regenerated epidermis. Genetically modified mouse models (GEMMs) are a valuable tool to study the implication of a specific gene or genetic alteration in a biological function or a disease. Some genetic alterations may affect stem cell function. This aspect can be addressed using the abovementioned methods to measure stem cell frequency and function. Here we describe in detail a method to measure stem cell potential by means of a competitive long-term in vivo regenerative assay. We used this approach to answer the question whether in the K5-myrAktL84 [9] mice (myrAkt mice hereafter) the expression in basal keratinocytes of a permanently active form of Akt confers increased stem cell potential. The general guidelines for the method are as follows. First, we isolated epidermal keratinocytes from the dorsal skin of adult myrAkt mice, whereas the wild-type competitor keratinocytes were isolated from beta-actin-EGFP mice [10]. MyrAkt keratinocytes can be traced due to the fact that the myrAkt construction bears a hemagglutinin tag (myrAkt-HA keratinocytes from here on), and wild-type keratinocytes can be followed by the expression of the EGFP protein and the fluorescent properties it confers them (WT-GFP keratinocytes from here on). Adult keratinocytes were isolated using a method described in a previous edition of Epidermal Cells Methods and Protocols [5]. The cells can be stored frozen in liquid nitrogen until used. Next, the cell mixture (keratinocytes + fibroblasts) was transplanted into silicon chambers implanted onto the back skin of immunodeficient mice, and the transplant was left for 7 weeks. At this time, initially transplanted transient amplifying cells and their progeny are no longer present in the graft and only true long-term repopulating stem cells and their progeny remain. The description of the method and its use to measure stem cell frequency in epidermis has been described by Strachan and colleagues in a previous

Competitive Repopulation Assay of Epidermal Stem Cell Potential

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edition of Epidermal Cells Methods and Protocols [6] and in Stem Cells [7]. Here we describe a variant of this method that we used to answer the question whether the expression of a permanently active form of Akt in the keratinocytes of the basal layer of the epidermis [1] confers increased stem cell potential. In this context myrAktHA keratinocytes compete with WT-GFP keratinocytes to regenerate the epidermis in vivo, and their relative abundance is analyzed after 7–10 weeks using flow cytometry.

2 2.1

Materials Mice

1. K5-myrAktL84 mice [9]. 2. C57BL/6-Tg(CAG-EGFP)1Osb/J (beta-actin-EGFP) mice [10] (The Jackson Laboratory). 3. C57BL/6 wild-type mice (CIEMAT animal facilities). 4. ATHYM-Foxn1nu/nu (Athymic Nude) mice (Janvier Labs).

2.2 Reagents and Solutions

1. Sterile phosphate-buffered saline (PBS) (Sigma-Aldrich; cat. no. 806552). 2. Freezing media (for 100 mL): 75-mL heat-inactivated (56  C, 30 min) fetal bovine serum (FBS, Fisher Scientific; cat. no. SH3007103), 15-mL EMEM (BioWhittaker; cat. no. BE06-174G), 10-mL dimethyl sulfoxide (DMSO, SigmaAldrich; cat. no. 276855), 3% (w/v) glucose. 3. FACS buffer: PBS containing 5% heat-inactivated FBS and 0.09% (w/v) sodium azide, pH 7.4 (0.2 μM-pore filtered). 4. FACS-permeabilization buffer: PBS containing 5% heatinactivated FBS, 0.09% (w/v) sodium azide and 0.1% saponin, pH 7.4 (0.2 μM-pore filtered). 5. Cytofix/Cytoperm buffer: PBS containing 4% paraformaldehyde and 0.1% saponin. 6. Anti-HA-Alexa 647 antibody (Santa Cruz Biotechnology; cat. no. sc-805 AF647).

2.3

Tools and Tubes

1. For flow cytometry: 5-mL polypropylene round-bottom tubes (BD Falcon; cat. no. 352063). 2. 4  C centrifuge. 3. Samples were acquired with an LSRFortessa™ (BD Biosciences) flow cytometer and analyzed with FlowJo V7.6 software (Tree Star Inc.).

