The authors thank Andrea Axtell, Satoshi Itoh, MD, Jeff Velotta, MD, Grant Hoyt, Robert C. Robbins, MD, Jin Yu, MD, Tim Doyle, PhD, and the Stanford Small Animal Imaging Core for technical assistance. The authors also thank A.M. Bickford, Inc. for support of veterinary equipment. This research was supported by research grants from the National Institutes of Health (R01 HL-75774, R01 CA098303, R21 HL085743, 1K12 HL087746), the California Tobacco Related Disease Research Program of the University of California (15IT-0257 and 1514RT-0169 ), and the California Institute for Regenerative Medicine (RS1-00183).
N.H. is supported by a fellowship from the American Heart Association. can Heart Association.

1. Differentiation of murine ESCs into endothelial cells
<ol>
<li>The protocol for differentiating ESCs into endothelial cells is described elsewhere and is not the focus of this protocol2,3. Briefly, the cells are allowed to differentiate, and the cells that are positive for the endothelial markers such as CD31 or vascular endothelial cadherin (VE-cadherin) are then purified by fluorescence activated cell sorting (FACS).</li>
</ol>
2. Construction of the Double Fusion Reporter Gene...

<ol>
	<li>Niiyama, H., Huang, N. F., Rollins, M., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> JoVE. , (2008).</li><li>Levenberg, S., Golub, J. S., Amit, M., Itsakovitz-Eldor, J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+derived+from+human+embryonic+stem+cells.">Endothelial cells derived from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 4391-4396 (2002).</li><li>Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flk1-positive+cells+derived+from+embryonic+stem+cells+serve+as+vascular+progenitors.">Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.</a> Nature. 408, 926-926 (2000).</li><li>De, A., Yaghoubi, S. S., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+lentiviral+vectors+in+noninvasive+molecular+imaging.">Applications of lentiviral vectors in noninvasive molecular imaging.</a> Methods Mol Biol. 433, 177-202 (2008).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+laboratory+mouse.">The anatomy of the laboratory mouse.</a> , (1976).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nature Med. 4, 245-247 (1998).</li><li>Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Huang, N. F., Lee, R. J., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physical+regulation+of+stem+cells+and+progenitor+cells:+potential+for+cardiovascular+tissue+engineering.">Chemical and physical regulation of stem cells and progenitor cells: potential for cardiovascular tissue engineering.</a> Tissue Eng. 13, 1809-1823 (2007).</li><li>Cao, F., Lin, S., Xie, X., Ray, P., Patel, M., Zhang, X., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X., Weissman, I. L., Gambhir, S. S., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+embryonic+stem+cell+survival,+proliferation,+and+migration+after+cardiac+delivery.">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> Circulation. 113, 1005-1114 (2006).</li><li>Wilson, K., Yu, J., Lee, A., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+bioluminescence+reporter+gene+imaging+of+human+embryonic+stem+cells.">In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells.</a> J Vis Exp. , (2008).</li></ol>

ESCs are a promising cell source for the treatment of tissue ischemia because of their plasticity of differentiation and their ability to give rise to cell lineages comprising all three germ layers, including endothelial cells. To overcome the ethical concerns associated with ESCs, induced pluripotent stem cells (iPSCs) may be an alternative pluripotent stem cell source that overcomes the ethical concerns. Besides ESCs, adult stem cells such as endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs) can...

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Surgical tools</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Syringe needle</td>
<td>Tool</td>
<td>BD</td>
<td>&nbsp;</td>
<td>28G insulin syringe is preferred</td>
</tr>
<tr>
<td>Phosphate Buffered Saline </td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-luciferin</td>
<td>Reagent</td>
<td>Biosynth International, Inc</td>
<td>&nbsp;</td>
<td>Prepare D-luciferin in advance into filtered stock solutions of 15 mg/mL in PBS</td>
</tr>
<tr>
<td>IVIS 200 Bioluminescence imaging system and acquisition software </td>
<td>Equipment</td>
<td>Xenogen Corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

Watch this Scientific Journal Video about Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia at JoVE.com

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

embryonic-stem-cell-derived-endothelial-cells-for-treatment-hindlimb

Research

JoVE Journal

Biology

11.7K ビュー.  Stanford University. 胚性幹細胞の配信のための外科的処置は、虚血後肢への細胞由来の内皮細胞は、生物発光イメージングによる非侵襲的な追跡と、示されている。

ビデオ: Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

murine model of hindlimb ischemia 

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion...

Murine Model of Hindlimb Ischemia 

neisseria meningitidis infection of induced pluripotent stem-cell derived brain endothelial cells

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

methods for acute and subacute murine hindlimb ischemia

Methods for Acute and Subacute Murine Hindlimb Ischemia

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

isolation of murine embryonic hemogenic endothelial cells

Isolation of Murine Embryonic Hemogenic Endothelial Cells

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic...

migration, chemo-attraction, and co-culture assays for human stem cell-derived endothelial cells and gabaergic neurons

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic...

isolation of embryonic ventricular endothelial cells

Isolation of Embryonic Ventricular Endothelial Cells

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further...

cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells at the optimal stage

Author Spotlight: Development of an Efficient and Feasible Method of Retinal Pigment Epithelial Cell Freezing

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial...

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

preservation of human embryonic stem cell-derived retinal pigment epithelial cells with optimized cryopreservation

Preservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells with Optimized Cryopreservation

pluripotent stem cell derived cardiac cells for myocardial repair

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine...

endothelial cell co-culture mediates maturation of human embryonic stem cell to pancreatic insulin producing cells in a directed differentiation approach

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing...

microfluidic assay for the assessment of leukocyte adhesion to human induced pluripotent stem cell-derived endothelial cells (hipsc-ecs)

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

assessing therapeutic angiogenesis in a murine model of hindlimb ischemia

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic...

clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche co-culture

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic...

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...

isolation, in vitro expansion, and characterization of mesenchymal stem cells from mouse epididymal adipose tissue

Author Spotlight: Advancing Treatment Approaches with Adipose-Derived Stem Cells

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.

Primary Culture of Adipose Tissue Derived Stem Cells from Mouse Epididymis

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sub-retinal delivery of human embryonic stem cell derived photoreceptor progenitors in rd10 mice

Author Spotlight: Advancing Vision Restoration - Stem Cell-Based Therapy for Retinal Diseases 

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10...

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Advancing Cryostorage of Human Stem Cell-Derived Retinal Pigment Epithelial Cells

subretinal transplantation of human embryonic stem cell derived-retinal pigment epithelial cells into a large-eyed model of geographic atrophy

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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 ...

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...