Generation of hepatocyte like cells from mouse embryonic stem (ES) and bone marrow (BM) cells

Generation of Hepatocyte-like cells from Mouse Embryonic Stem (ES) and Bone Marrow (BM) Cells YIN YIJUN NATIONAL UNIVERSITY OF SINGAPORE 2004 Generation of Hepatocyte-like cells from Mouse Embryonic Stem (ES) and Bone Marrow (BM) Cells YIN YIJUN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF MEDICINE DEPARTMENT OF OBSTETRICS & GYNAECOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2004 DECLARATION This is to declare that the work reported in this thesis was done solely by the undersigned candidate, and has not been submitted to any University for admission to a degree, diploma or other qualification Yin Yijun i ACKNOWLEDGEMENT I would like to express my sincere gratitude to my supervisors Dr Sai Kiang Lim (Genome Institute of Singapore) and Professor Soon Chye Ng (Department of Obstetrics & Gynaecology, NUS) for all of their supervision, assistance, encouragement and support during the course of this project I would like to thank Dr Manuel Salto-Tellez (Department of Pathology, NUS) for experimental consultation Additional thanks must be extended to laboratory colleagues past and present – Yew Koon Lim, Shee Han Lee, Keng Suan Yeo, and others for their help with my experiment during my study Worth mentioning is my wife, Yonghong I would like to say genuinely gratefulness for her encouragement, understanding and support during my study Further extension of my appreciation is to my parents for their spiritual support This study was supported by a postgraduate scholarship awarded by The University of Singapore ii TABLE OF CONTENTS DECLARATION i ACKNOWLEDGEMENT ii TABLE OF CONTENTS iii LIST OF FIGURES vii LIST OF TABLES .x ABBREVIATION .xi CONFERENCE AND PAPER xiv ABSTRACT OF THESIS ENTITLED xv General Statement 1 Liver development Causes and treatment of liver failure 3 Particular cell response to different liver injury 4 Definition and classification of stem cells Part I AFP+, ES Cell-Derived Cells Engraft and Differentiate into Hepatocyte-like cells in vivo Abstract Introduction 10 1.1 General statement 10 1.2 Generation and experimental application of ES cell derivatives 10 1.3 Challenges in isolating ES cell derivatives 15 1.4 Specific challenges in isolating ES-derived hepatic progenitor cells 16 1.5 The objectives of the study 17 iii Materials and Methods 18 2.1 Gene Targeting 18 2.2 Maintenance of recombinant ES (rES) cell without differentiation 19 2.3 rES cell differentiation 19 2.3.1 Differentiation in vitro using suspension culture method (Robertson 1987)19 2.3.2 Differentiation in vitro using a modified two-step suspension culture method (Wiles 1993) 20 2.3.3 Analysis and assortment of GFP expressing cells in differentiated rES cells by FACS 21 2.3.4 Fluorescent-microscope analysis of rES and D12 differentiated rES cells 21 2.4 Partial Hepatectomy (PHx) and Cell Transplantation 21 2.5 Collection of tissues 24 2.6 β-gal assay – X-gal staining 25 2.7 Immunohistochemistry (IHC) 25 2.8 DNA and RNA extractions and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analyses 26 2.9 Immunoprecipitation 29 2.10 Western blot analysis 30 Results 32 3.1 Modification of ES cells by homologous recombination of GFP gene with AFP locus 32 3.2 AFP/GFP expression in in vitro differentiated rES cells 34 3.3 Engraftment and differentiation of GFP+ cells to hepatocyte-like cells 39 iv Discussion 47 Part II Derivation of Hepatocyte-like cells from BM cells without Hematopoietic Reconstitution 52 Abstract 52 Introduction 54 1.1 General Statement 54 1.2 Non-hematopoietic cell derivatives from BM cells 54 1.3 The purpose of this study 59 Materials and Methods 60 2.1 Preparation of mononuclear cells from BM 60 2.2 Experimental Transplantation 61 2.3 Isolation of NPCs from livers by using modified conventional