Characterization of the ground state Pluripotency in Planarian Dissertation zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakult¨aten der Universit¨atdes Saarlandes von Thao Anh Tran M.Sc. Saarbr¨ucken 2019 Tag des Kolloquiums: 30.07.2019 Dekan: Prof. Dr. Gregor Jung Berichterstatter: Prof. Dr. Heiko Zimmermann Prof. Dr. J¨ornWalter Vorsitz: Prof. Dr. Uwe Walldorf Akad. Mitarbeiter: Dr. Judith Becker ACKNOWLEDGEMENTS I am grateful to my supervisor, Dr. Luca Gentile for his skillful guidance and his stoic patience with my mistakes. We had spent a long period of time cross-checking all the errors of many experiments and his positive attitude helped me go through and solve all of those problems. I could not have reached this state without his great support. I would like to express my sincere thanks to Professor Heiko Zimmermann for his support in both financial and academic aspects. Thanks for spending time to organize a presentation club, giving valuable advice to students' projects and improving our presentation skills. Special thanks to Professor J¨ornWalter for his supervision. As an expert in the genetic field, his feedbacks were very helpful and my thesis could not have been completed without his suggestion in using RNA sequencing for comprehensive molecular information. I would also like to acknowledge the supports from Dr. Kerstin Bartscherer and her lab members for an intensive training course, especially Henning Schmitz for his kindness to explain all of my questions with dozen of back and forth emails. I really appreciate the opportunity working with Dr. Karen Smeet, Annelies Wouters, Dr. Yvonne Kohl, Michelle Hesler. Thanks for their contributions in this thesis. I am also thankful to Professor Hagen von Briesen and Dr. Julia Neubauer for their supports and advice on my projects. I express my appreciation to Dr. Hannes Drexler, Annemarie Jungmann and Dr. Karl Nordstr¨omfor their help in Mass Spec and RNA sequencing. It was a great pleasure to work with Michael Gepp, Ina Meiser, Sabine M¨uller, Katharina Schmidt, Julia Majer. I would like to recognize the important roles of Linda Elberskirch, Soon Kyung, Andr² Schulz, Benjamin Fisher, Alba Romero, Anna Meier who made IBMT more joyful. Many thanks to Duy and my friends in Vietnam and Saarbr¨ucken for their supports and immediate help whenever I needed. My deepest appreciation belongs to my family for their love. Saarbr¨ucken,2019 - Thao Anh Tran ABSTRACT Planarian is an excellent model for regenerative studies due to its ability to regenerate the entire body from a tiny fragment. Thanks to a large number of adult stem cells, the mechanism of stem cell compartment could be studied in vivo, in the context of an adult organism. How pluripotent stem cells commit to prime states to regenerate new tissues without tumor formation? To answer this fundamental question, it is essential to characterize the ground state of the stem cell compartment. In this project, we proposed a novel surface antibody 6-9.2 that isolated pluripotent cells from a heterogeneous stem cell population. We concluded that the 6-9.2 antigen is a new biomarker for stem cell commitment and 6-9.2 antibody could be a useful tool for a live cell tracking method. Mass spectrometry and RNAi data showed a high relation of the 6-9.2 antibody with the transmembrane protein TMEM215/128. Knocking down of TMEM128/215 significantly affected the stem cell population and impaired the planarian regeneration and homeostasis. Our data contributed to defining the fluidic identity of stem cell transition. The gained knowledge will lead to a better understanding of regeneration and stem cell biology. ZUSAMMENFASSUNG Planarier gelten als Meister der Regeneration. Aufgrund eines großen Vorrats pluripotenter Stammzellen k¨onnen sie ihren gesamten K¨orper aus einem winzigen Fragment regenerieren. Planarien sind derzeit die einzigen Bilateria, bei denen pluripotente Stammzellen in vivo im Kontext zu einem erwachsenen Organismus untersucht werden k¨onnen. Wie erneuern oder differenzieren sich pluripotente Zellen, ohne dass sich ein Tumor bildet? Um diese grundlegenden Frage zu beantworten, ist es wichtig, den Grundzustand der Stammzellen zu charakterisieren. Hierzu wird in der vorliegenden Arbeit ein neuartiger Oberfl¨achenantik¨orper mit der Bezeichnung 6-9.2 vorgeschlagen. Wir konnten zeigen, dass das 6-9.2-Antigen ein neuartiger Biomarker f¨ur das Stammzellkompartiment ist und der 6-9.2-Antik¨orper ein n¨utzliches Werkzeug f¨ur die Beobachtung lebender Zellen sein k¨onnte. Daten aus massenspektrometrischen Analysen zeigten den engen Bezug dieses Antik¨orpers zum Transmembranprotein TMEM128/215. Zusammenfassend l¨asstsich sagen, dass die Daten aus diesem Projekt zur Entwicklung eines Modells zur Beobachtung lebender Zellen verwendet werden k¨onnen,was zu einem besseren Verst¨andnisder