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  • Pluripotent Stem Cell Handbook

  • ii

    Contents

    Introduction .................................................................................................................... 1 References ........................................................................................................................ 1

    Reprogramming .............................................................................................................. 3 Introduction ....................................................................................................................... 4 Choosing a reprogramming method ................................................................................... 4 Reprogramming with episomal vectors ............................................................................... 7 Reprogramming with Sendai virus (SeV) ............................................................................. 8 References ...................................................................................................................... 10

    Engineering ................................................................................................................... 13 Introduction ......................................................................................................................14 Gene engineering tools .....................................................................................................17 CRISPR-Cas9 technology ................................................................................................ 19 TAL effectors ................................................................................................................... 24 Additional TAL functionalities ............................................................................................ 26 Screening methods for TAL and CRISPR .......................................................................... 28 Screening with the GeneArt Genomic Cleavage Selection Kit ........................................... 30 Screening with the GeneArt Genomic Cleavage Detection Kit ........................................... 31

    Culture ........................................................................................................................... 33 Introduction ..................................................................................................................... 34 Feeder-dependent culture systems .................................................................................. 35 Feeder-free culture systems ............................................................................................. 37 Choosing a culture system ............................................................................................... 40 Adapting to feeder-free culture systems ............................................................................ 43 Cryopreservation ............................................................................................................. 45 References ...................................................................................................................... 47

  • Pluripotent Stem Cell Handbook | iii

    Differentiation ............................................................................................................... 49 Introduction ..................................................................................................................... 50 Neural differentiation ........................................................................................................ 53 Cardiomyocyte differentiation ........................................................................................... 55 Definitive endoderm ........................................................................................................ 57 Differentiation functional assays ....................................................................................... 62

    Characterization ........................................................................................................... 67 Introduction ..................................................................................................................... 68 Detecting self-renewal marker expression ......................................................................... 70 Evaluating differentiation potential ......................................................................................74 References ...................................................................................................................... 78

    Applications for Cell Therapy ..................................................................................... 81 Introduction ..................................................................................................................... 82 Translating research into clinical evaluation ....................................................................... 83 Cell Therapy Systems (CTS) products ............................................................................... 84 Custom Services and scalability ....................................................................................... 85 References ...................................................................................................................... 85

    CellModel Services ...................................................................................................... 87

    Gibco Stem Cell Training Courses ............................................................................. 89

    Ordering Information ................................................................................................... 91 Products .......................................................................................................................... 92

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  • Pluripotent Stem Cell Handbook | 1

    The Stem Cell HandbookPluripontent Stem Cell Handbook

    Introduction Pluripotent stem cells (PSCs) is a term that encompasses both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

    Human ESCs (hESCs) are isolated from the inner cell mass of the blastocyst stage of a developing embryo and were first derived in 1998 by Dr. James Thomson at the University of Wisconsin, Madison1. iPSCs are generated via ectopic expression of one or more genes to reprogram an adult somatic cell. They are similar or equivalent to ESCs and were first derived by Dr. Shinya Yamanaka in 2007 in Kyoto, Japan2. PSCs are characterized by their ability to renew themselves indefinitely and differentiate into almost any cell type when exposed to the right microenvironment.

    iPSCs have revolutionized the field of stem cell research by simplifying the generation of patient- specific stem cells that can then be used to model diseases in a dish. These models can be valuable in defining the mechanisms of disease pathology and thereby play a vital role in drug discovery and identification of therapeutic targets.

    Below are the major areas in which PSCs and their derivatives have many potential applications:

    • Regenerative medicine: PSC-derived cells can be used to repair or replace diseased or damaged cells

    • Disease research: PSC-derived cells can be useful for modeling various disease conditions

    • Drug discovery and development: PSC-derived cells are excellent tools for testing the effects of experimental drugs

    • Developmental biology: PSCs and PSC-derived cells provide a system for studying normal development

    The applications mentioned above involve a variety of protocols and require different tools. This handbook serves as a resource for the pluripotent stem cell workflow and provides recommendations for the use of related tools.

    References 1. Takahashi K, Tanabe K, Ohnuki M et al. (2007).

    Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, (5):861 - 872.

    2. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, (5391): 1125 - 1147.

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  • Pluripotent Stem Cell Handbook | 3

    Reprogramming

    Section 1

    Reprogramming

  • 4

    1.1

    Introduction iPSCs are generated from somatic cells through the forced expression of specific transcription factors that reprogram the cells to a pluripotent state. To date, different sets of reprogramming factors have been tested, along with different types of gene delivery

    technologies that are associated with varying levels of efficiency and safety (Figure 1.1).

    To find the best solution for your reprogramming experiment, go to thermofisher.com/reprogramming.

    Figure 1.1. Safety and efficiency of various reprogramming technologies. Different reprogramming agents are classified as integrating, excisable or non-integrating technologies, which exhibit increasing levels of safety. Under each category, technologies are listed in order of decreasing efficiency.

    • Lentivirus • Retrovirus • PhiC31 integrase

    • Excisable lentivirus • Excisable transposon

    • Sendai virus • Episomal vector • Minicircle • Synthetic mRNA • Self-replicating RNA

    EXCISABLE NON-INTEGRATINGINTEGRATING

    Safety

    1.2

    Choosing a reprogramming method Different reprogramming technologies have their own advantages and disadvantages that must be weighed when planning an experiment. The main features to consider include a lab’s flexibility in working w

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