Embryo Models In Vitro: Methods and Protocols

Preface
Contents
Contributors
Generation of Human Blastoids from Naive Pluripotent Stem Cells
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
		2.3 Reagent Setup
			2.3.1 β-Estradiol (E2)
			2.3.2 Progesterone (P4)
			2.3.3 Sodium Pyruvate
			2.3.4 Gentamicin
			2.3.5 MEF Medium
			2.3.6 Immortalized Stromal Cell Culture Medium
			2.3.7 N2B27 Basal Medium
			2.3.8 Washing Medium
			2.3.9 4.5i/L/A Medium
			2.3.10 eHDM Medium
			2.3.11 eTDM Medium
			2.3.12 EBC1 Medium
			2.3.13 Fixation Buffer
			2.3.14 Holding Buffer
			2.3.15 Washing Buffer
			2.3.16 Permeabilization Buffer
			2.3.17 Blocking Buffer
			2.3.18 Antibody Buffer
	3 Methods
		3.1 Culturing of Naive hPSCs
			3.1.1 Preparation of iMEF Feeder Layers-Timing 1 h
				CRITICAL (See Notes 1-3)
			3.1.2 Thawing 4.5i/L/A Naive hPSCs-Timing 30 min
				CRITICAL (See Note 4)
			3.1.3 Confirmation of the Extraembroynic Lineage Plasticity of 4.5i/L/A Naive hPSCs-Timing 2 h
		3.2 Generation of Human Blastoid-Timing 7-9 Days
			3.2.1 Preparation of the AggreWell Plates-Timing 10-20 min
			3.2.2 Aggregation of Naive hPSCs in the AggreWell Plates-Timing 2-3 h
			3.2.3 Blastoid Development-Timing 7-8 Days
		3.3 Human Blastoid Handling-Timing 30 min
		3.4 Differential Immunofluorescent Staining of Human Blastoids-Timing 3 Days
			3.4.1 Human Blastoid Fixation-Timing 30 min
			3.4.2 Incubation with Primary Antibodies-Timing 2-3 h
			3.4.3 Incubation with Secondary Antibodies-Timing 2 h
		3.5 Peri-implantation Modeling of Human Blastoids (Fig. 3)-Timing 3-4 Days
			3.5.1 Preparation of the Extended Culture Plates-Timing 30 min
				CRITICAL (See Note 21)
			3.5.2 Blastoid Extended culture-Timing 3 Days
	4 Notes
	References
Human Pre-gastrulation Embryo Culture in 3D Condition
	1 Introduction
	2 Materials
		2.1 Embryo Culture
		2.2 Embryo Thawing and Zona Pellucida Removing
		2.3 Fixation and Immunostaining Medium
	3 Methods
		3.1 Embryo Thawing and Zona Pellucida Removal
		3.2 Evaluation of Embryo Quality
		3.3 In Vitro Three-Dimensional Culture of Human Embryos
		3.4 Frozen Section of Embryo
		3.5 Embryo Section Staining and Image Taking
		3.6 Whole Embryo Staining and Photo Taking
	4 Notes
	References
Protocol for the Generation of Human EPS-Blastoids Using a Three-Dimensional Two-Step Induction System
	1 Introduction
	2 Materials
		2.1 Mouse Embryonic Fibroblast (MEF) Cell Medium
		2.2 Human ESC Medium
		2.3 Human EPS Cell Derivation Medium
		2.4 TE-Like Cell Derivation Medium Using Human EPS Clls
		2.5 IVC1 Culture Medium
		2.6 Human EPS-Blastoid Culture Medium
		2.7 IVC2 Culture Medium
		2.8 Cell Lines
		2.9 Antibodies
		2.10 Other Reagents
	3 Methods
		3.1 Generation of Human iPSCs from Human Skin Fibroblasts with Episomal Vectors
		3.2 Preparation of MEF Feeders
			3.2.1 Thawing of ICR MEF Cells
			3.2.2 Maintenance of MEF Cells
			3.2.3 Preparation of Feeder Cells
