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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Comprehensive coverage of the entire induced pluripotent stem cell basic work flow Pluripotent stem cells (PSC) can divide indefinitely, self-renew, and can differentiate to functionally reconstitute almost any cell in the normal developmental pathway, given the right conditions. This comprehensive book, which was developed from a training course, covers all of the PSCs (embryonic, embryonic germ, and embryonic carcinoma) and their functions. It demonstrates the feeder-dependent and feeder-free culture of hESC and hiPSC, which will be referred to in all protocols as PSCs. It also addresses the methods commonly used to determine pluripotency, as defined by self-renewal marker expression and differentiation potential. Human Pluripotent Stem Cells: A Practical Guide offers in-depth chapter coverage of introduction to stem cell, PSC culture, reprogramming, differentiation, PSC characterization, and more. It also includes four appendixes containing information on reagents, medias, and solutions; common antibodies; consumable and equipment; and logs and forms. * Includes helpful tips and tricks that are normally omitted from regular research papers * Features useful images to support the technical aspects and results visually as well as diagrammatic illustrations * Presents specific sections (ie: reprogramming, differentiation) in a concise and easily digestible manner * Written by experts with extensive experience in stem cell technologies Human Pluripotent Stem Cells: A Practical Guide is an ideal text for stem cell researchers, including principal investigators, and others in university and industry settings, and for new graduate students in PSC labs.
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Veröffentlichungsjahr: 2017
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Table of Contents
Cover
Title Page
1 Introduction
1.1 Biosafety
1.2 Biosafety Levels
1.3 Aseptic Technique
1.4 Storage
1.5 Contamination
1.6 Pluripotent Stem Cells
1.7 Procedures
References
2 Pluripotent Stem Cell Culture
2.1 Introduction
2.2 Materials
2.3 Solutions
2.4 Methods
References
3 Reprogramming
3.1 Introduction
3.2 Materials
3.3 Solutions
3.4 Methods
References
4 Characterization
4.1 Introduction
4.2 Materials
4.3 Solutions
4.4 Methods
References
5 Differentiation
5.1 Introduction
5.2 Materials
5.3 Solutions
5.4 Methods
References
Index
End User License Agreement
List of Tables
Chapter 01
Table 1.1 Overview of pluripotent stem cell (PSC) culture manipulations per week.
Chapter 02
Table 2.1 Volume of attachment factor for iMEF culture.
Table 2.2 Media volume recommendations for seeding of iMEFs.
Table 2.3 iMEF cell concentrations per vessel configuration.
Table 2.4 Volume of Geltrex for coating of tissue culture vessels.
Table 2.5 Vitronectin volume recommendations for PSC culture.
Table 2.6 Laminin 521 volume recommendations for PSC culture (2.5 µg/mL).
Table 2.7 Volume of collagenase IV for PSC enzymatic passaging.
Table 2.8 Media volume recommendations for PSC culture.
Table 2.9 D‐PBS and EDTA volume recommendations for PSC culture.
Table 2.10 Essential 8 medium volume recommendations for PSC culture.
Table 2.11 Cryopreservation medium volume recommendations for cryopreserving feeder‐free PSCs.
Chapter 03
Table 3.1 Formula to calculate the volume of each virus needed to reach the target MOI(s).
Table 3.2 RT‐PCR primers to detect CytoTune Sendai vectors.
Table 3.3 Volumes for PCR reaction (Sendai vector detection).
Table 3.4 Conditions for PCR reaction (Sendai vector detection).
Table 3.5 Neon settings for Epi5 electroporation of human dermal fibroblasts or CD34+ cells.
Table 3.6 PCR primers for detection of Epi5 vectors.
Table 3.7 Volumes for PCR (Epi5 vector detection).
Table 3.8 Conditions for PCR (Epi5 vector detection).
Chapter 04
Table 4.1 Volume of collagenase IV for PSC enzymatic passaging.
Table 4.2 Volumes for 2× RT Master Mix.
Table 4.3 Thermal Cycler condition for RT reaction.
Table 4.4 TaqMan ScoreCard cycling parameters.
List of Illustrations
Chapter 01
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).
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).
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).
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
Figure 2.1 50× magnified phase contrast image of iMEFs showing expected density and uniformity of coating for feeder‐dependent PSC culture.
Figure 2.2 50 × magnified phase contrast image of uniform‐sized PSC clusters right after thaw, seeded onto iMEF‐coated dishes.
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.
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).
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.
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
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.
Figure 3.2 Schematics of reprogramming workflows for fibroblast and blood cells in feeder‐dependent (F) and feeder‐free conditions (ff).
Figure 3.3 BJ fibroblasts seeded at desired densities, at 50× magnification (a) and 100× magnification (b).
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.
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.
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).
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).
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).
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
Figure 4.1 Schematic of comprehensive characterization.
