Dr Hay was supported by a RCUK Fellowship, Dr West was supported by the Department of Surgery, Dr Medine was supported by a grant from the BHF Core Fund, Mr. Baltasar Lucendo-Villarin was supported by a MRC PhD Studenship. Dr Zhou was supported by a scholarship from the Chinese Government.

1. Initial preparation of all chemical stocks and coating of culture plasticware 
All steps to be carried out in a tissue culture hood under aseptic conditions.
<ol>
 <li>Preparation of human basic Fibroblast Growth Factor (hbFGF)
 <ol>
 <li>Prepare 10% BSA solution in PBS and filter through a 0.22 &mu;m filter.</li>
 <li>From the 10% BSA solution prepare a 0.2% BSA solution.</li>
 <li>Add 10 mL 0.2% BSA solution/100 &mu;g hbFGF.</li>
 <li>Pre-wet a 0.22 &mu;m filter by filtering 5 mL 10% B...

<ol>
	<li>Asgari, S., Pournasr, B., Salekdeh, G. H., Ghodsizadeh, A., Ott, M., Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+a+new+era+for+hepatology.">Induced pluripotent stem cells: a new era for hepatology.</a> J. Hepatol. 53, 738-751 (2010).</li><li>Hay, D. C., Pernagallo, S., Diaz-Mochon, J. J., Medine, C. N., Greenhough, S., Hannoun, Z., Schrader, J., Black, J. R., Fletcher, J., Dalgetty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+Screening+of+Polymer+Libraries+to+Define+Novel+Substrates+for+Functional+Hepatocytes+with+Inducible+Drug+Metabolism.">Unbiased Screening of Polymer Libraries to Define Novel Substrates for Functional Hepatocytes with Inducible Drug Metabolism.</a> Stem Cell Research. 6, 92-101 (2011).</li><li>Payne, C. M., Samuel, K., Pryde, A., King, J., Brownstein, D., Schrader, J., Medine, C. N., Forbes, S. J., Iredale, J. P., Newsome, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+Functional+Hepatocyte+Like+Cells+in+Immune+Compromised+Mice.">Persistence of Functional Hepatocyte Like Cells in Immune Compromised Mice.</a> Liver International. 31, 254-262 (2011).</li><li>Greenhough, S., Medine, C., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+Stem+Cell+Derived+Hepatocyte+Like+Cells+and+their+Potential+in+Toxicity+Screening.">Pluripotent Stem Cell Derived Hepatocyte Like Cells and their Potential in Toxicity Screening.</a> Toxicology. 278, 250-255 (2010).</li><li>Medine, C. N., Greenhough, S., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Stem+Cell+Derived+Hepatic+Endoderm+in+Human+Drug+Discovery.">The Role of Stem Cell Derived Hepatic Endoderm in Human Drug Discovery.</a> Biochemical Society Transactions. 38, 1033-1036 (2010).</li><li>Hannoun, Z., Fletcher, J., Greenhough, S., Medine, C. N., Samuel, K., Sharma, R., Pryde, A., Black, J. R., Ross, J. A., Wilmut, I., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Comparison+between+Conditioned+Media+and+Serum+Free+Media+in+Human+Embryonic+Stem+Cell+Culture+and+Differentiation.">The Comparison between Conditioned Media and Serum Free Media in Human Embryonic Stem Cell Culture and Differentiation.</a> Cellular Reprogramming. 12, 133-140 (2010).</li><li>Dalgetty, D. M., Medine, C., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+Future+Challenges+in+Stem+Cell-Derived+Liver+Technologies.">Progress and Future Challenges in Stem Cell-Derived Liver Technologies.</a> American Journal of Physiology - Gastrointestinal and Liver Physiology. 297, 241-248 (2009).</li><li>Hay, D. C., Fletcher, J., Payne, C., Terrace, J. D., Gallagher, R. C. J., Snoeys, J., Black, J., Wojtacha, D., Samuel, K., Hannoun, Z., Pryde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Efficient+Differentiation+of+hESCs+to+Functional+Hepatic+Endoderm+Requires+ActivinA+and+Wnt3a+Signalling.">Highly Efficient Differentiation of hESCs to Functional Hepatic Endoderm Requires ActivinA and Wnt3a Signalling.</a> Proceedings of the National Academy of Sciences. 105, 12301-12306 (2008).</li><li>Fletcher, J., Cui, W., Samuels, K., Black, J. R., Currie, I. S., Terrace, J. D., Payne, C., Filippi, C., Newsome, P., Forbes, S. J., Ross, J. A., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Inhibitory+Role+of+Stromal+Cell+Mesenchyme+on+Human+Embryonic+Stem+Cell+Hepatocyte+Differentiation+is+Overcome+by+Wnt3a+Treatment.">The Inhibitory Role of Stromal Cell Mesenchyme on Human Embryonic Stem Cell Hepatocyte Differentiation is Overcome by Wnt3a Treatment.</a> Cloning and Stem Cells. 10, 331-340 (2008).</li></ol>