			3.2.4 Preparation of MEF-Coated Plates
		3.3 Generation of Human EPS Cells from Human iPSCs
		3.4 The 3D, Two-Step Generation of Human EPS-Blastoids In Vitro
			3.4.1 Preparation of TE-Like Cells
			3.4.2 Prepare the AggreWell400 Culture Plate (See Note 10)
			3.4.3 Generation of Human EPS-Blastoids (Fig. 1)
			3.4.4 Human EPS-Blastoid Collection
		3.5 In Vitro Extended Culture of EPS-Blastoids for 8 and 10 Days
		3.6 Immunofluorescence of Human EPS-Blastoids (See Note 15)
	4 Notes
	References
Generation of Human Trophoblast Stem Cell-Dependent Placental In Vitro Models
	1 Introduction
	2 Materials
		2.1 Reagents and Equipment
		2.2 Inhibitor Cocktail
		2.3 hTSC Basal Medium
		2.4 hTSC Culture Medium
		2.5 Directed Differentiation Basal Medium
		2.6 STB Differentiation Medium
		2.7 EVT Differentiation Medium
	3 Methods
		3.1 Maintenance of hTSC (Fig. 1)
			3.1.1 Culture of hTSC
			3.1.2 Passaging of hTSC
			3.1.3 Cryopreservation of hTSC
			3.1.4 Thawing of hTSC
		3.2 Directed Differentiation of hTSC (Fig. 1)
			3.2.1 Differentiation of hTSC into STB (See Note 3)
			3.2.2 Differentiation of hTSC into EVT
		3.3 Generation and Differentiation of hTSC-Organoid (Fig. 2)
			3.3.1 Generation of hTSC-Organoid (Fig. 2a-c)
			3.3.2 Passaging of hTSC-Organoid
			3.3.3 Differentiation of hTSC-Organoids to Generate EVT Lineage (Fig. 2a, d)
			3.3.4 Validation of hTSC-Organoids
			3.3.5 Collection of hTSC-Organoid
	4 Notes
	References
Workflow for Performing Genetic Manipulation in Human Trophoblast Stem Cells Using CRISPR/Cas9 Technology
	1 Introduction
	2 Materials
		2.1 Reagents and Equipment
		2.2 Inhibitor Cocktail
		2.3 hTSC Basal Medium
		2.4 hTSC Culture Medium
		2.5 HEK293T Culture Medium
	3 Methods
		3.1 CRISPR/Cas9-Dependent Gene Knockout in hTSCs (Fig. 1a, c, d)
			3.1.1 sgRNA Design (See Notes 1-4)
			3.1.2 Construction of sgRNA Expression Vectors
			3.1.3 Lentivirus Production
			3.1.4 Lentiviral Infection and Drug Resistance Selection
			3.1.5 Selection of Monoclonal hTSC by Gradient Dilution (See Note 9)
			3.1.6 Design Primers for Genotyping
			3.1.7 Genomic DNA Extraction and Genotyping
		3.2 CRISPR/dCas9-VP64-Dependent Transcriptional Activation in hTSCs (Fig. 1b-d)
			3.2.1 sgRNA Design (See Notes 2 and 4)
			3.2.2 Construction of sgRNA Expression Vectors
			3.2.3 Lentiviral Infection and Drug Resistance Selection
			3.2.4 Identification of Target Gene Expression
	4 Notes
	References
Endometrial Assembloids to Model Human Embryo Implantation In Vitro
	1 Introduction
	2 Materials
		2.1 Digestion Medium
		2.2 Dextran-Coated Charcoal-Stripped Fetal Bovine Serum (DCC-FBS)
		2.3 Growth Medium (Stromal Monolayers)
		2.4 Expansion Medium (Epithelial Organoids)
		2.5 Differentiation Medium
		2.6 Embryo Co-culture Medium
	3 Methods
		3.1 Mincing and Digestion of Human Endometrium Tissue
		3.2 Separation, Plating, Expansion, and Passage of the Stromal Cell Fraction