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).
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).
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.
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.
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).
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.
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).
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.
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
Figure 5.1 Workflow schematic for induction of definitive endoderm differentiation from monolayer of pluripotent stem cells.
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).
Figure 5.3 Workflow schematic for induction of neural stem cells from monolayer of pluripotent stem cells.
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).
Figure 5.5 Workflow schematic for induction of cardiomyocytes from monolayer of pluripotent stem cells.
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).
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.
Guide
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Human Pluripotent Stem Cells
A Practical Guide
Uma LakshmipathyChad C. MacArthurMahalakshmi SridharanRene H. Quintanilla
Cell Biology, Life Sciences Solutions, Thermo Fisher Scientific, USA
This edition first published 2018© 2018 John Wiley & Sons, Inc.
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The right of Uma Lakshmipathy, Chad C. MacArthur, Mahalakshmi Sridharan and Rene H. Quintanilla to be identified as the authors of this work has been asserted in accordance with law.
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Names: Lakshmipathy, Uma, author. | MacArthur, Chad C., author. | Sridharan, Mahalakshmi, author. | Quintanilla, Rene H., author.Title: Human pluripotent stem cells : a practical guide / Uma Lakshmipathy, Chad C. MacArthur, Mahalakshmi Sridharan, Rene H. Quintanilla.Description: Hoboken, NJ : John Wiley & Sons, 2018. | Includes bibliographical references and index. |Identifiers: LCCN 2017036447 (print) | LCCN 2017046356 (ebook) | ISBN 9781119394341 (pdf) | ISBN 9781119394365 (epub) | ISBN 9781119394334 (cloth)Subjects: LCSH: Multipotent stem cells. | Stem cells.Classification: LCC QH588.S83 (ebook) | LCC QH588.S83 L35 2018 (print) | DDC 616.02/774–dc23LC record available at https://lccn.loc.gov/2017036447
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1Introduction
Successful execution of any cell‐based project relies on a setting up a robust cell culture laboratory. Guidelines under the Guidance on Good Cell Culture Practice provides an overview of the critical parameters in establishing facilities and training personnel [1]. This is even more important for a stem cell laboratory where primary cells from donor tissue or their derivatives are cultured for extended periods of time [2].
1.1 Biosafety
In addition to the safety risks common to most workplaces, such as electrical and fire hazards, a cell culture laboratory has a number of specific hazards associated with handling and manipulating human or animal cells and tissues, as well as toxic, corrosive or mutagenic solvents and reagents. The most common of these hazards are accidental inoculations with syringe needles or other contaminated sharps, spills and splashes onto skin and mucous membranes, ingestion through mouth pipetting, animal bites and scratches, and inhalation exposures to infectious aerosols.
The fundamental objective of any biosafety program is to reduce or eliminate exposure of laboratory workers and the outside environment to potentially harmful biological agents. The most important element of safety in a cell culture laboratory is strict adherence to standard microbiological practices and techniques.
1.2 Biosafety Levels
The regulations and recommendations for biosafety in the United States are contained in the document Biosafety in Microbiological and Biomedical Laboratories, prepared by the Centers for Disease Control (CDC) and the National Institutes of Health (NIH), and published by the US Department of Health and Human Services. The document defines four ascending levels of containment, referred to as biosafety levels 1 through 4, and describes the microbiological practices, safety equipment, and facility safeguards for the corresponding level of risk associated with handling a particular agent.
Biosafety Level 1 (BSL‐1)
: the basic level of protection common to most research and clinical laboratories. Appropriate for agents that are not known to consistently cause disease in normal, healthy human adults (examples:
Bacillus subtilis
,
E. coli
).
Biosafety Level 2 (BSL‐2)
: appropriate for moderate‐risk agents known to cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. Most cell culture labs should be at least BSL‐2, and all stem cell labs have this as a requirement.
Biosafety Level 3 (BSL‐3)
: BSL‐3 is appropriate for indigenous or exotic agents with a known potential for aerosol transmission, and for agents that may cause serious and potentially lethal infections.
Biosafety Level 4 (BSL‐4)
: BSL‐4 is appropriate for exotic agents that pose a high individual risk of life‐threatening disease by infectious aerosols and for which no treatment is available. These agents are restricted to high‐containment laboratories.
For more information about the biosafety level guidelines, refer to Biosafety in Microbiological and Biomedical Laboratories, 5th edition, which is available for downloading at www.cdc.gov/biosafety/.
1.3 Aseptic Technique
Successful cell culture depends heavily on keeping the cells free from contamination by microorganisms such as bacteria, fungi, and viruses. Non‐sterile supplies, media, and reagents, airborne particles laden with microorganisms, unclean incubators, and dirty work surfaces are all sources of biological contamination.
