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Stem cell laboratory techniques : a guide for researchers and students /

Stem Cell Laboratory Techniques: A Guide for Researchers and Students introduces the reader to stem cell culture, handling techniques and versatile applications used by researchers. Sections introduce stem cells, including definitions, types and basic use of stem cells in biomedical science research...

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Bibliographic Details
Corporate Author: ScienceDirect (Online service)
Other Authors: Subbiah, Suresh Kumar
Format: eBook
Language:English
Published: London ; San Diego, CA : Academic Press, an imprint of Elsevier, [2023]
Subjects:
Stem cells > Research > Methodology.
Stem Cells
Cellules souches > Recherche > Méthodologie.
Cellules souches.
Electronic books.
Online Access:Connect to the full text of this electronic book
Table of Contents:
  • Intro
  • Stem Cell Laboratory Techniques: A Guide for Researchers and Students
  • Copyright
  • Contents
  • Contributors
  • About the editors
  • Chapter 1: Introduction: Stem cells and their application in research and therapy
  • 1. Definition and characteristics of stem cells
  • 2. Classification of stem cells
  • 2.1. Classification based on differentiation potential
  • 2.2. Classification based on cell sources
  • 2.3. The classical types of stem cells
  • 2.3.1. Embryonic stem cells (ESCs)
  • 2.3.2. Mesenchymal stem cells (MSCs)
  • 2.3.3. Induced pluripotent stem cells (iPSCs)
  • 3. Research and application of stem cells
  • 3.1. Developmental biology research
  • 3.2. Application in drug screening and development
  • 3.3. Application in regenerative medicine
  • 4. Conclusion
  • References
  • Chapter 2: Animal stem cells: Extraction, expansion, and cryopreservation
  • 1. Introduction
  • 1.1. Embryonic stem cells (ESCs)
  • 1.2. Adult stem cells (ASCs)
  • 2. Materials, reagents, chemicals, apparatus, and kits
  • 2.1. Embryonic stem cells(ESCs) isolation
  • 2.2. Mesenchymal stem cells (MSCs) isolation from umbilical cord
  • 2.3. Stem cell isolation from bone marrow
  • 2.4. Isolation ofHSCs derived from bone marrow or umbilical cord stem cells
  • 2.5. Stem cell isolation from peripheral blood
  • 2.6. Stem cell isolation from adipose tissue
  • 2.7. Neural stem cell isolation
  • 2.8. Stem cell isolation from epidermis
  • 2.9. Cryopreservation
  • 3. Procedure
  • 3.1. Embryonic stem cells (ESCs) isolation
  • 3.2. Stem cell isolation from umbilical cord
  • 3.3. Stem cell extraction from bone marrow
  • 3.4. Isolation of HSCs derived from bone marrow or umbilical cord stem cells
  • 3.5. Stem cell isolation from peripheral blood
  • 3.6. Stem cell isolation from adipose tissue
  • 3.7. Neural stem cell isolation
  • 3.8. Stem cell isolation from epidermis.