		3.3 Plating, Expansion, and Passage of the Epithelial Cell Fraction
		3.4 Establishment and Growth of Assembloid Cultures
		3.5 Differentiation of Assembloid Cultures
		3.6 Assembloid and Embryo Co-cultures
	4 Notes
	References
Stem Cell-Derived Microfluidic Amniotic Sac Embryoid (μPASE)
	1 Introduction
	2 Materials
		2.1 Human Pluripotent Stem Cells
		2.2 Geltrex
	3 Methods
		3.1 Microfluidic Devices
		3.2 Geltrex Injection
		3.3 μPASE Generation
		3.4 Immunofluorescence Staining of μPASEs
		3.5 Live Cell Retrieval
	4 Notes
	References
Generation of 3D Trophoblast Organoids from Human Naïve Pluripotent Stem Cells
	1 Introduction
	2 Materials
		2.1 Maintenance of Naïve and Primary hTSCs
		2.2 Generation and Maintenance of SC-TOs
		2.3 Differentiation of SC-TOs into Specialized SC-EVTOs
		2.4 Characterization of Cellular Identity
		2.5 In vitro Human Endometrium Invasion Assay
	3 Methods
		3.1 Derivation of hTSCs from Naïve hPSCs (See Note 1)
		3.2 Generation of SC-TOs from Naïve and Primary hTSCs
		3.3 Detection of Secreted hCG with hCG Pregnancy Test Strips
		3.4 Expression Analysis of Trophoblast-Specific mRNAs and miRNAs
		3.5 Immunofluorescence Analysis and Whole Organoid Imaging
		3.6 Preparing SC-TOs for Single Cell RNA-Sequencing
		3.7 Analysis of HLA Surface Antigen Expression by Flow Cytometry
		3.8 Differentiation of SC-TOs into Specialized 3D EVT Organoids (SC-EVTOs)
		3.9 Quantifying Proteins Secreted from SC-TO and SC-EVTO Culture by ELISA
		3.10 Invasion Assay in the Presence of Human Endometrial Cells (Fig. 2e-g)
	4 Notes
	References
Induction of Human Extraembryonic Mesoderm Cells from Naive Pluripotent Stem Cells
	1 Introduction
	2 Materials
		2.1 General Culture Reagents
		2.2 General Culture Media
		2.3 Characterization of Cellular Identity
	3 Methods
		3.1 Preparation of Tissue Culture Plate Coated with MMC-MEF (Day 0)
		3.2 Seeding Naive Human Pluripotent Stem Cells (Day 1)
		3.3 Conversion of Naive Pluripotent Stem Cells to Extraembryonic Mesoderm Cells (Day 2-5)
		3.4 Purification and Validation of EXMCs by Flow Cytometry (Day 17-18)
			3.4.1 Purification Using Fluorescence-Activated Cell Sorting (FACS)
			3.4.2 Validation by Immunofluorescence (IF)
		3.5 Routine Culture and Maintenance of EXMCs
	4 Notes
	References
Modeling Human Paraxial Mesoderm Development with Pluripotent Stem Cells
	1 Introduction
	2 Materials
		2.1 Mediums and Solutions
		2.2 Factors and Supplements
		2.3 Culture Vessels and Equipment
		2.4 DICL Medium
		2.5 DICLFBR Medium
		2.6 SIM Medium
		2.7 N2B27 Medium
		2.8 Matrigel-Coated Plates
	3 Methods
		3.1 Dissociation and Passage of Human iPSC
		3.2 2D Differentiation
		3.3 Somitoid
		3.4 Segmentoid
	4 Notes
	References
Generation of Stem Cell-Based Mouse Embryo-Like Structures
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Experimental Plates
		2.3 Cell Culture Media
