Contents -- Protocol list -- Abbreviations -- Contributors -- 1 Introduction -- 1 Aims and scope of the volume -- 2 Control of differentiation -- 3 Future perspectives -- 2 Procedures for deriving ES cell lines from the mouse -- 1 Introduction -- 2 Derivation of mES cell lines -- 3 General principles of mES cell culture -- 3.1 Culture media and supplements -- 3.2 Preparation of MEFs -- 3.3 Preparation of feeder layers -- 3.4 Fetal-calf serum (FCS) -- 3.5 Routine culture of mES cells -- 4 Methods for the derivation of mES cell lines -- 4.1 Derivation of mES cells from intact blastocysts -- 4.2 Derivation of mES cells from isolated epiblast -- 5 Validation of new mES cell lines -- 5.1 Karyotype -- 5.2 Pluripotency -- 6 Female mES cell lines -- 3 Production of ES cell-derived mice -- 1 Introduction -- 2 Maintenance of mES cell lines and embryo culture -- 3 Reconstituting tetraploid embryos with mES cells -- 3.1 Tetraploid embryos -- 3.2 Mouse ES cell lines for tetraploid-embryo reconstitution -- 3.3 Methods for reconstituting tetraploid embryos with mES cells -- 4 Hyperthermic treatment of diploid host blastocysts -- 5 Nuclear transfer using mES cells as karyoplasts -- 5.1 Mouse strains for mES-cell NT -- 5.2 Cell-cycle synchronization in mES cells -- 5.3 Enucleation of MII-phase oocytes -- 5.4 Activation of nuclear-transfer embryos -- 6 Analysis of distribution of mES-cell derivatives in chimeras -- 7 Perinatal mortality and developmental abnormalities in mES cell-derived mice -- 4 Generating animal models of human mitochondrial genetic disease using mouse ES cells -- 1 Background -- 1.1 Mitochondrial genetics and biochemistry -- 1.2 Deleterious mtDNA mutations in hereditary disease -- 1.3 Generation, identification, and recovery of mtDNA mutations from mice -- 2 Introduction of mtDNA mutations into the female mouse germline.

2.1 Screening for mutations in mtDNA -- 2.2 Preparation of ρ[sup(°)] cells for use as recipient cells for mtDNA transfer -- 2.3 Preparation of cytoplasts from cell lines harbouring a mtDNA polymorphism -- 2.4 Generation of brain synaptosomes from mice harbouring mtDNA polymorphisms -- 2.5 Recovery of mtDNA mutations by electrofusion of cytoplasts or synaptosomes with ρ[sup(°)] or mES cells -- 2.6 Culture of mES cells -- 2.7 Depletion of mitochondria in mES cells by treatment with R6G -- 2.8 Culture and selection of trans-mitochondrial mES cybrids -- 2.9 PCR-based allele genotyping of the nuclear genome of cybrids -- 2.10 Use of X-linked GFP transgenic mice to select female blastocysts for microinjection with trans-mitochondrial mES cells -- 2.11 PCR-based allele genotyping of mice -- 3 Phenotypic analysis of trans-mitochondrial, chimeric mice -- 4 Concluding comments -- 5 Controlling the differentiation of mouse ES cells in vitro -- 1 Introduction -- 2 Experimental rationale -- 3 Methods -- 3.1 General considerations for controlled mES-cell differentiation -- 3.2 Recommendations for CDM preparation and experimental design -- 3.3 Neuroectoderm differentiation -- 3.4 Mesoderm and haematopoietic differentiation -- 3.5 Mesoderm and neuroectoderm differentiation -- 4 Evaluation of mES-cell differentiation -- 5 RNA isolation -- 6 Conclusions -- 6 In vitro differentiation of mouse ES cells into muscle cells -- 1 Introduction -- 2 Culture of mES cell lines for differentiation into muscle cells -- 3 Differentiation of mES cells into muscle cell lineages -- 3.1 Differentiation of mES cells into cardiac muscle cells -- 3.2 Differentiation of mES cells into skeletal myocytes -- 3.3 Differentiation of mES cells into smooth-muscle cells -- 4 Isolation techniques for differentiated muscle-cell derivatives.