Aseptic technique, designed to provide a barrier between the microorganisms in the environment and the sterile cell culture, depends upon a set of procedures to reduce the probability of contamination from these sources. The elements of aseptic technique are a sterile work area, good personal hygiene, sterile reagents and media, and sterile handling.
1.3.1 Maintaining a Sterile Work Area
The simplest and most economical way to reduce contamination from airborne particles and aerosols (e.g., dust, spores, shed skin, sneezing) is to use a cell culture hood.
The cell culture hood should be properly set up, and located in an area that is restricted to cell culture, is free from drafts from doors, windows, and other equipment, and with no through traffic.
The work surface should be uncluttered and contain only items required for a particular procedure; it should not be used as a storage area.
Before and after use, the work surface should be disinfected thoroughly, and the surrounding areas and equipment should be cleaned routinely.
For routine cleaning, wipe the work surface with 70% ethanol before and during work, especially after any spillage.
Using a Bunsen burner for flaming is not necessary or recommended in a cell culture hood.
Leave the cell culture hood running at all times, turning it off only when it will not be used for extended periods of time.
Practice good personal hygiene. Wash your hands before and after working with cell cultures. In addition to protecting you from hazardous materials, wearing personal protective equipment also reduces the probability of contamination from shed skin as well as dirt and dust from your clothes.
1.3.2 Aseptic Work Area
The major requirement of a cell culture laboratory is to maintain an aseptic work area that is restricted to cell culture work. Although a separate tissue culture room is preferred, a designated cell culture area within a larger laboratory can be used for sterile handling, incubation, and storage of cell cultures, reagents, and media. The simplest and most economical way to provide aseptic conditions is to use a cell culture hood (i.e., biosafety cabinet).
1.3.3 Cell Culture Hood
The cell culture hood provides an aseptic work area while allowing the containment of infectious splashes or aerosols generated by many microbiological procedures. Three kinds of cell culture hoods, designated as Class II, III, and I, have been developed to meet varying research and clinical needs.
Class I
cell culture hoods offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques, but they do not provide cultures with protection from contamination. They are similar in design and airflow characteristics to chemical fume hoods.
Class II
cell culture hoods are designed for work involving BSL‐1, ‐2, and ‐3 materials, and also provide an aseptic environment necessary for cell culture experiments. A Class II biosafety cabinet should be used for handling potentially hazardous materials (e.g., primate‐derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents).
Class III
biosafety cabinets are gas tight, and provide the highest attainable level of protection to personnel and the environment. A Class III biosafety cabinet is required for work involving known human pathogens and other BSL‐4 materials.
A cell culture hood should be large enough to be used by one person at a time, be easily cleanable inside and outside, have adequate lighting, and be comfortable to use without requiring awkward positions. Keep the workspace in the cell culture hood clean and uncluttered, and keep everything in direct line of sight. Disinfect each item placed in the cell culture hood by spraying it with 70% ethanol and wiping clean.
The arrangement of items within the cell culture hood usually adheres to the following right‐handed convention.
A wide, clear workspace in the center with your cell culture vessels.
Pipettor in the front right and glass pipettes in the left, where they can be reached easily.
Reagents and media in the rear right to allow easy pipetting.
Small container in the rear middle to hold liquid waste.
1.3.3.1 Airflow Characteristics of Cell Culture Hoods
Cell culture hoods protect the working environment from dust and other airborne contaminants by maintaining a constant, unidirectional flow of HEPA‐filtered air over the work area. The flow can be horizontal, blowing parallel to the work surface, or vertical, blowing from the top of the cabinet onto the work surface.
Depending on its design, a horizontal flow hood provides protection to the culture (if the air is flowing towards the user) or to the user (if the air is drawn in through the front of the cabinet by negative air pressure inside). Vertical flow hoods, on the other hand, provide significant protection to both the user and the cell culture.
1.3.3.2 Clean Benches
Horizontal laminar flow or vertical laminar flow “clean benches” are not biosafety cabinets; these pieces of equipment discharge HEPA‐filtered air from the back of the cabinet across the work surface toward the user, and may expose the user to potentially hazardous materials. These devices only provide product protection. Clean benches can be used for certain clean activities, such as the dust‐free assembly of sterile equipment or electronic devices, and they should never be used when handling cell culture materials or drug formulations, or when manipulating potentially infectious materials.
1.3.4 Incubator
The purpose of the incubator is to provide the appropriate environment for cell growth. The incubator should be large enough, have forced air circulation, and should have temperature control to within ±0.2 °C. Stainless steel incubators allow easy cleaning and provide corrosion protection, especially if humid air is required for incubation. Although the requirement for aseptic conditions in a cell culture incubator is not as stringent as that in a cell culture hood, frequent cleaning of the incubator is essential to avoid contamination of cell cultures.
1.3.4.1 Types of Incubators
There are two basic types of incubators, dry incubators and humid CO2