			2.3.1 N2B27 Medium (for 50 mL)
			2.3.2 Feeder Cell Medium (FC, for 1 Full Bottle)
			2.3.3 Trophoblast Stem Cell Medium (TS, for 50 mL)
		2.4 In Vitro Culture (IVC) Medium
			2.4.1 Base for IVC (ADF+++)
			2.4.2 IVC-20% FBS (10 mL)
			2.4.3 IVC-30% FBS (10 mL)
			2.4.4 DMEM/Rat Serum Medium/Human Cord Serum (DRH, for 1 mL)
			2.4.5 Human and Rat Serum Preparation
		2.5 Roller System Components
	3 Methods
		3.1 Culture of Mouse Embryonic Stem Cells (ESCs)
		3.2 Culture of Mouse Trophoblast Stem Cells (TSCs)
		3.3 Culture and Inactivation of Mouse Embryonic Fibroblasts (MEFs)
		3.4 Preparation and Seeding of Cells for ETiX-Embryoid Formation
	4 Notes
	References
Embryonic Spinal Cord Innervation in Human Trunk Organogenesis Gastruloids: Cardiac Versus Enteric Customization and Beyond
	1 Introduction
	2 Materials
		2.1 Pluripotent Stem Cells, Culture, and Passaging
		2.2 EMLO and EMLOC Gastruloid Formation (General Reagents)
		2.3 EMLO and EMLOC Gastruloid Formation (Differentiation Media)
		2.4 Immunofluorescence Buffers
		2.5 Single-Cell RNA Sequencing
		2.6 Equipment
		2.7 Software
	3 Methods
		3.1 Coating Cultureware with hESC-Qualified Matrigel
		3.2 hiPSC Culture and Pluripotency Maintenance
		3.3 Passaging of hiPSCs Prior to Induction
		3.4 2D Induction of hiPSC Colonies to Axial Trunk-Biased Phenotype
		3.5 Single-Cell Suspension, Transition to Shaking Culture, and Early Polarization
		3.6 EMLO Gastruloid Induction Continued (Neuro-enteric)
		3.7 EMLOC Gastruloid Induction (Neuro-cardiac)
		3.8 Multi-lineage Permissive Differentiation and Maturation in EMLO and EMLOC Gastruloids
		3.9 Biomarker Characterization of Cell Types by Immunofluorescence
		3.10 Gene Expression Analysis and Characterization by scRNAseq
		3.11 Future Directions
	4 Notes
	References
Use of Epigenetic Cues and Mechanical Stimuli to Generate Blastocyst-Like Structures from Mammalian Skin Dermal Fibroblasts
	1 Introduction
	2 Materials
		2.1 Mouse Embryonic Fibroblast (MEF) Thawing and Inactivation
		2.2 Isolation of Porcine Skin Dermal Fibroblasts
		2.3 Isolation of Human Skin Dermal Fibroblasts
		2.4 Seeding of Porcine and Human Dermal Fibroblasts
		2.5 Porcine and Human Fibroblast Exposure to 5-Azacytidine (5-aza-CR) and Trophoblast Induction
		2.6 Exposure of Porcine and Human Fibroblasts Encapsulated in PTFE Micro-bioreactor to 5-aza-CR
		2.7 Generation of Porcine and Human epiBlastoids
	3 Methods
		3.1 MEF Thawing and Inactivation
		3.2 Isolation of Porcine Skin Dermal Fibroblasts
		3.3 Isolation of Human Skin Dermal Fibroblasts
		3.4 Seeding of Porcine and Human Dermal Fibroblasts
		3.5 Porcine and Human Fibroblast Exposure to 5-aza-CR and Trophoblast Induction
		3.6 Exposure of Porcine and Human Fibroblasts Encapsulated in PTFE Micro-bioreactor to 5-aza-CR
		3.7 Generation of Porcine and Human epiBlastoids
	4 Notes
	References