  • 3.9. Cryopreservation
  • 4. Conclusion
  • References
  • Chapter 3: Protocols in stem cell culture
  • 1. Literature review
  • 1.1. Mesenchymal stem cells (MSCs)
  • 1.1.1. Bone marrow-derived MSCs (BMSCs)
  • 1.1.2. Human umbilical-cord-blood-derived mesenchymal stem cells (hUCB-MSCs)
  • 1.1.3. Adipose tissue-derived stem cells (ADSCs)
  • 1.1.4. Corneal stem cells
  • 1.2. Hematopoietic stem cells
  • 1.3. Biomaterials
  • 1.3.1. Stem cells cultivation and harvest on photo-responsive biomaterials
  • 1.3.2. Stem cell cultivation and harvest on thermoresponsive biomaterials
  • 1.3.2.1. Thermoresponsive polysaccharide interface
  • 1.3.2.2. Synthetic thermoresponsive polymer interface
  • 1.3.2.3. Thermoresponsive poly(N-isopropyl acrylamide) interface
  • 2. MSC-isolation of prime cells, cell culture and identification
  • 2.1. Bone marrow-derived MSCs
  • 2.1.1. Isolation of primary human BMSCs
  • 2.1.2. Cell culture and freezing
  • 2.2. Umbilical cord blood MSCs
  • 2.2.1. Isolation of primary human umbilical-cord-blood-derived mesenchymal stem cells (hUCB-MSCs)
  • 2.2.2. Cell culture and freezing
  • 2.3. Adipose tissue-derived stem cells
  • 2.3.1. Isolation of primary ADSCs
  • 2.3.2. Cell culture and freezing
  • 2.4. Corneal stem cells
  • 2.4.1. Isolation of primary corneal stem cells
  • 2.4.2. Cell culture and freezing
  • 2.5. Identification of MSCs
  • 2.5.1. Flow cytometry identification
  • 2.5.2. RNA extraction and qPCR
  • 3. Hematopoietic stem cells
  • 3.1. Apheresis
  • 3.1.1. Isolation of primary umbilical cord blood MSCs
  • 3.1.2. Cell culture and freezing
  • 3.1.3. Flow cytometry identification
  • 4. Materials, reagents, chemicals, apparatus, and kits
  • 4.1. Thermoresponsive poly(butylacrylate-co-N-isopropylacrylamide)-coated interface
  • 4.2. Procedure
  • 4.2.1. Preparation process of PNB-coated interface.
  • 4.2.2. Characterization of PNB-coated interface
  • 4.2.3. Preparation and culture of hESCs and hiPSCs
  • 5. Conclusion
  • References
  • Chapter 4: Isolation of human stem cells from full-term gestation amniotic fluid using immunoselection and one-stage cultur
  • 1. Introduction
  • 2. Materials, reagents, chemicals, apparatus, and kits
  • 2.1. Chemicals
  • 2.2. Kits
  • 2.3. Solution and reagent preparation
  • 2.4. Procedure
  • 2.4.1. Collection of the amniotic fluid (AF)
  • 2.4.2. Primary culture of amniotic fluid (AF) cells
  • 2.4.3. Immunoselection of c-kit+ human full-term amniotic fluid stem cells (hAFSCs)
  • 2.4.4. One-stage culture of human full-term AFSCs
  • 2.4.5. Propagation of full-term human AFSCs
  • 3. Characterization of c-kit cells/AFSCs
  • 3.1. Cell morphology
  • 3.2. Population doubling time
  • 3.3. Growth kinetic analysis
  • 3.4. RNA and protein expression
  • 3.5. In vitro differentiation into adipogenic, chondrogenic, and osteogenic lineage
  • 4. Conclusion
  • References
  • Chapter 5: Isolation and characterization of c-kit+ stem cells from rat full-term gestation amniotic fluid
  • 1. Introduction
  • 2. Materials, reagents, chemicals, apparatus, and kits
  • 2.1. Chemicals
  • 2.2. Preparation of solutions and reagents
  • 2.3. Procedure
  • 2.3.1. Collection of amniotic fluid
  • 2.3.2. Propagation of rat full-term amniotic fluid cells
  • 2.3.3. Isolation of c-kit+ cells/AFSCs
  • 2.3.4. Characterization of c-kit+ cells/AFSCs
  • 3. Conclusion
  • References
  • Further reading
  • Chapter 6: Human induced pluripotent stem cells (hiPSCs): Generation, characterization, and differentiation
  • 1. Introduction
  • 2. Literature review
  • 2.1. HiPSC generation method
  • 2.2. Generation of HiPSCs using small bioactive molecules
  • 2.3. Human iPSCs induced by small bioactive molecules in combination with a few transcription factors.
  • 2.4. Cell culture biomaterials for hiPSC culture
  • 2.5. HiPSC cultivation on ECM-immobilized biomaterials
  • 3. Characterization of hiPSCs
  • 4. Materials, reagents, chemicals, apparatus and kits
  • 5. Procedure
  • 5.1. Generation of hiPSCs using Sendai virus transfection kit
  • 5.2. Induction of hiPSC differentiation into cardiomyocytes
  • 5.3. Induction of hiPSC differentiation into MSCs
  • 6. Conclusion
  • References
  • Chapter 7: Confirmation of stem cells pluripotency via teratoma formation in an animal model**Important note: Animal rese ...