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Methods

3.1 Isolation of Adult Skin Keratinocytes and Neonatal Skin Fibroblasts: Storage and Retrieval of Frozen Stocks

The isolation of keratinocytes from the dorsal skin of adult mice was performed following the materials and method described in Epidermal Cells Methods and Protocols [5]. Similarly the isolation of fibroblasts from the skin of neonatal C57BL/6 wild-type mice was performed as reported in [6]. Keratinocytes and dermal fibroblasts were stored frozen in liquid nitrogen until the moment of their transplant into the silicon chambers. 1. Count the number of isolated keratinocytes or dermal fibroblast suspended in PBS (or in Cnt-07 keratinocyte cell grow media) using a hemocytometer and trypan blue to determine the percentage of dead cells (see Note 1). 2. Centrifuge at 190  g (see Note 2) for 7 min at 4  C, and suspend the pellet in freezing media. The optimal ratio is 1–5 million cells per 1 mL freezing media. 3. Store vials in liquid nitrogen until needed. 4. The day of the transplant rapidly thaw the number of vials with the amount of cells needed using a 37  C bath. 5. Wash the cells twice with ice-cold PBS to remove the freezing media, and suspend cells in PBS to a final volume of 8  107 cells/mL (4  106 cells per 50 μL PBS).

3.2 Chamber Implantation and Cell Transplantation

The materials and method used for the implantation of silicone chambers onto the back skin of immunodeficient mice are described by Strachan and Ghadially in Epidermal Cells Methods and Protocols [6]. For the competitive repopulation assay of epidermal stem cell potential, the procedure was modified as follows: 1. A total of 8  106 cells in a final volume of 100 μL PBS are transplanted in each silicon chamber. The cell mix is composed of (per 100 μL) 50 μL of fibroblasts (4  106 cells), 25 μL of myrAkt-HA test keratinocytes (2  106 cells), and 25 μL of WT-GFP competitor keratinocytes (2  106 cells) (Fig. 1a). 2. Prepare a master cell mix for your experiment allowing an excess of two to three animals. That is, if the number of animals to transplant is five, prepare at least 700 μL of cell mix. Keep the cell mix cold until transplanted. 3. The silicone chamber can be left in the animal for the whole duration of the experiment (7–10 weeks). Alternatively, we suggest removing the silicone chamber at week 2 after transplant just by pulling from it with forceps and leaving the circular gap to regenerate (see Note 3).

Competitive Repopulation Assay of Epidermal Stem Cell Potential

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B) KERATINOCYTE ISOLATION

A) CHAMBER IMPLANTATION AND CELL TRANSPLANTATION back skin keratinocytes 2ⴛ106 WT-GFP

+

2ⴛ106 myrAkt-HA

4ⴛ106 dermal fibroblasts

nu/nu receptor muscle fascia

7-10 weeks

C) FLOW CYTOMETRY

1

1

2

2

3

3

HA Alexa647

HA+

GFP+

GFP

Fig. 1 Competitive repopulating assay of long-term epidermal stem cell regeneration potential. (a) Adult telogen back skin keratinocytes from K5-myrAktL84 and beta-actin-EGFP mice were combined with dermal fibroblasts and implanted in silicon chambers that had been inserted onto the dorsal fascia of athymic nude mice. (b) After 7–10 weeks of the transplant skin from the recipient and from the graft is processed to isolate the epidermal keratinocytes. (c) Flow cytometry analysis of the keratinocyte populations labelled with anti-HA-Alexa647 (Table 1). Keratinocytes from the receptor can be gated out of the analysis (Tubes 1 and 2, quadrant Q4), and only HA+ (myrAkt) or GFP+ (WT) graft cells can be considered (quadrants Q1 and Q3, respectively)

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Table 1 Summary of the tubes and antibodies used Tube

Sample

Cells

Antibody

Populations

1

Negative control

Recipient keratinocytes

None

GFP /HA

2

Negative control

Recipient keratinocytes

15-μL anti-HA-Alexa 647

GFP /HA

3

Positive sample

Graft + recipient keratinocytes

15-μL anti-HA-Alexa 647

WT-GFP+ myrAkt-HA+ GFP /HA

3.3 Keratinocyte Isolation and Flow Cytometry Analysis of the myrAkt-HA Versus Competitor WT-GFP Long-Term Repopulating Keratinocytes and Their Progeny 3.3.1 Isolation of Keratinocytes from Grafts

