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Stem Book is an open access collection of invited, original, peer-reviewed chapters covering a range of topics related to stem cell biology written by top researchers in the field at the Harvard Stem Cell Institute and worldwide. Stem Book is aimed at stem cell and non-specialist researchers.

In addition to the contributions of the editorial board and the stem cell research community, the project is being done in collaboration with several other enterprises including Harvard’s Initiative in Innovative Computing. The Initiative in Innovative Computing created the Scientific Collaboration Framework (SCF), the extensible software infrastructure used for the project. SCF and the Stem Book project were funded, in part, by a generous grant from an anonymous foundation and also appreciates the input of WormBase's Textpresso team.



      Member: Girard, Lisa, Ph.D.
      Role: Associate Director of Strategic Alliances, Scientific Editor



    • Analyzing the genomic integrity of stem cells ( Protocol )

      "Prolonged culture of pluripotent and multipotent stem cells exposes the cells to strong selection pressures, often resulting in genomic alterations. Any genetic manipulation of the cells may further jeopardize their genomic stability. Genomic aberrations affect the differentiation capacity of stem cells, their stem cell identity and their tumorigenicity, and should thus be routinely evaluated for their proper use in basic research and in clinical trials. Here we review the common methods currently available to analyze the genomic integrity of stem cells, and present a recently developed method for the evaluation of the genomic integrity of stem cells by their gene expression profiles. We describe the principles of this method, provide guidelines for its implementation, and discuss its advantages and limitations compared to other available methods."

    • Assessment of human pluripotent stem cells with PluriTest ( Protocol )

      "PluriTest is a tool for rapid assessment of pluripotency in human in vitro stem cell preparations. We expect that researchers will initially screen for promising stem cell colonies after derivation or experimental manipulation by using RTPCR or immunocytochemistry for detecting pluripotency-associated markers before confirming pluripotency with whole genome expression analysis for PluriTest. However, with near-perfect sensitivity and specificity, PluriTest can function as the sole means to determine pluripotency in any given culture. We envision that further developments of PluriTest platform will have utility for not only testing for pluripotency, but also identifying differentiation propensity and genomic integrity in stem cell preparations."

    • Basic pluripotent stem cell culture protocol ( Protocol )

      "Stem cell research is a rapidly expanding field with the potential to develop therapeutic agents to treat diseases as well as study disease development from early stages. The culture of human pluripotent stem cells shares many of the same protocols as standard mammalian cell culture. However, the successful culture and maintenance of human pluripotent stem cells (hPSCs) in an undifferentiated state requires additional considerations to ensure that cells maintain their key characteristics of self-renewal and pluripotency. There are several basic techniques needed for the culturing of mammalian cells, including thawing frozen stocks, plating cells in culture vessels, changing media, passaging and cryopreservation. The protocols in this document represent a subset of the standard operating procedures used to maintain and culture stem cells at the Massachusetts Human Stem Cell Bank, and have been thoroughly testing and verified."

    • Blood - SeV derived fibroblast generated iPSCs ( Protocol )

      "CytoTune™-iPS Reprogramming System uses vectors based on replication in competent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTune™ Reprogramming System uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint). In addition, the host cell can be cleared of the vectors and reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the functional temperature sensitivity mutations introduced into the key viral proteins. The CytoTune™-iPS Reprogramming Kit contains four SeV-based reprogramming vectors, each capable of expressing one of the four Yamanaka factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and are optimized for generating iPSCs from human somatic cells. The reprogramming vectors in this kit have been engineered to increase biological and environmental safety."

    • Chondrocyte protocol ( Protocol )

      "We have developed for hESc a new 3-Stage directed differentiation protocol (DDP) to generate chondrocytes, the specialized cells that form cartilage tissue. The protocol is segmented into stages that mimic the developmental processes that occur in cell lineage specification during embryogenesis. Pluripotent hESc, are established as feeder-free cultures4 prior to DDP being carried out in monolayer culture on a sequence of matrix protein-coated surfaces with a chemicallydefined medium sequentially supplemented with growth factors to directdifferentiation through developmental intermediate cell populations of primitive streak/mesendoderm (Stage 1), mesoderm (Stage 2) and then chondrocytes (Stage 3) (Figure 1). This unique protocol is highly efficient, scalable and completely chemically-defined, thus making it appropriate for translation towards clinical applications of chondrocytes, as well as providing a defined system for characterizing the molecular mechanisms regulating hESc differentiation."

