We thank Brad Lackford for reading and editing the manuscript. This research was supported by the National Institute of Environmental Health Sciences, National Institutes of Health Intramural Research Program Z01ES102745 (to G. H.).

1. Oct4GiP Mouse ES Cell Maintenance
Oct4GiP cells were kindly provided by Dr. Austin Smith. They were derived from the 129/Ola mice carrying an Oct4-GFPiresPac transgene 4,5 . They are maintained in gelatin-coated tissue culture plates in the ESGRO complete plus clonal grade medium (Millipore), or in the M15 medium: DMEM (Invitrogen) supplemented with 15% FBS, 1000 U/ml ESGRO (Millipore), 1x Non-essential amino acids (Invitrogen), 1x EmbryoMax Nucleasides (Millipore), and 10 &mu;M &beta;-mercaptoethanol.
<ol>
	<li>Coat plates with 0.1% gelatin (Sigma) at room temperature for 30 minutes (0.1 ml gelatin solution/cm2).</li>
	<li>Plate Oct4GiP cells in gelatin-coated plates at ~2 x 104/cm2.</li>
	<li>Split the cells every two days with 0.05% trypsin.</li>
</ol>
2. siRNA Transfection in Oct4GiP Cells
The Oct4GiP reporter assay is most conveniently carried out in gelatin-coated flat bottom 96- or 384-well plates (Corning or BD) in the M15 medium. The overall procedure is outlined in Figure 1.
<ol>
	<li>siRNA-lipid complexes assembly:
	<ol>
		<li>For transfection in 96-well plates, assemble siRNA-lipid complexes in U-bottom 96-well plates.</li>
		<li>In each well, mix 10 &mu;l OptiMEM (Invitrogen) and 0.3 &mu;l Lipofectamine 2000 (Invitrogen) and incubate at room temperature for 5 min.</li>
		<li>Add 5 pmol siRNA to the lipid-OptiMEM mixture and incubate for another 15 minutes. The siRNA : lipid ratio is 100 pmol : 6ul.</li>
		<li>Transfer the siRNA-lipid complexes in OptiMEM from the U-bottom plate to the gelatin-coated flat-bottom plate with a multi-channel pipette.</li>
		<li>For transfection in 384-well plates, prepare the siRNA-lipid complexes using 0.1 &mu;l Lipofectamine 2000 and 2 pmol siRNA in 10 &mu;l OptiMEM.</li>
	</ol>
	</li>
</ol>
Note: The final siRNA concentration in the transfections is 50 nM. Carry out 3-4 biological replicates for each siRNA transfection. Set up master mixtures of the siRNA-lipid complexes in the U-bottom plate and aliquot into the flat-bottom gelatin-coated plate.
<ol start="2">
	<li>Oct4GiP cell plating and transfection:
	<ol>
		<li>For transfection in 96-well plates, collect the Oct4GiP cells and resuspend them in fresh M15 medium to 9 x 104 cells/ml.</li>
		<li>Add 100 &mu;l of the cell suspension to each well using a multi-channel pipette.</li>
		<li>Mix the cell suspension with the pre-aliquoted siRNA-lipids complexes in the well by pipeting up and down 3-5 times. Change tips for different siRNA transfections.</li>
		<li>For Transfection in 384-well plates, resuspend Oct4GiP cells to 6 x 104 cells/ml and aliquot 30 &mu;l cell suspension to each well.</li>
	</ol>
	</li>
</ol>
Note: The optimal cell plating density is usually 3 x 105 cells/cm2, but it may require further optimization for siRNAs that dramatically affect cell growth or viability. Low plating density will lead to poor cell survival during transfection. High plating density will lead to high background due to high cell confluence induced differentiation. If necessary, test plating density between 2-4 x 105 cells/cm2.