After 7–10 weeks of transplantation, the keratinocytes were isolated from the dorsal skin of adult mice as described in Epidermal Cells Methods and Protocols [5] with some important considerations. 1. Cut off an area of dorsal skin that includes the graft plus some of the surrounding recipient skin. This surrounding skin from the recipient can be used to hold the tissue with the forceps during the process of scraping the epidermis (keratinocytes) with the blade of the scalpel (Fig. 1b). 2. Cut off an area of dorsal skin without the graft and proceed to isolate the keratinocytes. These cells from the recipient are the negative controls for cytometry (Tubes 1 and 2, Table 1, Fig. 1b). 3. The epidermis scraped from the graft and from the recipient must be processed in separate tubes following the indications described in “Isolation of keratinocytes from skin samples” in Epidermal Cells Methods and Protocols [5]. 4. At the end of the process, both samples should be suspended in 2-mL FACS buffer and maintained in ice unless otherwise indicated.

3.3.2 Preparation of the Control and Labelled Sample Tubes for Flow Cytometry Analysis

The procedure can be done in 5-mL polypropylene round-bottom tubes. Cells must be kept cold at all times except when otherwise indicated. 1. The tube from Subheading 3.3.1, step 1, is Tube 3 for flow cytometry analysis (Table 1, Fig. 1b). It contains transplanted keratinocytes as well as recipient keratinocytes. Transplanted keratinocytes can include WT-GFP-positive cells (WT-GFP+) and myrAkt-HA-positive cells (myrAkt-HA+). Cells expressing myrAkt with the HA tag are recognized by the anti-HAAlexa647 antibody. Recipient keratinocytes are GFP negative and HA negative (GFP /HA ). 2. Divide the content of the tube from Subheading 3.3.1, step 2, into two tubes. These are Tube 1 and Tube 2 for flow cytometry analysis (Table 1, Fig. 1b). Both tubes contain keratinocytes

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from the recipient that are negative for GFP and HA (GFP / HA ). 3. Centrifuge Tubes 1, 2, and 3 at 190  g for 7 min at 4  C to pellet the cells. Discard the supernatant, and resuspend the cells in 150 μL of Cytofix/Cytoperm buffer (see Note 4). 4. Incubate in Cytofix/Cytoperm buffer for 15 min at room temperature. 5. Centrifuge Tubes 1, 2, and 3 at 190  g for 7 min at 4  C to pellet the cells. Discard the supernatant and resuspend the cells in 50-μL FACS-permeabilization buffer. 6. Add 15 μL of anti-HA-Alexa647 antibody to Tubes 2 and 3 and incubate for 30 min. 7. Wash the cells by adding 5 mL of FACS-permeabilization buffer to the tubes and centrifuge at 190  g for 7 min at 4  C to pellet the cells. 8. Discard the supernatant and resuspend the cells in 300 μL of FACS buffer. 9. Keep tubes at 4  C and proceed to the flow cytometry analysis of the samples. 3.3.3 Overall Guidelines for the Flow Cytometry Analysis of the Samples

Flow cytometry analysis of the samples allows the quantification of the amount of myrAkt-HA+ cells versus the competitor WT-GFP+ cells in the graft. We describe the method for an LSRFortessa flow cytometer and FlowJo software to analyze the samples. 1. Cell debris is excluded from the analysis through gating in a forward versus side scatter linear scale plot (see Note 4). 2. Assessment of fluorescence is performed using logarithmic scale dot plots of 488Blue 530_30-A versus 640Red 670_30-A fluorescence, to detect GFP (GFP+ cells) and Alexa647 (HA+ cells) fluorescence, respectively. 3. Use Tubes 1 and 2 negative control samples to adjust the negative/positive threshold of GFP (Tube 1) and Alexa647 (Tube 2) fluorescence (Fig. 1c). 4. Use Tube 3 to quantify the number of myrAkt-HA+ cells (upper left quadrant, Q1) and WT-GFP+ cells (lower right quadrant, Q3) in the graft (Fig. 1c). 5. The number of double-positive events (upper right quadrant, Q2) should be close to zero (Fig. 1c). 6. Double-negative events (lower left quadrant, Q4) are cells from the recipient (Fig. 1c).