4.1 Genetically modified mES cells for enrichment of muscle-cell derivatives -- 5 Assessing muscle-gene expression during in vitro differentiation of mES cells -- 5.1 RNA isolation from cell extracts -- 5.2 Reverse-transcription PCR, and quantitative reverse-transcription PCR -- 5.3 Non-radioactive in situ detection of transcripts in EBs -- 6 Assessing expression and function of muscle-specific proteins -- 6.1 Immunofluorescence -- 6.2 Functional analyses of muscle derivatives -- 6.3 Electrophysiological measurements in mES cell-derived cardiac cells -- 7 In vitro differentiation of mouse ES cells into haematopoietic cells -- 1 Introduction -- 1.1 Haematopoietic development -- 1.2 Haematopoietic differentiation in mouse ES cells -- 1.3 The mES/OP9-cell coculture system -- 2 Routine maintenance of cell lines -- 2.1 Maintenance of mES cells -- 2.2 MEF culture and feeder-layer preparation -- 2.3 Maintenance of OP9 stromal cells, and preparation for coculture -- 2.4 Fetal-calf serum (FCS) -- 3 mES/OP9-cell coculture system for haematopoietic differentiation -- 3.1 Primitive erythroid cell differentiation -- 3.2 Definitive erythroid cell differentiation -- 3.3 Definitive haematopoietic progenitors and CFU-OP9 -- 4 Analysis of definitive haematopoietic cells produced using the mES/OP9-cell coculture system -- 5 Evaluation of the mES/OP9-cell coculture system as a model for haematopoietic differentiation -- 8 Lineage selection and transplantation of mouse ES cell-derived neural precursors -- 1 Introduction -- 1.1 Neural differentiation potential of mES cells in vitro -- 2 Controlled differentiation of mES cells into neural precursors -- 3 Selection of lineage-restricted cell populations from differentiated mES cells -- 3.1 Transfection of mES cells with selectable marker genes, for neuronal and oligodendroglial lineage selection.

3.2 Neuronal lineage selection -- 3.3 Oligodendroglial lineage selection -- 4 Transplantation models for studying functional integration of mES cell-derived neural precursors -- 4.1 Neural precursor integration in organotypic slice cultures -- 4.2 In utero transplantation of mES cell-derived neural progenitors -- 4.3 Identification and characterization of transplanted cells using donor-specific markers -- 4.4 Immunohistochemical detection of transplanted cells with donor-specific antibodies -- 5 Translation to human ES cell-derived neural precursors -- 9 In vitro differentiation of mouse ES cells into pancreatic and hepatic cells -- 1 Introduction -- 2 Maintenance of undifferentiated mES cells -- 3 In vitro differentiation procedures -- 3.1 Generation of mES cell-derived, heterogeneous lineage progenitor cells -- 3.2 Induction of lineage-specific differentiation -- 4 Characterization of differentiated, pancreatic and hepatic cell phenotypes -- 4.1 RT-PCR -- 4.2 Immunofluorescence staining -- 4.3 ELISA -- 4.4 Microscopic analysis and imaging of samples labelled by immunofluorescence -- 5 Animal transplantation models -- 5.1 Pancreatic islet regeneration models -- 5.2 Liver reconstitution models -- 10 Isolation and characterization of human ES cells -- 1 Introduction -- 2 Isolation of hES cells -- 2.1 Human embryos -- 2.2 Initiation of primary cultures -- 3 Human ES cell maintenance: serum-supplemented versus serum-free media -- 4 Cryopreservation of human ES cells -- 5 Characterization of hES cells -- 5.1 Immunochemical characterization of hES cells -- 5.2 FACS analysis of hES-cell antigen expression -- 5.3 Immunomagnetic isolation of viable hES cells -- 5.4 Gene expression in hES cells -- 5.5 Biological assay of hES-cell pluripotentiality -- 11 Differentiation of human ES cells -- 1 Introduction -- 2 Maintenance of hES cell lines.

2.1 Spontaneous differentiation of hES cells -- 3 In vivo differentiation of hES cells -- 4 In vitro differentiation of hES cells -- 4.1 Differentiation via the formation of embryoid bodies -- 4.2 Vascular morphogenesis within EBs -- 4.3 Cardiac differentiation in EBs -- 5 Guided differentiation -- 5.1 Haematopoietic differentiation -- 5.2 Non-EB-mediated hES-cell differentiation: vascular development -- 6 Scale-up procedures -- 6.1 Dynamic systems for EB production -- 6.2 Production of hES cell-derived EBs within three-dimentional matrices -- 7 Epilogue: future clinical applications and limitations -- 12 ES cell lines from the cynomolgus monkey (Macaca fascicularis) -- 1 Introduction -- 1.1 Primate versus murine ES cells -- 2 Establishment and maintenance of cES cell lines -- 2.1 Preparation of feeder layers for cES-cell derivation -- 2.2 Culture of ICMs from cynomolgus monkey blastocysts -- 2.3 Expansion and routine maintenance of undifferentiated cES cells -- 3 Characterization of cES cell lines -- 3.1 Expression of cES-cell markers and karyotype analysis -- 4 Genetic modification of cES cells -- 5 Differentiation of cES cells -- 6 Directed differentiation of cES cells induced by coculture -- 6.1 Neural differentiation of cES cells induced by coculture with PA6 cells -- 6.2 Haematopoietic differentiation of cES cells induced by coculture with OP9 cells -- 7 Conclusion -- List of Suppliers -- Index -- A -- B -- C -- D -- E -- F -- G -- H -- I -- K -- L -- M -- N -- O -- P -- Q -- R -- S -- T -- U -- V -- W -- X -- Z.