Germ Cell Development: Methods and Protocols

Preface
Contents
Contributors
Part I: Isolation and Purification of Primordial (and) Germ Cells
	Chapter 1: Isolation and Purification of Viable PGCs from Mouse Embryos
		1 Introduction
		2 Materials and Instruments
			2.1 Isolation of PGCs by MiniMACS
			2.2 Isolation of PGCs from OG2 Mice by FACS
		3 Methods
			3.1 Dissection of AGM and GRs
			3.2 Isolation of 10.5-12.5 dpc PGCs by MiniMACS
			3.3 Isolation of 10.5-12.5 dpc PGCs by FACS
		4 Notes
		References
	Chapter 2: Purification and Transfection Methods of Chicken Primordial Germ Cells
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Equipment
		3 Methods
			3.1 Preparation Embryos to Isolate of PGCs
			3.2 Isolation of PGCs from Bloodstream (bPGCs)
			3.3 Isolation of PGCs from Gonads (gPGCs)
			3.4 Purification and Identification of PGCs
				3.4.1 Percoll Purification
				3.4.2 Identification of PGCs
					PAS Staining
					Immunocytochemistry
			3.5 Transfection Methods and Culture of PGCs
				3.5.1 Electroporation
				3.5.2 Lipofection
		4 Notes
		References
	Chapter 3: Isolation and In Vitro Propagation of Human Spermatogonial Stem Cells (SSCs)
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Equipment
		3 Methods
			3.1 Before Isolation
			3.2 Isolation
			3.3 Cell Culture
			3.4 Cell Cryopreservation
		4 Notes
		References
	Chapter 4: Purification by STA-PUT Technique of Male Germ Cells from Single Mouse and RNA-Extraction for Transcriptomic Analys...
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 BSA Gradients
			2.3 STA-PUT Apparatus
			2.4 Testicular Germ Cells Preparation
			2.5 Fractions Collection and Analysis of Germ Cells Nuclear Morphology
			2.6 RNA Purification
		3 Methods
			3.1 Assembly of the STA-PUT Apparatus
			3.2 Isolation of Male Germ Cells from Single Mouse
			3.3 STA-PUT Sedimentation
			3.4 STA-PUT Fractionation and Analysis of Fractions
			3.5 RNA Extraction for Transcriptomic Analysis
		4 Notes
		References
	Chapter 5: Isolation of Mouse Germ Cells by FACS Using Hoechst 33342 and SYTO16 Double Staining
		1 Introduction
		2 Materials
			2.1 Single Cell Suspension Generation
			2.2 Cell Staining
			2.3 FACS
			2.4 Microscopic Quality Control
		3 Methods
			3.1 Single Cell Suspension Generation
			3.2 Staining of Cell Suspension
			3.3 FACS of Stained Cells
			3.4 Quality Control of Sorted Cells by Immunofluorescence
				3.4.1 Preparation of Meiotic Spermatocytes (Based on)
				3.4.2 Preparation of Haploid Spermatids
				3.4.3 Immunofluorescence
		4 Notes
		References
	Chapter 6: Isolation and In Vitro Culture of Germ Cells and Sertoli Cells from Human Fetal Testis
		1 Introduction
		2 Materials
			2.1 General Reagents, Disposables, and Equipment
				2.1.1 Reagents
				2.1.2 Disposables
				2.1.3 Equipment
				2.1.4 Facility Rooms
			2.2 Preparation of Human Fetal Testis for Enzymatic Digestion
				2.2.1 Fresh Human Fetal Testis
				2.2.2 Cryopreservation and Thawing Human Fetal Testis
			2.3 Enzymatic Digestion of Human Fetal Testis Pieces into Single Cells
			2.4 Isolation of FGCs and Sertoli Cells by FACS Sorting
			2.5 Culture of Sorted FGCs on Sorted Sertoli Cells
			2.6 Immunofluorescence of Cultured Sorted FGCs and Sertoli Cells
		3 Methods
			3.1 Preparation of Human Fetal Testis for Enzymatic Digestion
				3.1.1 Using Freshly Isolated Human Fetal Testis
				3.1.2 Cryopreservation and Thawing of Human Fetal Testis
				3.1.3 Using Cryopreserved-Thawed Human Fetal Testis
			3.2 Digestion of Human Fetal Testis Pieces into Single Cells
			3.3 Isolation of FGCs and Sertoli Cells by FACS Sorting
				3.3.1 Testis Sample
				3.3.2 Compensation Beads (If Using)
			3.4 Culture of FGCs on Sertoli Cells
			3.5 Immunofluorescence of Sorted FGCs on 18 Well μ-Slide
		4 Notes
		References
Part II: Establishing In Vitro Systems for Studying Primordial (and) Germ Cells