two-step collagenase perfusion (Seglen 1973) 63 2.4 Immunohistochemical staining of liver paraffin sections 64 2.5 Assay for serum ApoE in transplanted ApoE-deficient mice by western blot analysis 65 2.6 Analysis of NPCs by fluoresence microscopy 65 2.7 Isolation of Thy-1+ and Thy-1− cells from Rosa26 BM cells by MACS 66 Results 67 3.1 Donor BM cell engraftment in PHx mice 67 3.2 Characterization of BM-derived cells in recipient livers with surface markers 67 3.3 Engrafted BM-derived cell differentiation into hepatocyte-like cells in second PHx recipients 70 v 3.4 Derivation of hepatocyte-like cells from BM cells in mice with chronic hepatic deficiency induced by Fas-mediated apoptosis 73 3.5 BM cell type in generating engrafted Thy-1+ cells in recipient livers 76 Discussion 77 Future study 84 Summary 86 References 87 vi LIST OF FIGURES Part I Figure GFP recombination at the AFP locus Figure Southern blot hybridization of genomic DNA from WT and recombinant ES (rES) cell clones Figure 33 33 Fluorescent microscopy of GFP expression in EBs during in vitro differentiation using suspension culture method 36 Figure rES-derived EBs at day 36 Figure FACS analyses of AFP/GFP rES cells by using a two-step in vitro differentiation method Figure Fluorescent microscopy results in undifferentiated and differentiated ES cells Figure 41 X-gal and H&E staining showed the teratoma formed by injection of rES cells into PH× mice Figure 10 38 The Rosa26 mouse liver (a transgenic mouse) and 129sv mouse liver (a wild type mouse) were stained with X-gal Figure 37 RT-PCR pictures demonstrate the mRNA expressions of AFP and Albumin in recombinant and differentiated ES cells Figure 37 41 Pictures showed the X-gal staining of liver sections from GFP+ cell transplanted Rosa26 mice that ubiquitously express beta-galactosidase in all tissue (X-gal staining positive) 44 vii Figure 11 Genomic DNA was extracted and amplified by PCR from rES-derived AFP/GFP+ cell transplanted mouse liver samples Figure 12 44 Immunohistochemical staining showed that albumin and ApoE expressions could be demonstrated in X-gal negative cells that should be derived from transplanted AFP/GFP+ cells Figure 13 45 RT-PCR analysis demonstrated the presence of ApoE or haptoglobin mRNA in ApoE- or Hp-deficent mice after GFP+ cell transplantation 45 Figure 14 Immunohistochemical staining demonstrated the presence of ApoE in ApoE-deficent mice after GFP+ cell transplantation Figure 15 Western-blot showed ApoE protein in ApoE-deficient mouse after AFP/GFP+ cell transplantation Figure 16 46 46 Immunoprecipitation and Western-blot analyses showed haptoglobin protein in Hp-deficient mouse with LPS treatment after GFP + cell transplantation 46 Part II Figure liver section from Rosa26 BM transplanted C57BL/6J mice were perfused and fixed glutaraldehyde and stained by X-gal staining and H&E Figure 69 Immunofluroscent pictures overlaying with transmission picture of β galactosidase and antibody staining of isolated NPCs from Rosa26 BM transplanted C57BL/6J mice antibody Figure 69 NPCs are isolated from MTnLacZ mouse BM-transplanted livers and double stained with β-gal substrate and Thy-1 antibody 70 viii 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Nat Biotechnol 19(12): 1129-33 106 .. .Generation of Hepatocyte- like cells from Mouse Embryonic Stem (ES) and Bone Marrow (BM) Cells YIN YIJUN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF MEDICINE DEPARTMENT OF. .. ENTITLED Generation of Hepatocyte- like cells from Mouse Embryonic Stem (ES) and Bone Marrow (BM) Cells The paucity of hepatocytes available for hepatocyte replacement therapy requires consideration of. .. type of stem cell is the adult stem cells Like ES and EG cells, adult stem cells can self-renew, but unlike ES and EG cells, they give rise to specific sets of mature cell types Adult stem cells