We have developed a simple, homogeneous and highly reproducible in vitro model to generate scalable levels of human HLCs. Our model has been validated by a number of external collaborating laboratories. We routinely characterise stem cell derived HLCs using our in house tool box of developmental markers and liver specific functional assays (most of which are commercially available). The critical stages in our process are: the maintenance of stem cell pluripotency; the ability to direct stem cell differentiation ...

Matrigel coating plates and flasks 
<ol>
<li> Matrigel (10 mL, BD Biosciences, UK); store at -20&deg;C.</li>
<li>KO-DMEM (500 mL, Gibco, Invitrogen, UK); store at 4&deg;C.</li>
<li>Tissue culture plates (6 well, 12 well, Corning, UK) </li>
<li>Tissue culture flask (25 cm2 vented, Corning, UK)</li>
</ol>
hESC Maintenance 
<ol>
<li>Mouse embryonic fibroblast conditioned medium (MEF-CM) (100 mL, R &amp; D Systems, USA); store at -20&deg;C.</li>
<li>BSA solution (50 mL, Sigma Aldrich, UK); store at 4&deg;C.</li>
<li>Human basic fibroblast growth factor (100 &mu;g, Peprotech, USA); store at -20&deg;C.</li>
</ol>
Passaging hESCs with collagenase
<ol>
<li>Confluent well or flask of hESCs.</li>
<li>Matrigel coated wells or flasks as appropriate.</li>
<li>Phospate buffered saline (-MgCl2, -CaCl2) (500 mL, Gibco, Invitrogen, UK); store at room temperature.</li>
<li>Collagenase IV (1 g, Gibco, Invitrogen, UK); store at 4&deg;C.</li>
<li>Mouse embryonic fibroblast conditioned medium (MEF-CM) (100 mL, R &amp; D Systems, USA).</li>
<li>Human basic fibroblast growth factor (100 &mu;g, Peprotech, USA).</li>
</ol>
Differentiation of hESCs to hepatic endoderm
<ol>
<li>RPMI 1640 (500 mL, Gibco, Invitrogen, UK); store at 4&deg;C.</li>
<li>B27 Supplement (10 mL, Gibco, Invitrogen, UK); store at -20&deg;C.</li>
<li>Activin A (2 &mu;g, Peprotech, USA); store at -20&deg;C.</li>
<li>Recombinant mouse Wnt3a (2 &mu;g, R &amp; D Systems, USA); store at -20&deg;C.</li>
<li>KO-DMEM (500 mL, Gibco, Invitrogen, UK); store at 4&deg;C.</li>
<li>KO-SR (500 mL, Gibco, Invitrogen, UK); store at -20&deg;C.</li>
<li>Non-essential amino acids (100 mL, Gibco, Invitrogen, UK); store at 4&deg;C.</li>
<li>&beta;-Mercaptoethanol (10 mL, Gibco, Invitrogen, UK); store at 4&deg;C.</li>
<li>DMSO (Sigma Aldrich, UK); store at room temperature</li>
<li>Leibovitz L-15 culture medium (500 mL, Sigma Aldrich, UK); store at 4&deg;C.</li>
<li>Tryptose phosphate broth (100 mL, Sigma Aldrich, UK); store at 4&deg;C.</li>
<li>Foetal bovine serum, heat inactivated (500 mL, Gibco, Invitrogen, UK); store at -20&deg;C.</li>
<li>Hydrocortisone 21-hemisuccinate (100 mg, Sigma Aldrich, UK); store at -20&deg;C.</li>
<li>Insulin (bovine pancreas) (100 mg, Sigma Aldrich, UK); store at -20&deg;C.</li>