Directed Differentiation of Human Pluripotent Stem Cells to Cytotrophoblast and Syncytiotrophoblast
	1 Introduction
	2 Materials
		2.1 Cell Banking and Storage
		2.2 Reconstitution of Recombinant Growth Factors and Inhibitors
			2.2.1 hbFGF
			2.2.2 hBMP-4
			2.2.3 ALK 4/5/7 Inhibitor-SB431542
			2.2.4 ALK 4/5/7 Inhibitor-A 83-01
			2.2.5 MEK/ERK Pathway Inhibitor (FGFR-1/3/2/4 Inhibitor)-PD173074
			2.2.6 MEK/ERK Pathway Inhibitor (FGFR-1 Inhibitor)-SU5402
			2.2.7 ROCK Inhibitor-Y27632
		2.3 Preparation of Extracellular Matrices
			2.3.1 Vitronectin
			2.3.2 Matrigel
		2.4 Media Preparation, Aliquoting, and Storage
			2.4.1 Maintenance Medium (Essential 8 (E8) Media)
			2.4.2 Differentiation Medium (N2B27)
				N2B27 medium (50 mL)
		2.5 Reagents for Cell Dissociation
			2.5.1 EDTA (0.5 mM)
			2.5.2 Accutase
		2.6 Reagents and Materials for qRT-PCR
	3 Methods
		3.1 Extracellular Matrix Coating of Cell Culture Plates
			3.1.1 Vitronectin (VTN-N) Coating
			3.1.2 Matrigel Coating
		3.2 Passaging and Seeding of hPSCs
			3.2.1 Chemical Method (Dissociation into Clumps Using 0.5 mM EDTA)
			3.2.2 Enzymatic Method (Single Cell Suspension by Accutase Treatment)
		3.3 Differentiation of hPSCs to Trophoblast Lineage
			3.3.1 Differentiation Using Chemical (Clumps) Method
			3.3.2 Differentiation Using Enzymatic (Single Cell) Method
		3.4 Quantitative Real-Time PCR (qRT-PCR)
	4 Notes
	References
Single-Cell mRNA-sncRNA Co-sequencing of Preimplantation Embryos
	1 Introduction
	2 Materials
		2.1 General
		2.2 Small-Seq
		2.3 Smart-Seq2
		2.4 Consumables
		2.5 Equipment
		2.6 Oligonucleotides
		2.7 Software
	3 Methods
		3.1 Single-Cell Collection
		3.2 Day 1: Cell Lysis, 3′ Adapter Ligation (Small-Seq), Reverse Transcription, and PCR Amplification (Smart-Seq2)
		3.3 Day 2: 3′ Adapter Digestion, 5′ Adapter Ligation, Reverse Transcription and PCR Amplification (Small-Seq), and Purificatio...
		3.4 Small-Seq PCR 2 and Final Library Prep
		3.5 Smart-Seq2 Tagmentation, PCR 2, and Final Library Prep
		3.6 Data Analysis
	4 Notes
	5 Strengths and Limitations
	References
Evaluation of Stem-Cell Embryo Models by Integration with a Human Embryo Single-Cell Transcriptome Atlas
	1 Introduction
	2 Materials
		2.1 Library
		2.2 Download Human Blastoid Data
	3 Methods
		3.1 Analyze Blastoid Data
			3.1.1 Create Seurat Object
			3.1.2 Quality Control of Single-Cell Data
			3.1.3 Selection of Highly Variable Genes
			3.1.4 Data Normalization and Scaling, and Linear Dimension Reduction
			3.1.5 Non-linear Dimensional Reduction
			3.1.6 Annotate Clusters by Marker Gene Expression
		3.2 Format Data for Integration
		3.3 Integration of Blastoid and Embryo Data
		3.4 Analyze Blastoids Integrated with Embryos
			3.4.1 Non-linear Dimensional Reduction of Integrated Data
			3.4.2 Change Annotation
			3.4.3 Highlight Specific Cells of Interest
	4 Notes
	References
Inferring Gene Regulatory Networks and Predicting the Effect of Gene Perturbations via IQCELL
