Stepwise Culture of Human Adult Stem Cells

Cover
Half Title
Series Page
Title Page
Copyright Page
Table of Contents
Preface
List of Contributors
List of Figures
List of Abbreviations
Introduction: Core Techniques in Stem Cell Culture
	Introduction
	Adult Stem Cell
	Fetal Stem Cell
	Cancer Stem Cell (CSC)
	Other Supporting Methods
	References
Chapter 1: Isolation of Hematopoietic Stem Cells
	1.1: Introduction
	1.2: Materials
	1.3: Method
	1.4: Notes
	References
Chapter 2: Isolation Protocol for CD133+ and CD34+ Very Small Embryonic-like Cells (VSEL) from Human Umbilical Cord Blood
	2.1: Introduction
	2.2: Materials
		2.2.1: Human umbilical cord blood (hUCB)
		2.2.2: Reagents for hUCB CD34+ and CD133+ VSEL isolation
		2.2.3: Antibodies
		2.2.4: Plastics and equipment
	2.3: Methods
		2.3.1: Isolation of MNCs
		2.3.2: Staining for CD133+ or CD34+ VSELs
		2.3.3: Sorting protocol
	2.4: Notes
	References
Chapter 3: Isolation and Expansion of Mesenchymal Stem Cells from the Adult Bone Marrow
	3.1: Introduction
	3.2: Materials
	3.3: Methods
		3.3.1: Bone marrow collection (also Method 13)
		3.3.2: Plating bone marrow aspirates
		3.3.3: Red blood cell removal
		3.3.4: Culturing of MSCs
	3.4: Notes
	References
Chapter 4: Manual Isolation and Expansion of Adipose-derived Stem Cells from Adipose Tissue
	4.1: Introduction
	4.2: Materials
	4.3: Methods
		4.3.1: Preparation
		4.3.2: Lipoaspirate preparation
		4.3.3: Block adipose preparation
		4.3.4: Enzymatic digestion of adipose tissue (optional)
		4.3.5: ASC culture and expansion
	4.4: Notes
	References
Chapter 5: Isolating Compact Bone-derived Mesenchymal Stem Cells from Rodent and Rabbit Femurs
	5.1: Introduction
	5.2: Materials
		5.2.1: Reagents for CBM-MSC isolation
		5.2.2: Animals
		5.2.3: Materials
	5.3: Methods
		5.3.1: Isolating CBM MSCs
	5.4: Notes
	References
Chapter 6: Isolating Dental Pulp Stem Cells
	6.1: Introduction
	6.2: Materials
		6.2.2: Dental instruments
		6.2.3: DPSCs culture media
		6.2.4: Flow cytometry for phenotype
	6.3: Methods
		6.3.1: Tooth preparation
		6.3.2: Dental pulp extraction
		6.3.3: Dental pulp preparation
	6.4: Notes
	References
Chapter 7: A Murine System for Somatic Cellular Reprogramming into Induced Pluripotent Stem Cells
	7.1: Introduction
	7.2: Material
		7.2.1: Reprogrammable mouse model and reagents to prepare murine embryonic fibroblasts (MEFs)
		7.2.2: Preparation of 2i/LIF media supplemented with vitamin C
		7.2.3: Quantification of iPSC colonies by alkaline phosphatase (AP) staining
	7.3: Method
		7.3.1: Harvesting somatic cells from reprogrammable mouse model (see Notes 1 and 2)
		7.3.2: Somatic cell reprogramming into iPSCs using 2i/LIF media supplemented with Vitamin C
	7.4: Notes
	References
Chapter 8: Isolation and Expansion of Placental Stem Cells (P-MSCs)
	8.1: Introduction
	8.2: Reagents and Materials
		8.2.1: Placenta sample
		8.2.2: Reagents
		8.2.3: Materials and surgical accessories
		8.2.4: Equipment
	8.3: Method
		8.3.1: Placental collection and preparation
		8.3.2: Handle the placentas under standard conditions in a laminar flow hood
		8.3.3: Mechanical separation
		8.3.4: Explant method
		8.3.5: Type-II collagenase digestion method (Note 3)
		8.3.6: MSC characterization
		8.3.7: P-MSC multilineage differentiation
	8.4: Notes
	References
Chapter 9: Expanding Human Mesenchymal Stem Cells from the Umbilical Cord
	9.1: Introduction
	9.2: Method and Reagents
		9.2.1: Procuring the umbilical cord (UC)
		9.2.2: Reagents
		9.2.3: Tissue culture
	9.3: Notes
	References
Chapter 10: Laboratory Isolation of Leukemia Stem Cells
	10.1: Introduction
	10.2: Materials