<li>L-Glutamine (100 mL, Gibco, Invitrogen, UK); store at -20&deg;C.</li>
<li>Ascorbic acid (25 g, Sigma Aldrich, UK); store at -20&deg;C.</li>
<li>Human HGF (10 &mu;g, Peprotech, USA); store at -20&deg;C.</li>
<li>Recombinant Human Oncostatin M (OSM) (50 &mu;g, R &amp; D Systems, USA); store at -20&deg;C.</li>
<li>Syringe driven filter unit 0.22 &mu;m (Millipore, UK)</li>
</ol>
Characterisation of hESC derived Hepatic Endoderm
Immunostaining 
<ol>
<li>Phosphate buffer saline (-MgCl2, -CaCl2) (500 mL, Gibco, Invitrogen, UK); store at room temperature.</li>
<li>PBST, PBS made up with 0.1% TWEEN 20 (Sigma-Aldrich, UK).</li>
<li>Paraformaldehyde (PFA) (Sigma-Aldrich, UK) is made up in PBS, store -20&deg;C.</li>
<li>Glycerol (Sigma-Aldrich, UK), store at room temperature.</li>
<li>Tris Base (Sigma-Aldrich, UK), store at room temperature.</li>
<li>Ethanol</li>
<li>Serum (AbD Serotech, UK), store at -20&deg;C.</li>
<li>Secondary Antibody, Alexa Fluorophores (Molecular Probes, Invitrogen, UK).</li>
<li>MOWIOL 4-88 (Polysciences Inc, USA) is made up in Tris HCL and glycerol as per manufacturers instructions. DAPI (Pierce, Thermo Fisher Scientific, UK) is added to the MOWIOL solution at a 1:1000 dilution.</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Primary antibodies</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antigen*</td>
 <td>Type</td>
 <td>Supplier</td>
 <td>Dilution</td>
 </tr>
 <tr>
 <td>ALB</td>
 <td>Mouse Monoclonal</td>
 <td>Sigma Aldrich</td>
 <td>1/500</td>
 </tr>
 <tr>
 <td>E-Cadherin</td>
 <td>Mouse Monoclonal</td>
 <td>Millipore</td>
 <td>1/100</td>
 </tr>
 <tr>
 <td>&alpha;-fetoprotein</td>
 <td>Mouse Monoclonal</td>
 <td>Sigma</td>
 <td>1/500</td>
 </tr>
 <tr>
 <td>SSEA-4 FITC</td>
 <td>Mouse Monoclonal</td>
 <td>Biolegend</td>
 <td>1/100</td>
 </tr>
 <tr>
 <td>IgG</td>
 <td>Mouse Monoclonal</td>
 <td>DAKO</td>
 <td>1/500</td>
 </tr>
 <tr>
 <td colspan="2">Secondary antibodies</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse FITC conjugate</td>
 <td>Goat Monoclonal</td>
 <td>Invitrogen</td>
 <td>1/400</td>
 </tr>
 </tbody>
</table>
Table 2. The antibodies used for hESC derived hepatic endoderm immunostaining, the concentrations used, the species developed in and the companies they are purchased from.
Functional Analysis of Hepatic Endoderm and Normalisation (per mg protein)
Cytochrome P450 Assays
<ol>
<li>p4-GLO CYP3A4, CYP1A2, Kits and luminometer (Promega, USA).</li>
<li value="1">White flat bottom 96 well assay plate (BD Biosciences, UK).</li>
<li>BCA Assay Kit (Pierce, Thermo Fisher Scientific, UK).</li>
<li>Transparent 96 well assay plate (IWAKI, UK)</li>
</ol>

robust generation of hepatocyte-like cells from human embryonic stem cell populations