Regeneration und der Stammzellbiologie f¨uhrt. Project Scope The main objective of my Ph.D project is to unravel the complex biology behind planarian of pluripotent stem cells. Tracing the fate of individual stem cells during homeostasis and regeneration in the only animal that posses pluripotent stem cells in the adult will help identifying conserved mechanisms of pluripotency-based cell turnover and regeneration. Project objectives To achieve this goal, we need to find a novel way to characterize stem cell sub-populations and developing an in vivo tracking method via non-genetic labeling (e.g. FluoSpheres). The project included three major steps: - The development of surface antibody to recognize stem cell sub-populations. - A cell labeling system to transplant into planarians. - An immobilization method to trace fluorescent cells in vivo. Project achievement - We successfully developed a surface antibody 6-9.2 that can recognize sub- populations of stem cells: naive and committed stem cells. - We found the putative name for 6-9.2 antigen, the transmembrane protein TMEM215/128 by Mass Spec and RNAi methods. - We developed a labeling system using fluospheres that are biocompatible for planarians. - We showed the possibility to immobilize planarian in alginate hydrogels for at least five days and did not affect the animal's homeostasis and regeneration. - Our data provided an ability to trace the fluorescent signals of different stem cell sub-groups in planarians. - For applied researches, planarians can be used as a model system for tumorigenesis. We found that MMPB plays an essential role in controlling tumor formation in planariran. - Planarians were also used for environmental toxicity assessment of iron oxide nanoparticles (IOPs). Our data showed the non-toxic effect of IOPs and encourage the use of these particles in medical applications. iv Contents Project Scope iv Contentsv List of Figures ix List of Abbreviations xi 1 Introduction and Motivation1 1.1 Planarian: An excellent model for regenerative medicine research .1 1.2 Planarian overview . .6 1.2.1 What planarians are? . .6 1.2.2 Planarian structures . .7 1.2.3 Planarian neoblasts . .8 1.2.4 Planarian germ cells . 11 1.3 Planarian stem cell compartment - State of the Art- . 11 1.3.1 The heterogeneity of neoblast . 11 1.3.2 Pathway underlying pluripotent state transition . 15 1.3.3 A lineage CLOUD for neoblast . 17 1.3.4 Tissue remodeling in planarian . 21 1.3.5 Planarian stem cell compartment is more and more similar to other models . 22 2 Materials and Methods 25 2.1 Materials . 25 2.1.1 Chemicals and reagents . 25 2.1.2 Antibodies . 27 2.1.3 Solutions and buffers . 28 2.1.4 List of primers used in this study . 32 v 2.1.4.1 List of primer sequences for qPCR . 32 2.1.4.2 List of primers for RNAi . 36 2.2 Methods . 37 2.2.1 Planarian maintenance . 37 2.2.2 Planarian irradiation . 37 2.2.3 Alginate preparation . 37 2.2.4 Cell dissociation and Flow cytometry . 37 2.2.5 Live immune staining with 6-9.2 antibody and cell sorting byFACS............................ 38 2.2.6 Immobilization using Alginate hydrogels . 38 2.2.7 RNA extraction . 39 2.2.8 Reverse Transcriptase PCR – cDNA synthesis . 39 2.2.9 Polymerase chain reaction (PCR) . 39 2.2.10 Quantitative Real-Time PCR (qRT-PCR) . 39 2.2.11 RNAi synthesis . 40 2.2.12 In vitro transcription . 40 2.2.13 Agarose gel electrophoresis . 41 2.2.14 RNAi injection . 41 2.2.15 RNA sequencing (RNAseq) . 42 2.2.16 Whole mount in situ hybridization (WISH) . 45 2.2.17 Fluorescent in situ hybridization (FISH) . 46 2.2.18 Protein extraction . 47 2.2.19 Western Blot . 47 2.2.20 Protein membrane protein isolation . 47 2.2.21 Immunoprecipitation . 48 2.2.22 Mass Spectrometry . 49 2.2.23 Bioinformatics . 53 2.2.24 Statistical analysis . 55 3 Results 57 3.1 Stem cell classification using novel surface antibody 6-9.2. 57 3.2 Surface antibody 6-9.2 recognized committed stem cells in neoblasts X1 FSC/SSC population. 58 3.3 The characterization of 6-9.2 antigen. 68 3.4 Live cell tracking method using 6-9.2 surface antibody. 81 3.5 Planarian - From basic science to applied researches . 85 vi CONTENTS 3.5.1 Planarian - A beautiful model for environment toxicological study . 85 3.5.2 Planarian - A suitable model for tumorigenesis . 91 4 Discussion and Future direction 97 References 105 vii viii List of Figures 1.1 Planarian Schmidtea mediterranea anatomy. .8 1.2 Planarian mucus and cilia under the Scanning electron microscope (SEM). .9 1.3 Planarian regeneration. .9 1.4 t-distributed stochastic neighbor embedding (t-SNE) for eye markers. 19 1.5 Planarian stem cell commitment models. 20 1.6 A lineage CLOUD for neoblasts on the badlands landscape. 20 2.1 Planarian injection experiment. 42 3.1 The overview of live cell tracking method using surface antibody and fluorescent bead-based labeling approach.