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Notes 1. Mix equal volumes of cell sample and Trypan blue solution, pipet the mixture gently a couple of times, and immediately place cells in a Neubauer or Malassez chamber for counting. Viable cells exclude trypan solution; however, after a time dye will also enter and positively stain viable cells. 2. 190  g is approximately 1200 rpm in a 12-cm radius floating rotor centrifuge. 3. The removal of the silicone chambers at week 2 allows the formation of a continuous skin layer with that of the recipient. This is useful for the full and optimal isolation of the keratinocytes that make up the graft. Since transplanted keratinocytes are tagged (HA or GFP), they can be easily distinguished from those of the recipient mouse. 4. Due to the fact that the cells are permeabilized to allow intracellular HA detection, it is not possible to gate live/dead cells based on DAPI exclusion. Instead gating based on FSC/SSC is used.

Acknowledgments Work in our laboratory is supported by FEDER cofounded ISCIII grants [CB16/12/00228 and PI18/00263] and Foundation of Fanconi Anemia grant [2018/127]. References 1. Segrelles C, Garcia-Escudero R, Garin MI, Aranda JF, Hernandez P, Ariza JM et al (2014) Akt signaling leads to stem cell activation and promotes tumor development in epidermis. Stem Cells 32(7):1917–1928. https:// doi.org/10.1002/stem.1669 2. Lorz C, Garcia-Escudero R, Segrelles C, Garin MI, Ariza JM, Santos M et al (2010) A functional role of RB-dependent pathway in the control of quiescence in adult epidermal stem cells revealed by genomic profiling. Stem Cell Rev 6(2):162–177. https://doi.org/10. 1007/s12015-010-9139-0 3. Wu WY, Morris RJ (2005) Method for the harvest and assay of in vitro clonogenic keratinocytes stem cells from mice. Methods Mol Biol 289:79–86 4. Blanpain C, Lowry WE, Geoghegan A, Polak L, Fuchs E (2004) Self-renewal, multipotency, and the existence of two cell

populations within an epithelial stem cell niche. Cell 118(5):635–648. https://doi.org/ 10.1016/j.cell.2004.08.012 5. Lorz C, Segrelles C, Garin M, Paramio JM (2010) Isolation of adult mouse stem keratinocytes using magnetic cell sorting (MACS). Methods Mol Biol 585:1–11. https://doi. org/10.1007/978-1-60761-380-0_1 6. Strachan LR, Ghadially R (2010) Limiting dilution analysis of murine epidermal stem cells using an in vivo regeneration assay. Methods Mol Biol 585:421–432. https://doi.org/ 10.1007/978-1-60761-380-0_29 7. Strachan LR, Scalapino KJ, Lawrence HJ, Ghadially R (2008) Rapid adhesion to collagen isolates murine keratinocytes with limited long-term repopulating ability in vivo despite high clonogenicity in vitro. Stem Cells 26(1):235–243. https://doi.org/10.1634/ste mcells.2007-0534

Competitive Repopulation Assay of Epidermal Stem Cell Potential 8. Schneider TE, Barland C, Alex AM, Mancianti ML, Lu Y, Cleaver JE et al (2003) Measuring stem cell frequency in epidermis: a quantitative in vivo functional assay for long-term repopulating cells. Proc Natl Acad Sci U S A 100(20):11412–11417. https://doi. org/10.1073/pnas.2034935100 9. Segrelles C, Lu J, Hammann B, Santos M, Moral M, Cascallana JL et al (2007)

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Deregulated activity of Akt in epithelial basal cells induces spontaneous tumors and heightened sensitivity to skin carcinogenesis. Cancer Res 67(22):10879–10888. https://doi.org/ 10.1158/0008-5472.CAN-07-2564 10. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407 (3):313–319

Methods in Molecular Biology (2020) 2109: 55–65 DOI 10.1007/7651_2019_241 © Springer Science+Business Media New York 2019 Published online: 04 June 2019