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.

Watch this Scientific Journal Video about Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations at JoVE.com

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

robust-generation-hepatocyte-like-cells-from-human-embryonic-stem

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Biology

47.6K ビュー.  University of Edinburgh. この記事では、ヒト胚性幹細胞集団からヒト肝内胚葉の発生に焦点を当てます。

ビデオ: Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell...

defined and scalable generation of hepatocyte-like cells from human pluripotent stem cells

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver...

stabilizing hepatocellular phenotype using optimized synthetic surfaces

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human...

an efficient method for directed hepatocyte-like cell induction from human embryonic stem cells

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm...

semi-automated production of hepatocyte like cells from pluripotent stem cells

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human...

using human induced pluripotent stem cell-derived hepatocyte-like cells for drug discovery

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic...

generation of first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem...

generation of human primordial germ cell-like cells at the surface of embryoid bodies from primed-pluripotency induced pluripotent stem cells

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells

Primordial germ cells (PGCs) are common precursors of both sperm and eggs. Human embryonic PGCs are specified from pluripotent epiblast cells through interactions of cytokines. Here, we describe a 13-day protocol of inducing human cells transcriptomally resembling PGCs at the surface of embryoid bodies from primed-pluripotency induced pluripotent stem...

application of live mitochondria staining in cell-sorting to purify hepatocytes derived from human induced pluripotent stem cells

Author Spotlight: Advancing Hepatocyte Purification from Human Induced Pluripotent Stem Cells for Regenerative Medicine

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166)...

Purification of Human iPSC-Derived Hepatocytes by Mitochondrial-ALCAM Sorting

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generation of spheroids from human pluripotent stem cells

This video demonstrates the transformation of human pluripotent stem cells (hPSCs) into spherical brain organoids. By using specific inhibitors, the hPSCs are guided to differentiate towards a neuronal lineage, forming neuroectodermal colonies. Special growth factors are then introduced, which encourage the self-organization and proliferation of the colonies, ultimately leading to the formation of spherical brain...

Generation of Spheroids from Human Pluripotent Stem Cells

chromatin immunoprecipitation from human embryonic stem cells

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation. <br...

culturing h9-embryonic stem cells for generation of human neural retina

Culturing H9-Embryonic Stem Cells for Generation of Human Neural Retina

generation of natural killer cells from human expanded potential stem cells

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

The present protocol shows how to differentiate CD3&#8722;/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.

vascular organoid generation from human-induced pluripotent stem cells

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

This protocol presents a method for generating vascular organoids using human pluripotent stem...

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

generation of airway epithelial cell air-liquid interface cultures from human pluripotent stem cells

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface...

derivation of hematopoietic stem cells from murine embryonic stem cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice.  Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic...

Add fresh N2 media supplemented with dual SMAD inhibitors daily to each well for the next 3 days. For generating 3D neuroectodermal spheroids from induced 2D neuroectodermal colonies, remove 2 milliliters of N2 medium from the 6-well plate, and wash one time with HBSS to ensure that all of the N2 medium is...

propagation of human embryonic stem (es) cells

Propagation of Human Embryonic Stem ES Cells

Propagation of Human Embryonic Stem (ES) Cells

generation of human blood vessel organoids from pluripotent stem cells

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement...

efficient neural differentiation using single-cell culture of human embryonic stem cells

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine...

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.