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Cover
Title Page
1 Introduction
1. 1.1 Biosafety
2. 1.2 Biosafety Levels
3. 1.3 Aseptic Technique
4. 1.4 Storage
5. 1.5 Contamination
6. 1.6 Pluripotent Stem Cells
7. 1.7 Procedures
8. References
2 Pluripotent Stem Cell Culture
1. 2.1 Introduction
2. 2.2 Materials
3. 2.3 Solutions
4. 2.4 Methods
5. References
3 Reprogramming
1. 3.1 Introduction
2. 3.2 Materials
3. 3.3 Solutions
4. 3.4 Methods
5. References
4 Characterization
1. 4.1 Introduction
2. 4.2 Materials
3. 4.3 Solutions
4. 4.4 Methods
5. References
5 Differentiation
1. 5.1 Introduction
2. 5.2 Materials
3. 5.3 Solutions
4. 5.4 Methods
5. References
Index
End User License Agreement

List of Tables

Chapter 01
1. Table 1.1 Overview of pluripotent stem cell (PSC) culture manipulations per week.
Chapter 02
1. Table 2.1 Volume of attachment factor for iMEF culture.
2. Table 2.2 Media volume recommendations for seeding of iMEFs.
3. Table 2.3 iMEF cell concentrations per vessel configuration.
4. Table 2.4 Volume of Geltrex for coating of tissue culture vessels.
5. Table 2.5 Vitronectin volume recommendations for PSC culture.
6. Table 2.6 Laminin 521 volume recommendations for PSC culture (2.5 µg/mL).
7. Table 2.7 Volume of collagenase IV for PSC enzymatic passaging.
8. Table 2.8 Media volume recommendations for PSC culture.
9. Table 2.9 D‐PBS and EDTA volume recommendations for PSC culture.
10. Table 2.10 Essential 8 medium volume recommendations for PSC culture.
11. Table 2.11 Cryopreservation medium volume recommendations for cryopreserving feeder‐free PSCs.
Chapter 03
1. Table 3.1 Formula to calculate the volume of each virus needed to reach the target MOI(s).
2. Table 3.2 RT‐PCR primers to detect CytoTune Sendai vectors.
3. Table 3.3 Volumes for PCR reaction (Sendai vector detection).
4. Table 3.4 Conditions for PCR reaction (Sendai vector detection).
5. Table 3.5 Neon settings for Epi5 electroporation of human dermal fibroblasts or CD34+ cells.
6. Table 3.6 PCR primers for detection of Epi5 vectors.
7. Table 3.7 Volumes for PCR (Epi5 vector detection).
8. Table 3.8 Conditions for PCR (Epi5 vector detection).
Chapter 04
1. Table 4.1 Volume of collagenase IV for PSC enzymatic passaging.
2. Table 4.2 Volumes for 2× RT Master Mix.
3. Table 4.3 Thermal Cycler condition for RT reaction.
4. Table 4.4 TaqMan ScoreCard cycling parameters.