<ol start="3">
	<li>Medium change:
	<ol>
		<li>Remove old medium using multi-channel aspirator (Corning) or vacuum wand (VP-Scientific). Be careful not to scratch the cells at the bottom of the plates.</li>
		<li>Feed the cells with fresh ES cell medium (100 &mu;l for 96-well plate and 30 &mu;l for 384-well plate) using multi-channel pipette every day.</li>
	</ol>
	</li>
</ol>
3. FACS Analysis
<ol>
	<li>Cell dissociation:
	<ol>
		<li>Four days after transfection, remove medium using multi-channel aspirator (Corning) or vacuum wand (VP-Scientific).</li>
		<li>For 96-well plates, rinse cells once with 100&mu;l PBS.</li>
		<li>Add 25 &mu;l 0.25% trypsin to each well using a multi-channel pipette.</li>
		<li>Incubate at room temperature for 5minutes with occasional agitation, and visually inspect to ensure complete detachment of the cells.</li>
		<li>Add 90 &mu;l of PBS with 10% FBS to inactivate the trypsin and dissociate cells into a single cell suspension by repeated pipetting.</li>
		<li>For 384-well plates, disassociate cells with 10 &mu;l trypsin and quench with 30 &mu;l PBS with 10% FBS.</li>
	</ol>
	</li>
	<li>FACS analysis:
	<ol>
		<li>Analyze the GFP fluorescence on the BD LSRII FACS analyzer equipped with the HTS unit or other similarly equipped FACS analyzer (such as Accuri or Intellicyt).</li>
		<li>Use the high-throughput mode on the HTS and analyze 10 &mu;l of cell suspension. Set the count threshold to 1.0 x 104 cells.</li>
		<li>Adjust PMT voltage and threshold to capture the cells in the forward vs. side-scatter plot. Gate for the live cell population in the forward vs. side-scatter plot.</li>
		<li>Create a histogram plot for the GFP channel. Set the gate in the GFP channel so that ~10% of the cells appear to be GFP-negative in the mock or control-siRNA transfected cells.</li>
		<li>Determine % GFP-negative cells from each treatment: % Differentiated cells = % GFP-negative cells. Compare the % Differentiated cells between experimental- and control-siRNA transfections using 2-tailed t-test, and score the positive hits with p-values &lt; 0.01.</li>
	</ol>
	</li>
</ol>
4. Representative Results
Oct4, Nanog, and Sox2 are three genes that play critical roles in the maintenance of ES cell self-renewal 6,7,9-11. Figure 2 shows that the Oct4GiP reporter assay can readily detect the differentiation caused by silencing these factors in the Oct4GiP ES cells.
Figure 2A shows the Oct4GiP ES cells are GFP-positive when maintained as ES cells. Figure 2B shows the forward vs. side scatter plot and the histogram of the GFP channel of the Oct4GiP cells transfected with the control- or Oct4-siRNAs. It is necessary to gate for live cells in the forward vs. side scatter plot, as the dead cells and debris are GFP-negative and will increase background. On the other hand, doublet discrimination to exclude possible cell clumps is not always needed. In the control-siRNA transfected cells, the vast majority of the cells should be GFP-positive. If obvious GFP-negative populations are present in the control-siRNA transfected wells, the starting Oct4GiP cells or the transfection procedure may have been compromised and the result may not be interpretable.
Figure 2C shows the bar graph of % Differentiated cells (% Differentiated cells = % GFP-negative cells) from control-, Oct4-, Nanog-, and Sox2-siRNA transfected cells.
<img alt="Figure 1" src="/files/ftp_upload/3987/3987fig1.jpg" /> 
Figure 1. Outline of the Oct4GiP reporter assay.
<img alt="Figure 2" src="/files/ftp_upload/3987/3987fig2.jpg" /> 
Figure 2. Oct4GiP reporter assay can detect ES cell differentiation caused by Nanog, Oct4, or Sox2 silencing. A) E14Tg2a (wild-type, black line) and Oct4GiP (green line) cells were analyzed by FACS. Histogram of the GFP-channel shows that Oct4GiP cells are GFP-positive. B) Oct4GiP cells were transfected in 96-well plates with the Control- or Oct4-siRNA and FACS analyzed 4 days after transfection. Forward vs. side scatter plots and histograms of the GFP-channel of the transfected cells are shown. C) Oct4GiP cells were transfected with the Control-, Nanog-, Oct4-, or Sox2-siRNA. The % Differentiated Cells was determined from the % GFP-negative cells 4 days after transfection, and was plotted as mean +/- standard error of the mean (n = 4). <a href="https://www.jove.com/files/ftp_upload/3987/3987fig2large.jpg" target="_blank"> Click here to view larger figure</a>.

