Neural Reprogramming: Methods and Protocols (Methods in Molecular Biology, 2352)

✍ Scribed by Henrik Ahlenius (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 267
• Category: Library

✦ Synopsis

This detailed book brings together a number of state-of-the-art protocols to generate different types of neural cells through the use of reprogramming technologies. Additionally, the volume explores different aspects of functional evaluation and applications of reprogrammed neural cells as well as in silico methods to aid reprogramming efforts. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, Neural Reprogramming: Methods and Protocols provides ample experimental experience and guidance for anyone, be it experienced researcher or beginner, to generate, validate, and apply reprogrammed neural cells in their research.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Isolation and Neuronal Reprogramming of Mouse Embryonic Fibroblasts
1 Introduction
2 Materials
2.1 MEF Isolation and Culture
2.1.1 Equipment
2.1.2 Solutions
2.1.3 Other Materials
2.2 Lentivirus Production
2.2.1 Equipment
2.2.2 Solutions
2.2.3 Other Materials
2.3 Neuronal Reprogramming
2.3.1 Equipment
2.3.2 Solutions
2.3.3 Other Materials
3 Methods
3.1 MEF Isolation
3.2 MEF Passaging and Cryopreservation
3.3 Lentiviral Production
3.4 MEF Transduction and Neuronal Reprogramming
4 Notes
References
Chapter 2: Direct In Vitro Reprogramming of Astrocytes into Induced Neurons
1 Introduction
2 Materials
2.1 Experimental Animals
2.2 Media and Solutions
2.2.1 Dissection of the Postnatal Mouse Neocortex
2.2.2 Primary Astroglia Culture: Preparation and Maintenance
2.2.3 Retroviral Transduction or Plasmid Transfection of Astroglial Cells
2.3 Equipment
3 Methods
3.1 Dissection of the Postnatal Mouse Neocortex
3.2 Preparation of Astroglia Primary Culture
3.3 Preparation of Glass Coverslips
3.4 Passaging and Plating of Astroglial Cells
3.5 Retroviral Transduction or Plasmid Transfection of Astroglial Cells
3.6 Astroglia-to-Neuron Conversion
3.7 Discussion and Prospects
4 Notes
References
Chapter 3: Direct Cell Reprogramming of Mouse Fibroblasts into Functional Astrocytes Using Lentiviral Overexpression of the Tr...
1 Introduction
2 Materials
2.1 Lentivirus Production and Validation
2.2 Cell Culture and Reprogramming
2.3 Immunofluorescence Staining
2.4 Calcium Imaging
3 Methods
3.1 Virus Production
3.2 Virus Validation
3.3 Reprogramming of MEFs into Induced Astrocytes
3.4 Immunofluorescence Staining
3.5 Calcium Imaging
4 Notes
References
Chapter 4: Direct Reprogramming of Fibroblasts to Astrocytes Using Small Molecules
1 Introduction
2 Materials
3 Methods
3.1 Isolation of MEFs
3.2 Direct Reprogramming Mouse Fibroblasts into Astrocytes
3.3 Characterization of Induced Astrocytes
3.3.1 Immunocytochemistry
3.3.2 Real-Time PCR
3.3.3 Glutamate Uptake Assay
3.3.4 Ca2+ Imaging
3.4 Astrocyte-Neuron Co-culture
3.4.1 Mouse Cortical Neuron Isolation
3.4.2 Astrocyte-Neuron Co-culture
3.5 Astrocyte Transplantation
4 Notes
References
Chapter 5: Bcl-2-Assisted Reprogramming of Mouse Astrocytes and Human Fibroblasts into Induced Neurons
1 Introduction
2 Materials
2.1 Animals
2.2 Astrocyte Isolation
2.3 Cell Culture and Reprogramming
2.3.1 Common Materials for Astrocytes and Fibroblasts
2.3.2 Materials Specifically Required for Astrocyte Culture and Reprogramming
2.3.3 Materials Specifically Required for Fibroblast Culture and Reprogramming
3 Methods
3.1 Isolation and Culture of Primary Cortical Astroglia from Postnatal Mice
3.2 Passaging and Plating Astroglial Cells
3.2.1 PDL Coating of Coverslips