List of Illustrations

Chapter 01
1. Figure 1.1 Morphology of intact PSC. A microscope with a 4× or 5× objective and phase contrast or DIC is ideal to observe PSC morphology. Bright‐field images at 50× magnification and 100× magnification are difficult to observe. Phase contrast images at 50× magnification and 100× magnification are preferable to be able to determine morphology (a). 50× magnified phase contrast image of PSC on mitotically inactivated MEF feeders (b). 100× magnified phase contrast image of feeder‐dependent PSCs (c).
2. Figure 1.2 Morphology of differentiating PSC. 100× magnified phase contrast image of feeder‐dependent PSC with early signs of differentiation, marked by stray edges and appearance of large flattened cells (a). 100× magnified phase contrast image of feeder‐dependent PSC with large areas of differentiating cells (b).
3. Figure 1.3 Monitoring PSC after passage. 50× magnified phase contrast images of feeder‐dependent PSC culture at 24 hours post passage (a), showing colonies with good morphologies (top panels i–iii) and bad morphologies (bottom panels iv–vi). 50× magnified phase contrast images of PSC colonies (b) at day 2 (i) and day 3 (ii) after passaging, showing clear increases in colony size while maintaining morphology. 50× magnified phase contrast images of PSC colonies at day 4 post passage (c) illustrating presence of large colonies (i) and areas with neighboring colonies beginning to grow into each other (ii).
4. Figure 1.4 Observing feeder‐free PSC cultures. 50× magnified phase contrast image of feeder‐free PSCs with ideal PSC morphology characterized by sharp edges and compact cells growing as a monolayer (a). 50× magnified phase contrast image of feeder‐free PSC with large areas of differentiated cells characterized by heterogeneous central core not typical of pluripotent stem cells and presence of large single cells or small flattened colonies (b).
Chapter 02
1. Figure 2.1 50× magnified phase contrast image of iMEFs showing expected density and uniformity of coating for feeder‐dependent PSC culture.
2. Figure 2.2 50 × magnified phase contrast image of uniform‐sized PSC clusters right after thaw, seeded onto iMEF‐coated dishes.
3. Figure 2.3 50× magnified phase contrast images of PSC colonies harvested in bulk using collagenase IV for 30–60 minutes, showing different stages of PSC colony detachment. First the edges of the colony will appear to thicken and start moving inwards (a). As incubation time increases, the edges will continue to move inwards (b,c), and then eventually the colony will curl up and detach from the dish (d,e), leaving behind iMEFs (f). The ideal time to harvest colonies is when the colonies just begin to curl up (b,c), and they should be gently dislodged with a 5 mL pipette.
4. Figure 2.4 50× magnified phase contrast image shows a PSC colony manually dissected using a needle and syringe (a). Image shows straight cuts in horizontal and vertical directions creating a checkerboard pattern of small cell clusters ready to be harvested, and then reseeded for expansion and passaging. 100× magnified phase contrast image of a PSC colony where the entire dish was scored using the StemPro EZPassage tool; the resulting clumps of the colony are ready to be harvested and transferred to a fresh iMEF‐coated dish (b).
5. Figure 2.5 50× magnified phase contrast images of feeder‐free PSCs grown in Essential 8 medium and vitronectin. Images are of cells immediately after passaging and seeding onto a fresh VTN‐coated plate (a), and at days 1–5 (b–f) after seeding.
6. Figure 2.6 50× magnified phase contrast images of PSCs from the same starting population, adapted using the double sedimentation protocol from feeder‐based cultures into different feeder‐free media and matrices, including KSR‐based medium and iMEFs (a), Essential 8 medium and vitronectin (b), and Essential 8 medium and Geltrex (c).
Chapter 03
1. Figure 3.1 The CytoTune‐iPS 2.0 Sendai reprogramming kit contains three SeV‐based reprogramming vectors that have been optimized for generating iPSC from human somatic cells; the first vector contains KOS (KLF4, OCT4, and SOX2); the second vector contains c‐MYC; and the third vector contains additional KLF4 to achieve higher reprogramming efficiency.
2. Figure 3.2 Schematics of reprogramming workflows for fibroblast and blood cells in feeder‐dependent (F) and feeder‐free conditions (ff).
3. Figure 3.3 BJ fibroblasts seeded at desired densities, at 50× magnification (a) and 100× magnification (b).
4. Figure 3.4 Time course of fibroblast reprogramming under feeder‐dependent conditions. 50× phase contrast images: after seeding CytoTune transduced fibroblasts onto iMEF‐coated dishes (day 7), and images of emerging iPSC colonies from day 9 to day 21.
5. Figure 3.5 Time course of fibroblast reprogramming under feeder‐free conditions. 50× phase contrast images: after seeding CytoTune transduced fibroblasts onto Geltrex‐coated dishes (day 7) and images of emerging iPSC colonies from day 9 to day 21.
6. Figure 3.6 50× magnification images of BJ HDFn cells transduced with the CytoTune‐EmGFP Sendai Fluorescence Reporter at the indicated MOI 1 (a,b) or MOI 5 (c,d) and imaged at the indicated time post‐transduction – 24 h (a,c) or 48 hours (b,d).