		10.2.1: Flow cytometers
		10.2.2: Reagents
		10.2.3: Monoclonal antibody panels
		10.2.4: Sample
	10.3: Methods
		10.3.1: Preparation of the bone marrow (BM) sample
		10.3.2: Staining of WBC
		10.3.3: Flow cytometry LSC assessment
		10.3.4: Gating and data analysis
	10.4: Notes
	References
Chapter 11: Isolation and Characterization of Cancer Stem Cells from Glioblastoma Multiforme and Breast Cancer Cell Lines
	11.1: Introduction
	11.2: Materials
		11.2.1: Reagents for isolation of Oct4a-GFP reporter vector from transformed bacteria
		11.2.2: Reagents for isolation of SORE6-GFP reporter vector from transformed bacteria
		11.2.3: DNA quantification
		11.2.4: Reagents for transfection of BC cell lines
		11.2.5: Reagents for lentiviral packaging of SORE6-GFP intended for transduction of GBM cell lines
		11.2.6: Reagents for transduction of GBM cell lines
		11.2.7: Selection of Oct4a-GFP positive cells
		11.2.8: Selection of SORE6-GFP positive cells
		11.2.9: Validation of transfection/transduction
		11.2.10: Reagents for sorting buffer used for sample preparation
		11.2.11: Sorting of CSC-enriched cells based on Oct4a-GFP or SORE6-GFP expression by flow cytometry
		11.2.12: Carboplatin (Thermo Scientific, Cat. No. J60433.03)
		11.2.13: In vitro serial dilution and tumorsphere assay
		11.2.14: Female immune deficient BALB/cJ mice (The Jackson Laboratory, Strain No. 000651)
	11.3: Methods
		11.3.1: DNA isolation from transformed bacteria with Oct4a-GFP/SORE6-GFP vector
		11.3.2: Transfection of breast cancer cells (BCCs)
		11.3.3: Selection of Oct4a-GFP positive cell clones
		11.3.4: Transduction of GBM cell lines
		11.3.5: Transduction of GBM cell lines with SORE6-GFP virus
		11.3.6: Selection of clones to create GBM SORE6 cell lines
		11.3.7: Sorting of CSCs (GFPhigh) from Oct4-aGFP (BC-CSCs) and SORE6-GFP (GBM-CSCs)
		11.3.8: In vitro serial dilution
		11.3.9: Treatment of BCCs with carboplatin to select for CSCs
		11.3.10: Injection of Oct4ahigh into NSG mice
	11.4: Notes
	References
Chapter 12: Bone Marrow Aspirate for Isolation of Mesenchymal Stem Cells
	12.1: Introduction
	12.2: Materials
	12.3: Methods
		12.3.1: Human donor
		12.3.2: Bone marrow aspiration
	12.4: Notes
	References
Chapter 13: Culturing Human Bone Marrow Stromal Cells
	13.1: Introduction
	13.2: Materials and Reagents
		13.2.1: BMSC tissue culture media
		13.2.2: Complete BMSC media
	13.3: Method
	References
Chapter 14: Isolating Mononuclear Cells by Ficoll-Hypaque Density Gradient
	14.1: Introduction
	14.2: Materials
	14.3: Methods
		14.3.1: Sample
		14.3.2: Loading onto Ficoll-Hypaque (Note 2)
	14.4: Notes
	References
Chapter 15: Phenotypic and Multipotent Characterization of Bone-Marrow-derived Mesenchymal Stem Cells
	15.1: Introduction
	15.2: Materials and Reagents
		15.2.1: Flow cytometry
		15.2.2: Tri-lineage differentiation
		15.2.3: Peptidergic neuronal transdifferentiation
	15.3: Methods
		15.3.1: Flow cytometry
		15.3.2: Tri-lineage differentiation capacity of the isolated MSCs
		15.3.3: Neuronal transdifferentiation of the MSCs
	15.4: Notes
	References
Chapter 16: Cryopreservation of Stem Cells
	16.1: Introduction
	16.2: Materials
	16.3: Methods
	16.4: Notes
	References
Chapter 17: Cryopreservation of Mobilized Peripheral Blood Stem Cells
	17.1: Introduction
	17.2: Method
		17.2.1: Reagents
		17.2.2: Samples
		17.2.3: Preparation of cells for cryopreservation
		17.2.4: Thawing cryopreserve cells
	17.3: Notes
	References
Chapter 18: Manual Cell Count
	18.1: Introduction
	18.2: Prerequisite Skills
	18.3: Reagents
	18.4: Method
	18.5: Notes
Index
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