    • Conditioning pluripotent stem cell media with mouse embryonic fibroblasts (MEF-CM) ( Protocol )

      "There are a number of methods to expand human pluripotent stem cells (hPSCs) without feeders. I prefer to culture my hPSCs with mouse embryonic fibroblasts (MEFs) but there are occasions when feeder free growth is required (e.g., nucleofection, viral transduction or karyotyping to name a few). To expand cells without feeders, I prefer to use mouse embryonic fibroblast-conditioned media (MEF-CM). This is simply human pluripotent stem cell (hPSC) media placed on MEFs overnight. The MEFs secrete factors that contribute to hPSC maintenance during the overnight incubation, and before use FGF2 is added to the preparation."

    • Cryopreservation of human pluripotent stem cells in defined conditions ( Protocol )

      "Cryopreservation is a critical step to preserve the integrity of human pluripotent stem cells, however, the recovery after cryopreservation is inefficient with traditional enzymatic methods, such as dispase and collagenase. Due to the technical difficulties of cryopreservation, regular passaging methods are often different from harvest methods used in cryopreservation. Even though individualization with ROCK inhibitor treatment has been successfully used to harvest cells in both passaging and cryopreservation, it is still desirable to have a method that could preserve stem cells without drug treatment. At the same time, serum or other animal products are often used in cryopreservation, which could potentially compromise the future applications of the cells. We developed an enzyme-free method to passage and cryopreserve pluripotent stem cells in chemically defined medium. High recovery efficiency could be achieved in dependent of ROCK inhibitor treatment.

      Here we describe how to cryopreserve hESCs and hiPSCs in E8 medium with EDTA dissociation method. For low passage and stock lines, we usually add 10 μM Y27632 to further insure the cryopreservation efficiency."

    • Feeder dependent (MEF) culture protocol - collagenase passaging ( Protocol )

      "This protocol is used for general maintenance and passaging of hES and iPS cells grown on MEFs (Feeder-Dependent). It assumes that the cells are grown in a 6-well plate format."

    • Feeder-independent culture protocol - EDTA splitting ( Protocol )

      "This protocol is used for general maintenance and passaging of hES and iPS cells in a feeder-independent culture system such as mTeSR1/Matrigel. It assumes that the cells are grown in a 6-well plate format."

    • Formation of embryoid bodies from Matrigel dots protocol ( Protocol )

      "This protocol was developed in the Salk STEM Cell Core to enable the formation of uniform and large embryoid bodies (EBs) from pluripotent human stem cells. It assumes the cells have previously been cultured on Matrigel and typically requires 1–2 wells of a fairly confluent 6-well plate as starting material. This is a low cost alternative to commercial products like the AggreWell that has proven especially useful for the formation of larger sized EBs that show good potential for differentiation to all 3 germ layers."

    • General spinfection ( Protocol )

      "This protocol was developed in the Salk STEM Cell Core to enable researchers to consistently and reproducibly produce reprogrammed iPS cells, the initial idea came via word of mouth reports of its effectiveness to increase the efficiency of viral transduction. It has most commonly been used on retrovirus and lentivirus factors. Initial evaluation of this method showed 5–10× increase in transduction efficiency."

    • Generation of human induced pluripotent stem cells from peripheral blood using the STEMCCA lentiviral vector ( Protocol )

      "Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxedexcisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described (Chou et al., 2011; van den Akker et al., 2010) in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood."

    • Genome editing in human pluripotent stem cells ( Protocol )

      "Genome editing is used to make targeted modifications to the genome of eukaryotic cells. There are many potential applications of genome editing in human pluripotent stem cells (hPSCs) including the generation of knockout and reporter cell lines. This protocol describes a system for efficient genome editing in hPSCs using engineered transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat (CRISPR) technology."

