Germline Stem Cells: Methods and Protocols (Methods in Molecular Biology, 2677)

Preface
Contents
Contributors
Chapter 1: Analysis of the C. elegans Germline Stem Cell Pool
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Web Resources
	3 Methods
		3.1 Identification of the Progenitor Zone in Wild-Type C. elegans Hermaphrodites
		3.2 Mounting Live Animals for Light Microscopy (See Notes 2 and 3)
		3.3 Immunohistochemistry of Extruded Gonads
		3.4 Visualization of DNA and Germline Apoptosis in Live Animals
		3.5 Scoring Progenitor Zone Length and Cell Number
		3.6 Characterization of the GSC Pool and GSC Number
		3.7 Markers for the GSC Pool, Progenitor Zone and Early Stages of Differentiation
		3.8 Markers for the Somatic Gonad
		3.9 Cell Cycle Analysis
		3.10 Mitotic Index with Anti-PH3
		3.11 EdU Labeling
		3.12 Label with EdU by Soaking
		3.13 Calculating Labeling Index and Identifying G1 and G2 Cells
		3.14 Estimating Total Length of the Cell Cycle
		3.15 To Measure Cell-Cycle Length by Measuring G2
		3.16 To Measure Cell-Cycle Length by Measuring G2 + M + G1
		3.17 Identification of Premeiotic Cells in the Progenitor Zone
		3.18 Characterization of Progenitor Zone Defects in Mutants
		3.19 Initial Characterization of Mutants
	4 Notes
	References
Chapter 2: Analysis of C. elegans Germline Small RNA Pathways
	1 Introduction
	2 Materials
		2.1 CRISPR-Cas9
		2.2 Synchronizing and Harvesting Nematode Populations
		2.3 Worm Pellet Lysis
		2.4 Immunoprecipitation
		2.5 RNA Extraction
		2.6 RNA 5′ Polyphosphatase Treatment
		2.7 Library Preparation, Sequencing, and Sequence Analysis
	3 Methods
		3.1 CRISPR-Cas9
			3.1.1 Selection of Epitope Tag
			3.1.2 Selection of Tag Location
			3.1.3 Functional Validation of Tagged Protein
			3.1.4 Generation of Ago Mutants
		3.2 Synchronizing and Harvesting Nematode Populations
			3.2.1 Bleaching to Synchronize the Population
			3.2.2 Plating Synchronized L1 Larvae
			3.2.3 Harvesting Nematode Pellets
		3.3 Nematode Pellet Lysis
		3.4 Immunoprecipitation
		3.5 RNA Extraction
		3.6 RNA 5′ Polyphosphatase Treatment
		3.7 Library Preparation
		3.8 Sequencing Analysis
			3.8.1 Choosing an Analysis Pipeline
			3.8.2 Quality Control
			3.8.3 Adaptor Trimming
			3.8.4 Genome Alignment
			3.8.5 Counting Reads
			3.8.6 Sequence Composition Analysis
			3.8.7 Differential Expression Analysis
	4 Notes
	References
Chapter 3: Lineage Tracing and Single-Cell RNA-seq in C. elegans to Analyze Transgenerational Epigenetic Phenotypes Inherited ...
	1 Introduction
	2 Materials
		2.1 Growing Worms
		2.2 scRNA-seq
		2.3 Preparation of Solutions and Equipment for Microscopy
		2.4 Website to Download Lineage Program Software
	3 Methods
		3.1 Cell Isolation for scRNA-seq
		3.2 Staging Worms and Collecting ~100 Cell Embryos
		3.3 Eggshell Removal and Single-cell Suspension
		3.4 Synchronization and Worm Dissection to Obtain 2-Cell Embryos for Lineage Tracing
	4 Notes
	References
Chapter 4: Methods to Analyze Nutritional and Inter-Organ Control of Drosophila Ovarian Germline Stem Cells
	1 Introduction
	2 Materials
		2.1 Fly Food Media
		2.2 Transgenic Lines
		2.3 Buffers and Reagents
		2.4 Tools
		2.5 Antibodies
	3 Methods
		3.1 General Diet Manipulation Paradigm
		3.2 Adult Adipocyte-specific Genetic Manipulations
		3.3 Initial Preparation for Ovary Dissection
		3.4 Ovary Dissection
		3.5 Fixation and Washing
		3.6 Blocking and Antibody Incubation
		3.7 Mounting Ovaries on Microscope Slides
		3.8 Identifying Ovarian Germline Stem Cells
	4 Notes
	References
Chapter 5: Targeting Endogenous Loci That Function in Drosophila Germline Stem Cells
	1 Introduction
	2 Materials