  • 1. Establishment of teratoma in small animal model
  • 1.1. Introduction
  • 1.2. Materials
  • 1.3. Protocols
  • 2. Histological examination on formed teratoma
  • 2.1. Introduction
  • 2.2. Materials
  • 2.3. Protocols
  • 2.3.1. Harvest and processing of teratoma
  • 2.3.2. Embedding of teratoma tissues
  • 2.3.3. Sectioning of teratoma tissues
  • 2.3.4. H&amp
  • E staining of teratoma slides
  • 3. Conclusion
  • References
  • Chapter 8: Gene editing for stem cells by CRISPR-Cas9
  • 1. Introduction
  • 1.1. CRISPR background
  • 1.2. CRISPR-Cas9 natural pathway
  • 1.3. Fundamental process of DNA repair
  • 1.4. Validation of CRISPR editing
  • 2. Materials
  • 2.1. Cell culture
  • 2.2. Transfection
  • 2.3. Validation of CRISPR editing
  • 2.3.1. T7 endonuclease 1 assay
  • 2.3.2. RT-qPCR
  • 2.3.3. Western blot
  • 3. Procedure
  • 3.1. Cell culture
  • 3.2. Gene editing of stem cells
  • 3.2.1. Cell seeding
  • 3.2.2. Transfection
  • 3.3. Validation of CRISPR editing
  • 3.3.1. T7 endonuclease 1 assay
  • 3.3.2. RT-qPCR
  • 3.3.3. Western blot
  • 4. Analyzation after gene editing: Transcriptomic analysis
  • 4.1. RNA-Seq and data processing pipelines
  • 5. Conclusion
  • References
  • Further reading
  • Chapter 9: Hypoxic environment: A new solution to stem cell culture
  • 1. Introduction.
  • 2. Influence of hypoxia condition on stem cell culture
  • 3. Procedural guide on choosing a hypoxic incubator
  • 4. Standard operation procedure/maintenance of a hypoxic incubator
  • 5. Conclusion
  • References
  • Chapter 10: Mouse embryonic stem cell-derived cerebral organoids
  • 1. Introduction
  • 2. Methods
  • 2.1. Preparation of media and reagents
  • 2.1.1. Media
  • Embryonic stem cell medium
  • Freezing medium
  • Knockout serum free medium
  • Cortical maturation medium
  • 2.1.2. Other reagents
  • Embedding medium
  • 2.2. Mouse embryonic stem cell culture
  • 2.2.1. Maintenance of embryonic stem cells
  • 2.2.2. Subculture
  • 2.2.3. Cryopreservation and thawing
  • 2.3. Cerebral organoids 3-dimensional (3D) culture
  • 2.4. Tissue processing
  • 2.4.1. Fixation
  • 2.4.2. Cryoprotection, embedding and cryosectioning
  • 2.5. Sources of materials
  • References
  • Chapter 11: Standardized practice of stem cell production
  • 1. Cell line identification and authentication by short tandem repeat (STR) DNA profiling
  • 1.1. Background and principle
  • 1.2. Protocol
  • 1.2.1. Cell harvest
  • 1.2.2. Spotting cells onto FTA card
  • 1.2.3. Amplification setup
  • 1.2.4. Thermal cycling
  • 1.2.5. Using the applied biosystems 3500 or 3500xL genetic analyzer for detection of amplified fragments
  • Preparation of sample
  • Instrument preparation and use
  • 1.2.6. Analysis of data
  • 1.2.7. Interpretation of data
  • 2. Human leukocyte antigen (HLA) analysis procedure-Polymerase chain reaction-based sequence-specific primers (PCR-SSP) m ...
  • 2.1. Background and principle
  • 2.2. Protocol
  • 2.2.1. Polymerase chain reaction (PCR)
  • 2.2.2. Gel electrophoresis
  • 2.2.3. Gel documentation
  • 2.2.4. Results evaluation and interpretation
  • 2.2.5. Interpretation of results on the gel based on reaction pattern
  • 3. Karyotyping
  • 3.1. Background and principle.