	Chapter 7: Human Primordial Germ Cell-Like Cell Induction from Pluripotent Stem Cells by SOX17 and PRDM1 Expression
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Preparation of Culture Medium
		3 Methods
			3.1 Transfection of Transgenes in 4i PSCs (see Fig. 2)
			3.2 Clonal Isolation of Transgene Transfected PSCs (see Fig. 2)
			3.3 Induction of PGCLCs with SOX17 and PRDM1 Transgenes from 4i and Pre-ME Cells
				3.3.1 Induction of Pre-ME Cells
				3.3.2 Induction of PGCLC by SOX17/PRDM1 from Pre-ME or 4i Cells
			3.4 Isolation and Detection of PGCLCs
		4 Notes
		References
	Chapter 8: Induction of Primordial Germ Cell-Like Cells from Rat Pluripotent Stem Cells
		1 Introduction
		2 Materials
			2.1 Maintenance of Rat Pluripotent Embryonic Stem Cells (rESCs)
			2.2 rEpiLCs Differentiation Via Spheroid Culture
			2.3 rPGCLCs Differentiation
			2.4 rPGCLCs Maturation Using Gonadal Somatic Cells
		3 Methods
			3.1 rESCs Maintenance
			3.2 rEpiLC Differentiation
			3.3 rPGCLC Differentiation and Validation by Flow Cytometry
			3.4 Preparation of Gonadal Somatic Cells
			3.5 rPGCLC Maturation by Aggregation with Gonadal Somatic Cells
			3.6 Validation of Matured rPGCLCs
		4 Notes
		References
	Chapter 9: Induction of Meiotic Initiation in Long-Term Mouse Spermatogonial Stem Cells Under Retinoid Acid and Nutrient Restr...
		1 Introduction
		2 Materials
			2.1 Establishment of Long-Term Mouse SSC Culture
			2.2 Meiotic Induction Media
			2.3 Meiotic Progression Media
		3 Methods
			3.1 Establishment of Mouse SSC Culture
			3.2 Induction of Meiotic Initiation
			3.3 Meiotic Progression
		4 Notes
		References
	Chapter 10: Lipofection-Based Delivery of CRISPR/Cas9 Ribonucleoprotein for Gene Editing in Male Germline Stem Cells
		1 Introduction
		2 Materials
			2.1 Testes Collection and Cryopreservation
				2.1.1 Compounds
				2.1.2 Equipment
			2.2 Cell Culture
				2.2.1 Compounds
				2.2.2 Equipment
			2.3 Lipofection of Cas9:gRNA Ribonucleoproteins
				2.3.1 Compounds
				2.3.2 Equipment
		3 Methods
			3.1 Testes Collection and Cryopreservation
			3.2 Initiation and Maintenance of a GS Cell Culture
			3.3 Preparation of CRISPR/Cas9 Transfection Complexes
			3.4 Reverse Transfection of GS Cells (Fig. 2)
		4 Notes
		References
	Chapter 11: Mouse In Vitro Spermatogenesis on 3D Bioprinted Scaffolds
		1 Introduction
		2 Materials
			2.1 Testis Collection and Cryopreservation
			2.2 Culture of 3D Constructs
				2.2.1 3D (bio)Printing Process and Parameters for Scaffolds and Mold Preparation
				2.2.2 Enzymatic Digestion of Testicular Tissue
				2.2.3 3D (bio)Printing
			2.3 Gross Histological Analysis and In-Depth Examination
		3 Methods
			3.1 Testes Collection and Cryopreservation
			3.2 Culture of TC/CFS Constructs
				3.2.1 3D Printing  CFS
				3.2.2 Fabrication TC/CFS
			3.3 Culture of CD49f/CLS Constructs
				3.3.1 Agarose Sockets
				3.3.2 Digestion Adult Mouse Testes
				3.3.3 3D Bioprinting  CLS
				3.3.4 MACS-Mediated Enrichment CD49f+ Epithelial Testicular Cells
				3.3.5 Fabrication CD49f/CLS
			3.4 Histochemistry and Immunofluorescence
				3.4.1 Fixation of Constructs
				3.4.2 Paraffin Embedding and Tissue Sectioning
				3.4.3 PAS Staining and Morphological Evaluation
				3.4.4 Fluorescence Staining and In-Depth Examination of Germ Cell Differentiation
		4 Notes
		References
	Chapter 12: Histological and Cytological Techniques to Study Perinatal Mouse Ovaries and Oocytes
		1 Introduction
		2 Materials
			2.1 Mouth Pipette Preparation
			2.2 Collection of the Ovaries
			2.3 Oocyte Collection and Culture
			2.4 Fixation and Immunofluorescence of Oocytes
			2.5 Fixation and Processing of Ovaries
			2.6 Sectioning
			2.7 Immunofluorescence of Ovarian Sections
				2.7.1 Deparaffinization-Rehydration
				2.7.2 Antigen Retrieval
				2.7.3 Immunofluorescence Staining
				2.7.4 PAS-Hematoxylin Staining of Ovarian Sections
		3 Methods
			3.1 Mouth Aspiration Unit Preparation
			3.2 Collection of Ovaries