Quantification of Melanosome Transfer Using Immunofluorescence Microscopy and Automated Image Analysis Aishwarya Sridharan, S. Y. John Lim, Graham D. Wright, and Leah A. Vardy Abstract The study of skin pigmentation requires determining the rate of melanin production in melanocytes and quantifying the rate of melanosome transfer to keratinocytes. Here, we describe a method to quantify melanosome transfer using immunofluorescence microscopy coupled with automated image analysis of in vitro human melanocytes and keratinocytes in co-culture. In this method, the number of melanin capped keratinocyte nuclei is quantified. Keywords Melanin transfer quantitation, Melanosome transfer, Melanocyte keratinocyte co-culture, Image quantification

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Introduction Melanins are naturally occurring pigments which determine the skin, hair, and eye color in humans. The process of melanin production is called melanogenesis, which occurs in highly specialized cells called melanocytes. Melanin production and distribution is the result of a complex relationship between melanocytes and the surrounding keratinocytes in the epidermis of the skin. In melanocytes, melanogenesis occurs within membrane-bound organelles called melanosomes, where melanin is synthesized and packaged. Melanosomes are then transferred from melanocytes to keratinocytes where they reside above the nucleus and form a protective cap called the melanin cap. Due to the optical properties of melanin, it can absorb UVB light [1], protecting the nuclei from UVB radiation-induced DNA damage and thus reducing the risk of cancer.

Electronic supplementary material: The online version of this chapter (https://doi.org/10.1007/7651_2019_ 241) contains supplementary material, which is available to authorized users.

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When studying skin pigmentation, it is important to determine how the rate of melanin transfer from melanocytes to keratinocytes is modulated under different experimental conditions. Here, we describe an image-based screen, coupled with an automated image analysis pipeline, developed within the open-source software CellProfiler, to determine the percentage of melanin capped keratinocytes in a co-culture of melanocytes and keratinocytes. This protocol involves staining the melanosomes, using a specific fluorescently labelled antibody to visualize the formation of melanin caps over keratinocyte nuclei. This protocol can be used to study the effect of different drugs or treatments on melanosome transfer. We present a protocol for the analysis of UVB-induced changes in melanin cap deposition using co-culture of human melanocytes and keratinocytes.

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Materials

2.1 Cells for Coculture

1. Human epidermal light-pigmented melanocytes-adult (HemaLP) (Thermo Fisher Scientific, Cat no: C0245C) 2. Human immortalized keratinocytes, N/TERT-1 [2]

2.2 Reagents for Coculture

1. Melanocyte media: 254 media (Thermo Fisher Scientific, Cat. no: M254CF500) supplemented with HMGS-2 (Thermo Fisher Scientific, Cat no: S0165) 2. Keratinocyte media: keratinocyte serum-free media (K-SFM) supplemented with 25 μg/ml bovine pituitary extract (BPE), 0.2 ng/ml epidermal growth factor (EGF 1–53) (supplied as a combo, Thermo Fisher Scientific, Cat no: 17005042), and 0.3 mM CaCl2 3. 10 cm dishes for maintaining melanocytes and keratinocytes, 24-well tissue culture plates for co-culture 4. Trypsin-EDTA (0.25%), phenol red (Thermo Fisher Scientific, Cat. no: 25200056) for keratinocytes, and Trypsin/EDTA Solution (TE) (0.025% trypsin and 0.01% EDTA) (Thermo Fisher Scientific, Cat no: R001100) for melanocytes. 5. Neutralizing medium (DMEM10), DMEM (Thermo Fisher Scientific, Cat no: 11965084) with 10% fetal bovine serum (Thermo Fisher Scientific, Cat no: 10082147)

2.3 Reagents for Immunofluorescence

1. 4% paraformaldehyde (PFA) (pH 7.4) 2. 1
phosphate-buffered saline (PBS) 3. Permeabilisation (perm) buffer: 0.2% TritonX100 in PBS 4. Blocking buffer: 3% bovine serum albumin in PBS 5. Primary antibody: melanoma gp100 mouse monoclonal [NKI/beteb] (Abcam, Cat no: ab34165)

Quantification of Melanosome Transfer Using Immunofluorescence Microscopy. . .