	1 Introduction
	2 Materials
		2.1 System Requirements
		2.2 Dataset Requirements
		2.3 Notebooks
	3 Methods
		3.1 Processing scRNA-seq Data (Notebook1)
		3.2 Selecting Informative Genes (Notebook2a and Notebook2b)
		3.3 GRN Inference (Notebook3)
		3.4 Run Boolean Simulations and GRN Perturbations (Notebook3)
	4 Notes
	References
ShapeMetrics: A 3D Cell Segmentation Pipeline for Single-Cell Spatial Morphometric Analysis
	1 Introduction
	2 Materials
		2.1 Software (All Publicly Available with No Cost)
	3 Methods
		3.1 Immunostaining of Cell Membranes
		3.2 Image Acquisition
		3.3 Creating a Prediction Map with Ilastik (Fig. 1, Part 1)
		3.4 MATLAB Watershed Segmentation Algorithm (Fig. 1, Part 2)
		3.5 Extraction of Morphological Features (Fig. 1, Part 3, and Fig. 2)
		3.6 Mapping of Cell Features Back into Spatial Context in the Original Images (Fig. 2)
		3.7 Pooling of Multiple Samples into One Heatmap (Fig. 3)
		3.8 Statistical Comparisons (Fig. 3)
		3.9 Extracting Morphometric Features of the Cells That Are Marked with Biomarkers (Fig. 4)
	4 Notes
	References
Guinea Pig Preimplantation Embryos: Generation, Collection, and Immunofluorescence
	1 Introduction
	2 Materials
		2.1 Guinea Pig Estrus Cycle Monitoring and Mating
		2.2 Euthanasia and Flushing In Vivo Embryos
		2.3 Processing Embryos: Immunofluorescence of Preimplantation Embryos
		2.4 In Vitro Culture from Morula to Blastocyst
	3 Methods
		3.1 Guinea Pig´s Estrus Cycle Monitoring and Mating
			3.1.1 Monitoring the Vaginal Membrane
			3.1.2 Mating
			3.1.3 Vaginal Flush and Smear
			3.1.4 Determining the Different Stages of the Estrous Cycle
		3.2 Euthanasia and Flushing In Vivo Embryos
			3.2.1 General Anesthesia of Guinea Pigs by Inhalation of Isoflurane
			3.2.2 Exsanguination by Cardiac Puncture
			3.2.3 Dissection
			3.2.4 Flushing
		3.3 Immunofluorescence of Preimplantation Embryos
			3.3.1 Fixation and Permeabilization
			3.3.2 Primary Antibody Incubation
			3.3.3 Secondary Antibody Incubation and Mounting
		3.4 In Vitro Culture from Compacted Morula to Blastocyst
	4 Notes
	References
Manual Dissociation of Mammalian Preimplantation Embryos for Single-Cell Genomics
	1 Introduction
	2 Materials
		2.1 Acquisition of Embryos
		2.2 Dissociation of Embryos into Single Cells
	3 Methods
		3.1 Acquisition of Embryos
			3.1.1 Superovulation Schedule
			3.1.2 Flushing
		3.2 Dissociation of Embryos into Single Cells
	4 Notes
	References
Whole-Mount RNA, Single-Molecule RNA (smRNA), and DNA Fluorescence In Situ Hybridization (FISH) in Mammalian Embryos
	1 Introduction
	2 Materials
		2.1 RNA/smRNA FISH of Embryos
		2.2 DNA FISH
	3 Methods
		3.1 RNA/mRNA FISH of Embryos
			3.1.1 Preparing Hybridization Buffer Stock
			3.1.2 Coverslip Cleaning
			3.1.3 Silanization of Coverslips
			3.1.4 Preparing Wash Solution
			3.1.5 RNA/sm RNA FISH
		3.2 DNA FISH
	4 Notes
	References
Index