				3.2.1 Ovaries´ Collection for Oocyte Culture
				3.2.2 Ovaries´ Collection for Fixation
			3.3 Oocyte Collection and Culture
			3.4 Fixation and Immunofluorescence of Cultured Oocytes
			3.5 Fixation and Processing of Ovaries
			3.6 Sectioning
			3.7 Immunofluorescence of Ovarian Sections
				3.7.1 Deparaffinization-Rehydration
				3.7.2 Antigen Retrieval
				3.7.3 Immunofluorescence Staining
			3.8 PAS-Hematoxylin Staining of Ovarian Sections
		4 Notes
		References
	Chapter 13: Method of Isolation and In Vitro Culture of Primordial Follicles in Bovine Animal Model
		1 Introduction
		2 Materials
			2.1 Manipulation Solution and Media
			2.2 Culture Medium
			2.3 Dual-Fluorescence Viability Assay
			2.4 Equipment
		3 Methods
			3.1 Isolation and Culture of Primordial Follicles
			3.2 Viability Assessment
		4 Notes
		References
Part III: Somatic Reprogramming to Studying Human Aneuploidy Features
	Chapter 14: Generation of iPSC Cell Lines from Patients with Sex Chromosome Aneuploidies
		1 Introduction
			1.1 Somatic Cell Reprogramming
			1.2 Klinefelter Syndrome and High-Grade Sex Chromosome Aneuploidies
			1.3 Disease Modeling of Klinefelter Syndrome and  SCAs
		2 Materials
			2.1 Fibroblast Culturing, Passaging, and Freezing
			2.2 Somatic Cell Reprogramming
			2.3 iPSC Picking and Replating
		3 Methods
			3.1 Isolation of Fibroblasts from Skin Biopsies (Fig. 1)
			3.2 Culture of Primary Fibroblasts
			3.3 Somatic Cell Reprogramming of Fibroblasts (Fig. 2)
			3.4 iPSC Picking and Expansion
		4 Notes
		References
Part IV: Informatic Tools for Studying Germ Cells Development and Recombination
	Chapter 15: Data Analysis Pipeline for scRNA-seq Experiments to Study Early Oogenesis
		1 Introduction
		2 Materials
			2.1 Required Resources
			2.2 Equipment
			2.3 Equipment Setup
				2.3.1 Software Requirements
				2.3.2 Software Installation
			2.4 Datasets
				2.4.1 Preparation of scRNA-seq Dataset
		3 Methods
			3.1 Overview of the scRNA-seq Pipeline
			3.2 Generation of Gene Expression Matrices Using CellRanger
			3.3 Constructing Seurat Object from CellRanger Output Files and Basic  QC
			3.4 Removing Doublets Using DoubleFinder
			3.5 Data Integration, Removal of the Batch Effect, and Nonlinear Dimensional Reduction Using  UMAP
			3.6 Cell-Type Characterization
			3.7 Subsetting Germ Cell Populations to Identify Fine-Scale Germ Cell Meiotic Transcriptome
		4 Notes
		References
	Chapter 16: Mapping Meiotic DNA Breaks: Two Fully-Automated Pipelines to Analyze Single-Strand DNA Sequencing Data, hotSSDS an...
		1 Introduction
		2 Materials
			2.1 Computing Resources
			2.2 Software Requirements
			2.3 Data
		3 Methods
			3.1 hotSSDS Pipeline
				3.1.1 Download the Pipeline
				3.1.2 Download Singularity Images
				3.1.3 Configure Computing Parameter Files
				3.1.4 Prepare Input File
				3.1.5 Edit/Create Parameter File
				3.1.6 Launch the Pipeline
				3.1.7 Run a Short Test
				3.1.8 Monitor the Pipeline
				3.1.9 Inspection of Results
			3.2 hotSSDS-extra Pipeline
				3.2.1 Download the Pipeline
				3.2.2 Download Singularity Images
				3.2.3 Configure Computing Parameter Files
				3.2.4 Edit/Create Parameter File
				3.2.5 Launch the Pipeline
				3.2.6 Monitor the Pipeline
				3.2.7 Inspect Results
		4 Notes
		References
	Chapter 17: ``MeiQuant´´: An Integrated Tool for Analyzing Meiotic Prophase I Spread Images
		1 Introduction
		2 Materials
			2.1 2D Chromosome Spreads
			2.2 Images
			2.3 Images Preprocessing
			2.4 Input Images File Format
			2.5 Versions and Plugins  Used
		3 Methods
			3.1 Stage Determination
			3.2 Reference ROI Module for Reference ROI Identification
			3.3 Axis Module for Axis Segmentation and Length Measurements
			3.4 Advanced User Parameters for Axis Identification
			3.5 Foci Module for Foci Identification and Counting
			3.6 Colocalization Module
			3.7 Intensities Module
			3.8 Input & Output Module
		4 Notes
		References
Index