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6. Secondary antibody: Alexa Fluor 488 donkey anti-mouse (Life Technologies Cat no: A21202) 7. Hoechst 33258 nuclear stain (Thermo Fisher Scientific Cat no: H3569) 2.4

Instruments

1. Stratalinker® UV Crosslinker or an equivalent instrument able to emit UVB radiation at 312 nm wavelength. 2. Motorized widefield fluorescence microscope. The instrument used in the present work is a motorized Olympus IX-81 microscope equipped with an Olympus LD-Achroplan 40
/0.6 objective lens, M€arzh€auser Wetzlar Scan IM motorized stage, Lumencore SOLA light engine solid-state light source, and a Photometrics CoolSNAP HQ2 CCD camera. The instrument is controlled using the MetaMorph software using the Screen Acquisition module.

2.5 Software for Analysis

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1. CellProfiler [3] for quantification (available from https://cel lprofiler.org)

Methods

3.1 UVB Treatment and Co-culturing Melanocytes and Keratinocytes

1. Culture melanocytes at a density of 5
103 viable cells/cm2 in 10 cm dishes in melanocyte media until they reach 80% confluence at 37  C and 5% CO2 in an incubator. 2. Culture N/TERT-1 at a density of 10
103 viable cells/cm2 in 10 cm dishes in keratinocyte media until they reach 80% confluence at 37  C and 5% CO2 in an incubator. 3. Discard the media and wash the cells with 10 ml PBS. 4. Expose the cells with 25 mJ/cm2 of UVB radiation, separately, with covers removed, in the presence of 3 ml PBS. Maintain equivalent unexposed additional plates for control. 5. Wash the cells with 10 ml of PBS. Trypsinize the N/TERT-1 cells for 5–10 min at 37  C using 1 ml of Trypsin-EDTA (0.25%), and trypsinize the Hema-LP cells for 1–3 min at room temperature using 1 ml of Trypsin/EDTA solution (TE) (0.025% trypsin and 0.01% EDTA). Neutralize the trypsin with 10 ml of DMEM10 and centrifuge N/TERT-1 at 290
g for 3 min and Hema-LP at 180
g for 7 min. Discard the media, count the cells, and plate UVB exposed and unexposed melanocytes and keratinocytes at a ratio 1:4 in the keratinocyte media with a density of 25
103 viable cells/cm2 in 24-well plates (see Note 1). 6. Co-culture the melanocytes and keratinocytes for 3 days at 37  C and 5% CO2 in an incubator without changing the media.

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3.2 Immunofluorescence Staining

1. On day 3, discard the media and wash the cells with PBS. 2. Fix the cells in 1 ml of 4% PFA at room temperature for 5 min. 3. Remove fixative and add 1 ml of ice cold perm buffer for 15 min at room temperature with mild agitation on an orbital shaker to permeabilize the membrane. 4. Remove perm buffer and add 1 ml of blocking buffer for 1 h at room temperature with mild agitation in an orbital shaker. 5. Remove blocking buffer and add 500 μl of primary antibody, 1:100 diluted in blocking buffer for 1 h at room temperature with no agitation. 6. Remove antibody solution and wash three times for 5 min each with 1 ml PBS with mild agitation on an orbital shaker. 7. Incubate with 500 μl of secondary antibody, 1:500 diluted in blocking buffer for 1 h at room temperature with no agitation. 8. Remove antibody solution and wash three times for 5 min each with 1 ml PBS with mild agitation in an orbital shaker. 9. Remove antibody solution and add 1 ml of Hoechst 33258 nuclear stain, 1:2000 diluted in blocking buffer for 10 min at room temperature with no agitation. 10. Remove the staining solution and wash three times for 3 min each with 1 ml PBS with mild agitation on an orbital shaker. 11. Add PBS and store at 4  C until imaging.

3.3 Image Acquisition

1. Configure a Screen Acquisition at 40
magnification with camera binning set to 2
2 using the widefield fluorescence microscope (see Note 2) as follows. (a) In the “Main” tab (Screen Shot 1), select “Multiple sites per well” and “Multiple wavelengths” to ensure 100 sites can be set (in the “Sites” tab) each with two channels (in the “Wavelengths” tab) for both Hoechst and gp100-Alexa Fluor 488. Configure autosaving such that the images are saved to the hard drive as the screen progresses under the “Directory” tab. (see Note 3). Hoechst was acquired in blue channel under the DAPI label, and gp100-Alexa Fluor 488 was acquired in green channel under GFP label.