7. Figure 3.7 Epi5 reprogramming kit comprising five vectors in two tubes. Reprogramming vector tube containing a mixture of three plasmids that code for OCT3/4, SOX2, KLF4, L‐MYC, and LIN28 (a); second tube containing a mixture of two plasmids that code for the p53 dominant negative mutant and EBNA1 (b).
8. Figure 3.8 100× magnification images of feeder‐dependent emerging iPSC colonies stained with AP Live Stain. Phase contrast (a), AP Live Stain (b), and merge of images (a) and (b) (c).
9. Figure 3.9 Terminal AP staining of BJ fibroblasts reprogrammed (day 21) with CytoTune 2.0, using feeders and KSR‐based medium (left) and vitronectin and Essential 8 medium (right). Colonies can be manually counted.
Chapter 04
1. Figure 4.1 Schematic of comprehensive characterization.
2. Figure 4.2 AP 100× magnification images of live staining of established iPSCs grown in feeder‐free conditions on Essential 8 medium and Geltrex. PSC colony morphology via phase contrast (a), AP Live Stain (b), and merge of images (a) and (b) (c).
3. Figure 4.3 Surface antibody staining with antibodies against surface antigens specific to the hPSC self‐renewal surface markers SSEA4 (a) and TRA‐1‐60 (b).
4. Figure 4.4 iPSC on feeders stained with a positive marker that specifically stains PSCs and not fibroblast feeders (SSEA4) and a negative marker that does not stain PSCs but does stain fibroblast feeders (CD44). Phase contrast (a), CD44‐AlexaFluor 488 (b), SSEA4‐AlexaFluor647 (c), merge of images (a–c) (d). All images were captured at 40× magnification.
5. Figure 4.5 Intracellular staining of hPSCs grown on feeders with Oct4, phase contrast (a), OCT4‐AlexaFluor 594 (b), counterstained with DAPI (c), and merge of (a–c) (d). All images were captured at 40× magnification.
6. Figure 4.6 Flow cytometry analysis of PSCs. Flow cytometry analysis of undifferentiated PSCs with >85% TRA‐1‐60 self‐renewal marker expression and less than 5% SSEA‐1 (differentiation marker) expression (a). Flow cytometry analysis of hPSCs and EBs differentiated from hPSCs demonstrates the loss of self‐renewal markers such as TRA‐1‐60 and the gain of differentiation markers such as SSEA‐1 during the differentiation process (b).
7. Figure 4.7 Embryoid bodies can be seeded on ECM‐coated dishes to facilitate further spontaneous differentiation and migration of cells that can mature into cell phenotypes indicative of the three developmental germ layers.
8. Figure 4.8 hPSCs spontaneously differentiated via EB formation and allowed to mature in culture for 21 days in order to characterize trilineage potential as a measure of pluripotency. Cultures are then probed with antibodies via ICC for cells indicative of the three somatic germ lineages: the neuronal marker beta III tubulin for ectoderm (a), the hepatic marker alpha‐fetoprotein (AFP) for endoderm (b), and the cardiac marker smooth muscle actin (SMA) for mesoderm (c).
9. Figure 4.9 Setting up your cDNA synthesis in eight wells of a 96‐well plate or in eight‐well PCR strips is highly recommended since it facilitates easy transfer of samples to 96‐well and 384‐well Scorecard plates. Follow the schematic.
10. Figure 4.10 Sample set up for cDNA synthesis. Top figure is for the 384‐well Scorecard; bottom figure is for the 96‐well FAST Scorecard.
Chapter 05
1. Figure 5.1 Workflow schematic for induction of definitive endoderm differentiation from monolayer of pluripotent stem cells.
2. Figure 5.2 Definitive endoderm cells derived from iPSCs hPSCs induced to definitive endoderm, at Day 3. ICC performed using FOXA2 (red), counterstained for nuclei (blue) with DAPI (a). Flow analysis for Sox17 staining of PSC (black) and PSC‐derived definitive endoderm (red) (b).
3. Figure 5.3 Workflow schematic for induction of neural stem cells from monolayer of pluripotent stem cells.
4. Figure 5.4 Neural stem cells derived from PSCs. hPSCs induced to NSCs. ICC performed using Nestin (green), Sox2 (red), counterstained for nuclei (blue) with DAPI (a). Flow analysis of Sox1 staining of PSCs (black) and PSC‐derived NSCs (red) (b).
5. Figure 5.5 Workflow schematic for induction of cardiomyocytes from monolayer of pluripotent stem cells.
6. Figure 5.6 Cardiomyocytes derived from PSCs. hPSCs induced to cardiomycotyes. ICC performed using TNNT2 (green) and NKX2.5 (red) counterstained for nuclei (blue) with DAPI (a). Flow analysis of TNNT2 staining of PSCs (black) and PSC‐derived cardiomyocytes (green) (b).
7. Figure 5.7 Flow cytometry data quantifying the level of TNNT2 expression in differentiated cardiomyocytes (e) in comparison to basal expression levels in the parental iPSCs (b) stained for TNNT2. Unstained populations for the parental iPSCs (a), cardiomyocytes (c) and a secondary antibody‐stained control (d) all serve as negative controls for the analysis.

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This edition first published 2018
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Library of Congress Cataloguing‐in‐Publication Data

Names: Lakshmipathy, Uma, author. | MacArthur, Chad C., author. | Sridharan, Mahalakshmi, author. | Quintanilla, Rene H., author.

https://lccn.loc.gov/2017036447

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Human Pluripotent Stem Cells

A Practical Guide