		2.1 Plasmids and Other DNAs
		2.2 Cloning Reagents
		2.3 Fly Rearing Components
		2.4 Ovary Dissection, Immunostaining, and Imaging Components
	3 Methods
		3.1 Cloning guideRNA Plasmids
		3.2 Cloning the Donor Plasmid
		3.3 Injection
		3.4 Screening
		3.5 Characterizing a Mutant
	4 Notes
	References
Chapter 6: Live Imaging of the Drosophila Testis Stem Cell Niche
	1 Introduction
	2 Materials
		2.1 Dissection of Testes From Adult Male Drosophila
		2.2 Live Imaging of Drosophila Testes
		2.3 Immunostaining Testes After Live Imaging
	3 Methods
		3.1 Preparing Imaging Dish
		3.2 Dissecting and Mounting Whole Testes
		3.3 Overnight Time-Lapse Imaging of the Testis Niche
		3.4 Immunolocalization After Time-lapse Imaging
	4 Notes
	References
Chapter 7: Enrichment of Undifferentiated Germline and Somatic Cells from Drosophila Testes
	1 Introduction
		1.1 Immunoprecipitation Using Cell Type-Specific Expression of Tagged Proteins
		1.2 Labeling of Protein or RNA Using Cell-Type-Specific Expression of Enzymes
		1.3 Usage of Mutant Gonad for Enrichment of Specific Cell Types
		1.4 Hand Picking of Specific Cell Types
		1.5 Flow Cytometry and Cell Sorting
	2 Materials
		2.1 Dissection
		2.2 Dissociation
		2.3 Flow Cytometry and Cell Sorting
	3 Methods
		3.1 Fly Culture
		3.2 Dissection of Testes (see Note 1)
		3.3 Dissociation of Cells (see Notes 4-7)
		3.4 Flow Cytometry and Cell Sorting
	4 Notes
	References
Chapter 8: Spermatogonial Dedifferentiation into Germline Stem Cells in Drosophila Testes
	1 Introduction
	2 Materials
	3 Methods
		3.1 Lineage-tracing Genetics and Husbandry
		3.2 Aging and Stress Assays
		3.3 Normal Aging
		3.4 Starvation
		3.5 Stress Through Mating
		3.6 Chronic Stress
		3.7 Dissection of Testes, Imaging, and Cell Characterization
	4 Notes
	References
Chapter 9: Standardization of Single-Cell RNA-Sequencing Analysis Workflow to Study Drosophila Ovary
	1 Introduction
	2 Materials
		2.1 Single-Cell RNA Sequencing Sample Preparation
	3 Methods
		3.1 Tissue and Cell Dissociation
		3.2 10X Genomics Library Preparation and Sequencing
		3.3 Data Pre-Processing in Linux-Based Command Line Interface
		3.4 Data Pre-Processing in R
		3.5 Dealing with Technical Noise and Confounding Factors
		3.6 Decomplicating Single-Cell Datasets by Linear Dimension Reduction
		3.7 Clustering and Visualization
		3.8 Identifying Cell States and Assigning Cell-Type Identities
		3.9 Inferring Enriched Markers
	4 Notes
	References
Chapter 10: Identification of Germline Stem Cells in Zebrafish
	1 Introduction
	2 Materials
		2.1 Tissue Fixation and Permeabilization
		2.2 Probe Synthesis
		2.3 nanos2 in Situ Hybridization
		2.4 Vasa Immunofluorescence
		2.5 Mounting and Confocal Microscopy
	3 Methods
		3.1 Preparation of Template DNA
		3.2 RNA Probe Synthesis
		3.3 Preparation of Gonads for in Situ Hybridization and Indirect Immunostaining
		3.4 Whole-Mount In Situ Hybridization
		3.5 Antibody Staining
		3.6 Mounting Ovaries for Confocal Analysis
	4 Notes
	References
Chapter 11: Characterization of the Postnatal Naked Mole-Rat Ovary: From Primordial Germ Cells to Meiotic Prophase I Oocytes
	1 Introduction
	2 Materials
		2.1 Collection of Ovaries
		2.2 Processing of Ovaries for Culture and Spreads
		2.3 Processing Ovaries for Ovarian Reserve Analysis and Histology Sections
		2.4 Oocyte Spreading for Immunofluorescence
		2.5 Meiotic Spreads Immunofluorescence
		2.6 Culture of Ovaries
		2.7 Fixation of Ovarian Cultures
		2.8 Immunofluorescence of Ovarian Cultures
		2.9 Clearing of Ovarian Cultures
		2.10 Embedding of Ovary in Paraffin for Histology Sections
		2.11 Immunofluorescence on Ovarian Histology Sections
