ausblenden:
Sprache(n):
eng - English
Datum:
2009-12-17
Publikationsstatus:
Angenommen
Seiten:
141
Ort, Verlag, Ausgabe:
Berlin : Freie Universität Berlin
Inhaltsverzeichnis:
Acknowledgement iii
Contents iv
Abbreviations viii
List of Figures xi
List of Tables xiii
Abstract xv
Abstract (German) xvi
1 Introduction 1
1.1 Human Embryonic Stem Cells (hESCs) and induced Pluripotent Stem Cells (iPSCs) – history and applications . . . . . . . . . . . . . . . . . . 1
1.2 Endoderm development in vivo . . . . . . . . . . . . . 2
1.2.1 Nodal signaling pathway – molecular mechanism of endoderm specification . . . . . . . . . . . . . . . . . . 3
1.2.2 Role of Wnt signaling in endoderm development . . . . . . . . . 7
1.2.3 Nodal and Wnt signaling in mesendoderm development - separating endoderm from mesoderm . . . . . . . . . . . . .7
1.3 Endoderm development in vitro - creating endoderm from human ES cells 8
1.3.1 Expandable endodermal progenitors . . . . . . . . . .9
1.4 Hepatogenesis in vivo. Regulatory phases of early liver development . . 10
1.4.1 Specifying the hepatic lineage by cardiac mesoderm 10
1.4.2 Specifying the hepatic lineage by septum transversum mesenchyme 12
1.4.3 Hepatic gene induction in embryonic endoderm cells .13
1.4.4 Growth of hepatic endoderm into the liver bud . . . 14
1.4.5 Other molecules affecting hepatogenesis . . . . . . 16
1.5 Transcriptional regulation of hepatogenesis . . . . . 16
1.5.1 Transcriptional regulation of early hepatogenesis . 16
Contents v
1.5.2 Transcriptional regulation of hepatic maturation . .18
1.6 Hepatogenesis in vitro - differentiation of human ES cells into hepatocytelike cells . . . . . . . . . . . . . 19
1.6.1 Induction of hepatic cell fate via addition of soluble factors (cytokines, growth factors, hormones) . . . . . . 19
1.6.2 Induction of hepatic cell fate via genetic alterations 20
1.6.3 Human ES cells differentiation into hepatocyte-like cells – protocols - overview . . . . . . . . . . . . . . 20
1.6.4 Present characterization strategies of human ES-derived hepatocytelike cells and their restrictions . 24
1.7 Human ES cells-derived hepatocyte-like cells – applications . . . . . . . 26
1.7.1 Regenerative medicine . . . . . . . . . . . . . . . 26
1.7.2 Pharmacology (hepatotoxicity tests) . . . . . . . . 27
1.8 Aim of this work . . . . . . . . . . . . . . . . . . 28
2 Materials and Methods 30
2.1 Mouse Embryonic Fibroblasts Culture . . . . . . . . . 30
2.1.1 Isolation of Mouse Embryonic Fibroblasts (MEFs) . . 30
2.1.2 Cryopreservation (freezing) of MEFs . . . . . . . . 31
2.1.3 Thawing and maintaining of MEFs . . . . . . . . . . 31
2.1.4 Inactivation and plating of MEFs (Feeders preparation) 31
2.2 Human ES cells culture in feeder-free conditions . . 31
2.2.1 The Human Basic Fibroblast Growth Factor preparation 32
2.2.2 Conditioned medium preparation . . . . . . . . . . .32
2.2.3 Measurement of Activin A in Conditioned Medium . . .32
2.2.4 Preparation of Matrigel R-coated plates . . . . . . 33
2.2.5 Passage of human ES cells on Matrigel R 33
2.2.6 Daily maintenance of human ES cells in feeder-free culture . . . 34
2.2.7 Freezing human ES cells . . . . . . . . . . . . . . 34
2.2.8 Thawing human ES cells . . . . . . . . . . . . . . .34
2.3 Defined Media to growth human ES cells . . . . . . . .35
2.4 Human ES cells differentiation . . . . . . . . . . . 35
2.4.1 Formation of Embryoid Bodies (EBs) . . . . . . . . 35
2.4.2 Human ES cells differentiation into hepatocyte-like cells . . . . . 35