    • Hematopoietic differentiation ( Protocol )

      "A protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin−CD34+CD43+CD45+ multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5–19 d of culture with cytokines, depending on the cell type."

    • iPSC derivation from fibroblast in chemically defined medium ( Protocol )

      "Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential to become the source materials for cell-based therapy, so the quality of the stem cells has great impact on how the cells could be utilized in future applications. Derivation and maintenance conditions have critical role determining the iPSC quality, mainly due to the involvement of animal products and feeder cells. We developed a simple procedure to derive and maintain hiPSCs in chemically defined media.

      Here we describe how to derive hiPSCs in E8 based growth conditions. This method has been successfully used on fibroblast, pre-adipocyte and HUVEC. The procedure can be adapted to most common reprogramming methods, such as Lentivirus, Episomal DNA and Sendai Virus. In companion with E8 medium and vitronectin (or synthetic surface), human pluripotent stem cells could be maintained in an enzyme-free, xeno-fee and chemically defined environment. To simplify the description, we use Sendai Virus to reprogram fibroblast, and use matrigel as the coating surface for cell culture in this protocol."

    • iPSC reprogramming from human peripheral blood using Sendai Virus mediated gene transfer ( Protocol )

      "This protocol allows efficient generation of integration-free iPS cells from a small amount of peripheral blood (<1 ml). Peripheral blood mononuclear cells (PBMCs) are cultured to expand the erythroblast (EB) population. They are then used to derive iPS cells using four recombinant Sendai viral vectors (CytotuneTM, Life Technologies), expressing the four reprogramming factors Oct4, Sox2, Kfl4 and c-Myc."

    • Midbrain dopamine neurons from hESCs ( Protocol )

      "The Kriks et al. protocol has finally achieved what we believe are genuine midbrain, dopamine neurons. Perrier et al. (2004) described a method to make dopamine neurons that we initially thought were mDA neurons. Later work by developmental biologists, however, discovered additional markers of these neurons that the Perrier cells lacked (e.g., FOXA2/TH). The Perrier protocol used neural rosettes as a patterning intermediate to make dopamine neurons. Later work in vivo demonstrated that rodent mDA neurons are derived from the floor plate, a cell type at the base of the neural tube that is not normally neurogenic – its usual role is to produce SHH that helps pattern the neural tube. Fasano et al. (2010) demonstrated a protocol to make floor plate from hESCs but the protocol was not efficient at making mDA neurons. Floor plate is achieved by very early, pre-rosette exposure to high levels of SHH. The Kriks et al. protocol modified this protocol to include early WNT activation, and this turned out to be the key for driving the progenitors into mature mDA neurons. Importantly, these neurons can be transplanted into the brain and retain the mDA neuron phenotype: something that the Perrier et al. cells could not do."

    • Monolayer endoderm differentiation from human ESCs ( Protocol )

      "When embryonic stem cells are differentiated in the presence of activin A in serum-free conditions, an endoderm progenitor population defined by the coexpression of either Brachyury, Foxa2 and c-Kit, or c-Kit and Cxcr4 is generated. Specification of these progenitors with bone morphogenetic protein-4 in combination with basic fibroblast growth factor and activin A results in the development of hepatic populations highly enriched (45–70%) for cells that express the alpha-fetoprotein and albumin proteins."

    • Neural induction - Dual SMAD inhibition ( Protocol )

      "Dual SMAD inhibition takes a confluent, feeder free culture of hPSCs and rapidly differentiates them into early neurectoderm (Chambers et al., 2009). This rapid differentiation is caused by blocking the two signaling pathways that utilize SMADs for transduction: BMP and TGFB. Oct4 is extinguished and Pax6 expression has begun by around day 7–8, depending on the line used. This neurectoderm can be passaged to become rosettes or can be patterning to become many other types of neural cells (for example, see Fasano et al., 2010)."