	3 Methods
		3.1 Collection of the Ovaries
		3.2 Oocyte Spreading for Immunofluorescence
		3.3 Meiotic Spread Immunofluorescence
		3.4 Culture of Ovaries
		3.5 Fixation of Ovarian Cultures
		3.6 Immunofluorescence of Ovarian Cultures
		3.7 Clearing of Ovarian Cultures
		3.8 Embedding of Ovary in Paraffin for Histology Sections
		3.9 Sectioning
		3.10 Immunofluorescence on Ovarian Histology Sections
	4 Notes
	References
Chapter 12: A Roadmap for Three-Dimensional Analysis of the Intact Mouse Ovary
	1 Introduction
	2 Materials
		2.1 General Supplies and Solutions
		2.2 BABB Clearing Solutions
		2.3 iDISCO Clearing Solutions
	3 Methods
		3.1 Whole-mount Immunofluorescence Staining for Embryonic Mouse Ovaries
		3.2 Whole-mount Immunofluorescence Staining for Postnatal and Adult Mouse Ovaries
		3.3 Imaging
		3.4 Image Analysis for Wholemount Stained Ovaries
		3.5 Spatial Analysis of Wholemount Stained Ovaries Using MATLAB
	4 Notes
	References
Chapter 13: Isolation and Culture Techniques for Fetal Mouse Germ Cells
	1 Introduction
	2 Materials
		2.1 Isolation of PGCs by Fluorescence Activated Cell Sorting (FACS)
		2.2 Isolation of PGCs by Magnetic-Activated Cell Sorting (MACS)
		2.3 Primary Fetal Germ Cell Culture
	3 Methods
		3.1 Isolation of PGCs by Fluorescence-Activated Cell Sorting (FACS)
		3.2 Isolation of PGCs by Magnetic-Activated Cell Sorting (MACS)
		3.3 Primary Fetal Germ Cell Culture
	4 Notes
	References
Chapter 14: Rattus norvegicus Spermatogenesis Colony-Forming Assays
	1 Introduction
	2 Materials
		2.1 Spermatogenesis Colony-Forming Assays
	3 Methods
		3.1 Preparing Laminin-Coated Culture Dishes
		3.2 Preparing Mouse Embryonic Fibroblast Feeder Layers
		3.3 Formulating Spermatogonial Culture Media
		3.4 Formulating Rat A-Single Spermatogonia Culture Media (As-SG Medium)
		3.5 Formulating Spermatogonial Differentiation Medium (SD Medium)
		3.6 In vitro Spermatogenesis Colony-Forming Assay
		3.7 Immunofluorescence Labeling on Spermatogonial Cultures
		3.8 Germline Gene Targeting in Rat Spermatogonial Lines by Transfection With CRISPR/Cas9 Ribonucleoprotein (RNP) Complexes
	4 Notes
	References
Chapter 15: Maintenance of Human Primordial Germ Cell-Like Cells in a Long-Term Culture System
	1 Introduction
	2 Materials
		2.1 Human Plasma Fibronectin (HPF)
		2.2 Basal Media
		2.3 iMeLC Complete Medium (See Table 1)
		2.4 hPGCLC Complete Medium (See Table 2)
		2.5 LTC-hPGCLC Complete Media (See Table 3)
		2.6 Mouse Embryonic Fibroblast (MEF) Media
		2.7 Fluorescent-Activated Cell Sorting Buffer
		2.8 Fluorophore Conjugated Antibodies
		2.9 hESC-Qualified Matrigel
	3 Methods
		3.1 Generation of iMeLCs
		3.2 Generation of hPGCLCs
		3.3 Isolating hPGCLCs from Aggregate by Fluorescent-Activated Cell Sorting
		3.4 Generation of LTC-hPGCLCs
	4 Notes
	References
Chapter 16: Derivation and Primordial Germ Cell Induction of Intermediate Pluripotent Stem Cells
	1 Introduction
	2 Materials
		2.1 Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Cells
		2.2 Derivation and Culture of Mouse FTW-ESCs
		2.3 Derivation and Culture of Human FTW-iPSCs
		2.4 PGC-LC Induction from FTW-ESCs
		2.5 Antibodies for Flow Cytometry and/or Immunostaining of PGC-LCs
	3 Methods
		3.1 Preparation of MEF Feeder Cells
		3.2 Derivation and Culture of Mouse FTW-ESCs
		3.3 Maintenance of Mouse FTW-ESCs
		3.4 Derivation and Culture of Human FTW-iPSCs
		3.5 Maintenance of Human FTW-iPSCs
		3.6 PGC-LC Induction from FTW-PSCs
		3.7 PGC-LC Induction from Mouse FTW-ESCs
		3.8 PGC-LC Induction from Human FTW-iPSCs
		3.9 Culture of Mouse and Human PGC-LCs
		3.10 Dissociation and Analysis of PGC-LCs
	4 Notes
	References
Index