2.5 Culture of hepatocellular carcinoma (HepG2) and human foreskin fibroblasts(HFF) cells . . . . . . . . . . . . . 38
2.6 Protein isolation . . . . . . . . . . . . . . . . . . 38
2.6.1 Protein quantification (Bradford Assay) . . . . . . 38
2.6.2 SDS-PAGE gel electrophoresis . . . . . . . . . . .. 39
2.6.3 Western blotting . . . . . . . . . . . . . . . . . .39
2.7 RNA isolation and reverse transcription-polymerase chain reaction . . . 40
2.7.1 Real-time polymerase chain reaction (Real-Time PCR) 40
2.8 Immunocytochemistry . . . . . . . . . . . . . . . . . 40
2.9 Genomic DNA isolation . . . . . . . . . . . . . . . . 42
2.9.1 RNA and genomic DNA quantification . . . . . . . . 42
2.9.2 Agarose gel electrophoresis . . . . . . . . . . . . 42
2.10 Illumina 8-Sample BeadChip hybridisation (Gene expression profiling) . 42
2.11 Data analysis . . . . . . . . . . . . . . . . . . . 43
2.12 Functional Assays for hepatocyte-like cells derived from human ES cells 44
3 Results 45
3.1 Optimization of human ES cells culture conditions . . 45
3.1.1 Feeder-dependent conditions . . . . . . . . . . . . 45
3.1.2 Feeder-free culture . . . . . . . . . . . . . . . . 46
3.1.3 Defined Media based culture conditions . . . . . . .46
3.2 Characterization of human ES cell lines (stem cell markers, in vitro differentiation)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.3 Derivation of endoderm lineage from human ES cells . .49
3.4 Multi-stage human ES cells hepatic differentiation . 51
3.5 Directed differentiation of definitive endoderm to hepatocytes . . . . . . 52
3.6 Human ES cell-derived hepatocyte-like cells exhibit hepatocyte-like functions
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.7 Global data analysis . . . . . . . . . . . . . . . . 61
3.7.1 Global gene expression and hierarchical cluster analysis . . . . . 61
3.7.2 Detailed analysis of common expressed genes in human ES cellsderived
hepatocyte-like cells and fetal liver . . . . . . . . . . 63
3.8 Time-resolved analysis of the hepatogenesis using reproducible in vitro system . . . . . . . . . . . . . . .72
3.8.1 Tracing genes expression patterns . . . . . . . . . 74
3.9 Sodium Butyrate (NaB) and its role in the differentiation protocol . . . 81
3.10 Comparison with available data sets . . . . . . . . 83
4 Discussion 89
4.1 Differentiation protocols imitate hepatogenesis in vivo 89
4.2 Epigenetic modifications during human ES cells differentiation into hepatocytelike cells . . . . . . . . 90
4.3 Maturation state of human ES cells-derived hepatocyte-like cells . . . . 92
4.4 Global gene expression profiling approach as a tool to trace dynamic changes during the process of hepatic differentiation . . . . . . . . . . . 93
5 Conclusion 95
References 96
Publications 114
Curriculum Vitae 115
A Supplementary protocols 117
A.1 MEF culture medium (components to make 500ml of media) 117
A.2 MEFs freezing media . . . . . . . . . . . . . . . . .117
A.3 Human Embryonic Stem Cells Media (UM, unconditioned medium) . . 117
A.4 Human ES cells freezing media . . . . . . . . . . . .117
A.5 Dispase solution . . . . . . . . . . . . . . . . . . 118
A.6 -Mercaptoethanol solution for hESCs media . . . . . 118
A.7 HepG2 and HFF culture medium . . . . . . . . . . . . 118
A.8 Defined (N2B27) culture media to growth hESCs (components to make 50ml) . . . . . . . . . . . . . . . 118
A.9 Buffers for SDS-PAGE gel electrophoresis . . . . . . 118
A.10 Buffers for Western blotting . . . . . . . . . . . .119
B Supplementary tables 121
Art der Begutachtung:
-
Identifikatoren:
eDoc: 451981
Art des Abschluß:
Doktorarbeit