    • Pancreatic differentiation ( Protocol )

      "The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells."

    • Protocol for directed differentiation of human pluripotent stem cells toward a hepatocyte fate ( Protocol )

      "The directed differentiation of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into hepatocytes could facilitate a rational study of the molecular mechanisms underlying human liver development as well as provide a renewable source of exogenous hepatocytes for drug toxicity testing and cell-based therapeutics. Moreover, if hepatocytes were produced from hiPSCs originating from patients with inborn errors of hepatic metabolism, such cells could be used for modeling liver disease. Here we present a step-wise protocol for efficiently and reproducibly inducing the differentiation of hepatocytes from human pluripotent stem cells under highly defined conditions."

    • Protocol for making retroviral reprogramming factors ( Protocol )

      "This protocol was developed as an efficient and consistent method to produce functional retroviral reprogramming factors. It avoids the need for concentrating virus via ultracentrifugation."

    • Robust generation of hepatocyte-like cells from human embryonic stem cell populations ( Protocol )

      "Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008; Fletcher et al 2008; Hannoun et al 2010; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies."

    • Splitting hESC/hiPSC lines on MEF using Accutase ( Protocol )

      "Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are known to be vulnerable to apoptosis upon various technical manipulation, such as single cell dissociation, freezing and thawing, etc., which hinder their use for clonal isolation in gene transfer, differentiation and FACS cell sorting.

      However, Y-27632, a selective inhibitor of p160-Rho-associated coiled-coil kinase (ROCK) was found to be an effective inhibitor of apoptosis and enhanced survival of hPSCs upon single cell dissociation.

      Here we describe how to propagate hPSCs in single cell dissociation using Accutase, a ready to use cell detachment solution of proteolytic and collagenolytic enzymes and a direct replacement for trypsin solution."

    • Splitting hESC/hiPSC lines with EDTA in feeder-free conditions ( Protocol )

      "Individualization leads to severe cell death in human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). This phenomenon leads to difficulties in handling cells in applications such as passaging, cryopreservation and transfection, and the treatment with ROCK inhibitors has been found to effectively improve cell survival. However, enzymatic dissociation and ROCK inhibitor treatment could bring potential complication in cell maintenance. An enzyme-free dissociation method was developed to passage stem cells and achieve robust cell survival in dependent of drug treatment.

      Here we describe how to propagate hPSCs as small aggregates using homemade EDTA/PBS solution. This method is suitable for feeder-free culture conditions, and has been used for StemPro, mTeSR and E8 media2. In companion with E8 medium and vitronectin (or synthetic surface), human pluripotent stem cells could be maintained in an enzyme-free, xeno-fee and chemically defined environment. For the simplicity, we used matrigel as the coating surface for cell culture in this protocol."

    • Splitting hPSCs with Dispase ( Protocol )

      "This protocol outlines the techniques used to routinely passage hPSCs in the SKI Stem Cell Research Facility at Sloan-Kettering. We prefer to culture our cells on mouse embryo fibroblasts (MEFs) but occasionally grow the cells feeder free for particular applications, such as nucleofection, viral transduction or karyotyping.

      We use Dispase for all applications requiring colony passage including feeder-dependent and independent expansion as well as for cryopreservation."

    • Teratoma formation: A tool for monitoring pluripotency in stem cell research ( Protocol )

      "Human embryonic stem cells and human induced-pluripotent stem cells are uniquely defined by their pluripotent differentiation potential and endless self-renewing ability. This capability to become any somatic cell type within the human body has garnered significant attention and interest in the fields of cell biology and regenerative medicine. In studying these promising cells, quality-control assays that can characterize their pluripotency and determine the tumorigenicity of their therapeutic progenies become critical. The most rigorous and arguably accurate among current assays is teratoma formation in vivo. This chapter will provide a brief description of teratoma biology, discuss its clinical relevance, and cover methods of forming, monitoring, and analyzing teratomas. The protocols outlined in this chapter have been extensively utilized in peer-reviewed literature."

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    Last updated: 2013-08-07T13:14:51.034-05:00

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