Quantification of Melanosome Transfer Using Immunofluorescence Microscopy. . .

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(b) In the “Plate” tab (Screen Shot 2), select the appropriate type of plate (e.g., 24-well) and establish the center position of well A1 (to ensure accurate positioning of each well during the screen).

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(c) In the “Sites” tab (Screen Shot 3), establish the setting to acquire a minimum 100 images per well/condition to help ensure statistical significance of results. A 10
10 array of images with 10 μm spacing was used in the present work.

(d) In the “Wavelengths” tab (Screen Shot 4a, b), set appropriate exposure conditions independently for both the nuclear (Hoechst, blue) and melanosome (gp100, green) channels, based on the criteria of utilizing ~75% of the dynamic range of the detector (~3000 counts for our 12-bit camera) for the objects of interest (see Note 4). In our experiments we typically used ~100 ms exposure for the blue channel and ~900 ms for the green channel, but this will inevitably vary for different experimental setups.

Quantification of Melanosome Transfer Using Immunofluorescence Microscopy. . .

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(e) In the “Auto Focus” tab (Screen Shot 5), establish the settings to ensure focal accuracy per channel, per site within each well. A higher bin setting (e.g., 4
4) should be used during autofocusing (configured in the “Auto Focus” tab) to allow for shorter exposures and to minimize photobleaching. In our experiments we utilized MetaMorph’s Low Signal Algorithm in combination with (1) “Find” with a 150 μm search range and 5 μm accuracy (this is applied in the first site of each well), (2) “Wide” with a 150 μm search range and 3 μm accuracy, and (3) “Narrow” with a 50 μm search range and 1 μm accuracy.

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Since DAPI is brighter and has a more robust fluorescence signal, it was the first channel to be acquired with both a wide and narrow autofocus. For gp100 only a narrow autofocus was applied. 2. Click acquire and run the experiment (see Note 5). 3. Once acquisition is complete, make an external backup of the data (e.g., network drive or external hard drive). 3.4 Image Quantitation

1. In the present study, images were analyzed using the opensource software, CellProfiler (version 2.2.0). This software is designed and optimized for high-throughput batch analysis. Within the software, images are processed by modules arranged into a pipeline. The pipeline developed here is described below. For more detailed explanation of each module, refer to the software manual (available online from https://cellprofiler. org). The overall flow of our pipeline is to identify specifically the keratinocyte nuclei and then analyze whether they have a melanin cap associated with them. A count is then made and intensities measured. The pipeline that was used for our experiment is attached along with this protocol for reference. 2. The first step of the pipeline loads the images into the software using “LoadImages.” The two channels were differentiated through the use of their filename suffixes “w1.TIF” (for wavelength 1, Hoescht) and “w2.TIF” (for wavelength 2, gp100).

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3. “CorrectIlluminationCalculate” creates an illumination function to correct for uneven illumination in nuclei image. The software allows iterative reviewing and fine-tuning of the parameters (e.g., block size and Gaussian smoothing radius) used for calculating the illumination function (see Note 6). “CorrectIlluminationApply” then applies the calculated illumination function to the nuclei image. This step helps to improve the subsequent segmentation results. 4. The module “IdentifyPrimaryObjects” is used to identify all the stained nuclei found in the co-culture (both keratinocyte and melanocytes) from the Hoechst images (see Notes 7 and 8). 5. Steps 2, 3, and 4 are repeated but applied to the gp100 images to correct for uneven illumination and to identify the melanocytes (bright green cells) (see Note 8). 6. To identify specifically the keratinocyte nuclei and exclude the melanocyte nuclei, “ConvertObjectToImage,” “MeasureObjectIntensity,” and “FilterObjects” are used. “ConvertObjectToImage” converts the object (whole melanocyte cells) to a binary image, and “MeasureObjectIntensity” measures the mean edge intensity for nuclei regions. “FilterObjects” identifies the keratinocytes nuclei based on the criteria of having a mean nucleus edge intensity