3.2.2 Laminin Coating of PDL-Coated Coverslips Required for the Fibroblast Reprogramming Procedures
3.2.3 Passaging Astroglial Cells
3.3 Transfection of Astroglial Cells for Neuronal Conversion
3.4 Plating and Passaging Human Skin Fibroblasts
3.4.1 Collagen IV Coating of T-75 Flasks and Multi-well 6 Plates
3.4.2 Culturing Human Fibroblasts in T-75 Flasks
3.4.3 Expanding Human Fibroblasts in T-75 Flasks
3.5 Neuronal Reprogramming of Human Fibroblasts
3.5.1 Retroviral Transduction of Human Fibroblasts
3.5.2 Using Astrocytes as Feeder Cells and to Produce Conditioned Medium for Fibroblast Reprogramming
3.5.3 Progression of Fibroblast-to-Neuron Conversion
4 Notes
References
Chapter 6: Direct Conversion of Human Fibroblasts to Induced Neurons
1 Introduction
2 Materials
2.1 Derivation of Dermal Fibroblast from Skin Punch Biopsy
2.2 Fibroblast Thawing, Splitting, and Freezing
2.3 UNA-Lentivirus Production
2.4 Fibroblast Transduction with UNA-Lentivirus
2.5 Direct Conversion to iNs
2.6 Live-Cell Fluorescence-Activated Cell Sorting (FACS)
3 Methods
3.1 Derivation of Dermal Fibroblasts from Skin Punch Biopsy
3.2 Fibroblast Thawing, Splitting, and Freezing
3.2.1 Thawing Fibroblasts
3.2.2 Passaging Fibroblasts (Splitting)
3.2.3 Freezing Fibroblasts
3.3 UNA-Lentivirus Production
3.3.1 HEK 293T Cell Preparation
3.3.2 Transfection of HEK293T
3.3.3 UNA-Lentivirus Harvesting
3.4 Fibroblast Transduction with UNA-Lentivirus
3.5 Direct Conversion to iNs
3.6 Live-Cell Fluorescence-Activated Cell Sorting (FACS)
4 Notes
References
Chapter 7: Generation of Induced Dopaminergic Neurons from Human Fetal Fibroblasts
1 Introduction
1.1 Direct Induction of Neurons
1.2 Direct Induction of iNs with Pan-Neuronal Properties
1.3 Direct Induction of Subtype-Specific Neurons
1.3.1 Induced Cholinergic and Spinal Motor Neurons
1.3.2 Peripheral Sensory Neurons
1.3.3 Serotoninergic
1.3.4 Medium Spiny Neurons
1.3.5 Forebrain GABAergic Neurons
1.3.6 Noradrenergic
1.4 Direct Induction of Dopaminergic Neurons
2 Materials
3 Methods
3.1 Seeding of Human Fetal Lung Fibroblasts for Conversion to Induced DA Neurons
3.2 Plating the Cells for Reprogramming
3.3 Fibroblast´s Freezing
3.4 Viral Transduction (Day 0) and Conversion Induction (Day 5)
3.5 Cell Maintenance During Reprogramming
4 Notes
References
Chapter 8: Direct Differentiation of Functional Neurons from Human Pluripotent Stem Cells (hPSCs)
1 Introduction
2 Materials
2.1 Lentivirus Production
2.2 Mouse Glial Cell Isolation Reagents
2.3 Cell Culture
2.4 Immunofluorescence
3 Methods
3.1 Lentivirus Production
3.2 Glial Cell Isolation
3.3 Transduction
3.4 iN Cell Maturation
3.5 Basic Characterization of iN Cells Using Immunofluorescence Analysis
4 Notes
References
Chapter 9: Generation of Motor Neurons from Human ESCs/iPSCs Using Sendai Virus Vectors
1 Introduction
2 Materials
2.1 Culture of ESCs/iPSCs
2.1.1 Cells
2.1.2 Reagents
2.1.3 Instruments
2.2 Generation of MNs
2.2.1 Reagents
2.2.2 Instruments
2.3 Immunostaining
2.3.1 Reagents
3 Methods
3.1 Culture of Human ESCs/iPSCs
3.2 Generation of MNs from Human ESCs/iPSCs
3.3 Immunostaining
4 Notes
References
Chapter 10: Transcription Factor Programming of Human Pluripotent Stem Cells to Functionally Mature Astrocytes for Monoculture...
1 Introduction
2 Materials
2.1 Equipment
2.2 Factors
2.3 Reagents
2.4 Solutions
2.5 Media
2.6 Plasmids
3 Methods
3.1 Lentivirus Production
3.2 Glass Coverslip Cleaning
3.3 Generation of iAs
3.4 Cryopreservation
3.4.1 Freezing Procedure
3.4.2 Thawing Procedure
3.5 Cocultures with iNs
4 Notes
References
Chapter 11: Single Transcription Factor-Based Differentiation Allowing Fast and Efficient Oligodendrocyte Generation via SOX10...
1 Introduction
2 Materials
2.1 Lab Materials
2.2 Buffers, Solutions, and Media
2.3 Equipment
3 Methods
3.1 Generation of Inducible SOX10 Lentiviral Particles
3.1.1 Lentiviral Particle Production
3.1.2 Lentiviral Particle Titration
3.2 Recombinase-Mediated Cassette Exchange (RMCE) to Generate Doxycycline-Inducible SOX10 Overexpression hPSC Line
3.3 Differentiation of hPSCs Toward O4+ Early Oligodendrocytes
3.3.1 MACS Purification
3.3.2 Cryopreservation of O4+ Cells
3.4 Generation of Cortical Neurons and Co-culturing with Early Oligodendrocytes
4 Notes
References
Chapter 12: Transcriptional Profiling During Neural Conversion
1 Introduction
2 Materials
3 Methods
3.1 Bulk RNA-Seq of Reprogrammed Cells
3.2 Single-Cell Transcriptomic Analysis of Neuronal Conversion
References
Chapter 13: Functional Assessment of Direct Reprogrammed Neurons In Vitro and In Vivo
1 Introduction
2 Materials
2.1 Lentiviral Production
2.1.1 Equipment
2.1.2 Reagents
2.2 Adeno-Associated Virus (AAV) Production
2.2.1 Equipment
2.2.2 Reagents
2.3 Cell Culture
2.3.1 Equipment
2.3.2 Reagents
2.4 Electrophysiology
2.4.1 Equipment
2.4.2 Solutions
3 Methods
3.1 Coverslip Preparation for Long-Term In vitro Cultures
3.2 Labeling of iNs In Vitro
3.2.1 Lentiviral Production
3.2.2 Lentiviral Titration
3.2.3 Viral Transduction
3.3 Labeling of iNs In Vivo
3.3.1 AAV Vector Cloning
3.3.2 AAV Vector Production
3.3.3 AAV Viral Vector Purification
3.3.4 AAV Titration
3.3.5 AAV Preparation for Surgery
3.4 Tissue Slice Preparation for In Vivo iNs
3.5 Whole-Cell Patch-Clamp Recordings
3.6 Post Hoc Identification of Cells
3.7 Analysis of Functional Characteristics of iNs
3.7.1 Post Hoc Analysis of iNs
4 Notes
References
Chapter 14: Neurotransmitter Release of Reprogrammed Cells Using Electrochemical Detection Methods
1 Introduction
2 Materials
2.1 Reference Electrodes
2.1.1 Equipment
2.1.2 Solutions
2.2 Cleaning of Electrodes
2.2.1 Equipment
2.2.2 Solutions
2.3 Application of Exclusion Layer
2.3.1 Equipment
2.3.2 Solutions
2.4 Preparation of Glutamate Oxidase
2.4.1 Equipment
2.4.2 Solutions
2.5 Application of Enzyme/Protein Matrix
2.5.1 Equipment
2.5.2 Solutions
2.6 Calibration of Electrodes
2.6.1 Equipment
2.6.2 Solutions
2.7 In Vitro Recordings
2.7.1 Equipment
2.7.2 Solutions
3 Methods
3.1 Reference Electrodes
3.1.1 Reference Electrode for Calibration
3.1.2 Reference Electrode for Recording
3.2 Cleaning of MEA Electrodes
3.2.1 Chemical Cleaning (New Electrodes)
3.2.2 Deep Cleaning (After Enzyme-Based Recording)
3.3 Application of Exclusion Layer
3.3.1 Nafion
3.3.2 mPD (1,3-Phenylenediamine)
3.4 Enzyme Preparation
3.5 Coating of Electrodes
3.5.1 Application of Protein Matrix (Glutaraldehyde/BSA Solution)
3.5.2 Application of Enzyme Layer
3.6 Calibration of Electrodes
3.7 In Vitro Recordings
3.8 Analysis
3.9 Future Outlook
4 Notes
References
Chapter 15: Combining Cell Fate Reprogramming and Protein Engineering to Study Transcription Factor Functions
1 Introduction
2 Materials
2.1 Fusion Construct Design
2.2 Molecular Cloning
2.2.1 Equipment
2.2.2 Solutions
2.2.3 Other Materials
3 Methods
3.1 Conceptual Design of the Fusion Constructs
3.2 Sequence Assembly and Primer Design
3.3 Fusion Construct Cloning: First PCR Reaction
3.4 Fusion Construct Cloning: Nested PCR Reaction
4 Notes
References
Chapter 16: CRISPR/Cas9 Genome Engineering in Human Pluripotent Stem Cells for Modeling of Neurological Disorders
1 Introduction
2 Materials
2.1 Media
2.2 Reagents
2.3 Plasmids
3 Methods
3.1 CRISPR/Cas9 Strategy and Design
3.2 Cloning of sgRNAs into the pX Vectors
3.3 Analysis of sgRNA Efficiency
3.4 ssODN Design
3.5 CRISPR/Cas9 Targeting
3.5.1 hPSC Transfection
3.5.2 Selection, Expansion, and Clone Analysis
4 Notes
References
Chapter 17: Derivation of Adult Human Cortical Organotypic Slice Cultures for Coculture with Reprogrammed Neuronal Cells
1 Introduction
2 Materials
2.1 Tissue Collection
2.2 Tissue Cutting
2.3 Slice Culture
3 Method
4 Notes
References
Correction to: Functional Assessment of Direct Reprogrammed Neurons In Vitro and In Vivo
Index