<ol>
	<li>Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+differentiation:+emergence+of+a+new+era+in+biology+and+medicine.">Embryonic stem cell differentiation: emergence of a new era in biology and medicine.</a> Genes Dev. 19, 1129-1155 (2005).</li><li>Murry, C. E., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+embryonic+stem+cells+to+clinically+relevant+populations:+lessons+from+embryonic+development.">Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.</a> Cell. 132, 661-680 (2008).</li><li>Hu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+RNAi+screen+identifies+a+new+transcriptional+module+required+for+self-renewal.">A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal.</a> Genes Dev. 23, 837-848 (2009).</li><li>Ying, Q. L., Nichols, J., Evans, E. P., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+potency+by+spontaneous+fusion.">Changing potency by spontaneous fusion.</a> Nature. 416, 545-548 (2002).</li><li>Ying, Q. L., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+conditions+for+neural+commitment+and+differentiation.">Defined conditions for neural commitment and differentiation.</a> Methods Enzymol. 365, 327-341 (2003).</li><li>Nichols, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+pluripotent+stem+cells+in+the+mammalian+embryo+depends+on+the+POU+transcription+factor+Oct4.">Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.</a> Cell. 95, 379-391 (1998).</li><li>Niwa, H., Miyazaki, J., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+expression+of+Oct-3/4+defines+differentiation,+dedifferentiation+or+self-renewal+of+ES+cells.">Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.</a> Nat. Genet. 24, 372-376 (2000).</li><li>Ding, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-scale+RNAi+screen+for+Oct4+modulators+defines+a+role+of+the+Paf1+complex+for+embryonic+stem+cell+identity.">A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity.</a> Cell Stem Cell. 4, 403-415 (2009).</li><li>Chambers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+cloning+of+Nanog,+a+pluripotency+sustaining+factor+in+embryonic+stem+cells.">Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.</a> Cell. 113, 643-655 (2003).</li><li>Masui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotency+governed+by+Sox2+via+regulation+of+Oct3/4+expression+in+mouse+embryonic+stem+cells.">Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells.</a> Nat. Cell Biol. 9, 625-635 (2007).</li><li>Mitsui, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+homeoprotein+Nanog+is+required+for+maintenance+of+pluripotency+in+mouse+epiblast+and+ES+cells.">The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.</a> Cell. 113, 631-642 (2003).</li><li>Fazzio, T. G., Huff, J. T., Panning, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNAi+screen+of+chromatin+proteins+identifies+Tip60-p400+as+a+regulator+of+embryonic+stem+cell+identity.">An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity.</a> Cell. 134, 162-174 (2008).</li><li>Pease, S., Braghetta, P., Gearing, D., Grail, D., Williams, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+embryonic+stem+(ES)+cells+in+media+supplemented+with+recombinant+leukemia+inhibitory+factor.">Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor.</a> 141, 344-352 (1990).</li><li>Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+the+embryonic+stem+cell+state.">Control of the embryonic stem cell state.</a> Cell. 144, 940-954 (2011).</li><li>Chambers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanog+safeguards+pluripotency+and+mediates+germline+development.">Nanog safeguards pluripotency and mediates germline development.</a> Nature. 450, 1230-1234 (2007).</li><li>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directly+reprogrammed+fibroblasts+show+global+epigenetic+remodeling+and+widespread+tissue+contribution.">Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.</a> Cell Stem Cell. 1, 55-70 (2007).</li><li>Rodda, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+nanog+by+OCT4+and+SOX2.">Transcriptional regulation of nanog by OCT4 and SOX2.</a> J. Biol. Chem. 280, 24731-24737 (2005).</li><li>Schaniel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+RNAs--triggers+of+gene+silencing--into+mouse+embryonic+stem+cells.">Delivery of short hairpin RNAs--triggers of gene silencing--into mouse embryonic stem cells.</a> Nat. Methods. 3, 397-400 (2006).</li><li>Toyooka, Y., Shimosato, D., Murakami, K., Takahashi, K., Niwa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+subpopulations+in+undifferentiated+ES+cell+culture.">Identification and characterization of subpopulations in undifferentiated ES cell culture.</a> Development. 135, 909-918 (2008).</li></ol>

No conflicts of interest declared.

The Oct4GiP reporter assay we describe above can quantitatively measure the extent of self-renewal vs. differentiation. Compared to other available methods, such as the morphology-based 12 and proliferation/viability-based assays, it offers higher sensitivity and throughput, as well as a more direct measurement of the ES cell state. It is therefore well suited for large-scale screens and genetic epistasis analysis. Indeed, we and others have successfully used the Oct4GiP reporter assay for genome-wide RNAi scr...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>ESGRO complete plus clonal grade medium</td>
			<td>Millipore</td>
			<td>SF001-500P</td>
		</tr>
		<tr>
			<td>DMEM (High glucose 1X)</td>
			<td>Invitrogen</td>
			<td>11965</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>1001817</td>
		</tr>
		<tr>
			<td>OPTI-MEM(reduce serum medium)</td>
			<td>Invitrogen</td>
			<td>31985</td>
		</tr>
		<tr>
			<td>ESGRO mLIF (107 units/1ml)</td>
			<td>Millipore</td>
			<td>DAM1776540</td>
		</tr>
		<tr>
			<td>MEM NEAA (Non-Essential Amino Acids)</td>
			<td>Invitrogen</td>
			<td>11140</td>
		</tr>
		<tr>
			<td>100x Nucleosides for ES cell</td>
			<td>Millipore</td>
			<td>10620-1</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522-100ml</td>
		</tr>
		<tr>
			<td>ES-qualified fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>10437</td>
		</tr>
		<tr>
			<td>Nanog siRNA</td>
			<td>Invitrogen</td>
			<td>MSS231181</td>
		</tr>
		<tr>
			<td>Oct4 siRNA</td>
			<td>Dharmacon</td>
			<td>D-046256-02</td>
		</tr>
		<tr>
			<td>Sox2 siRNA</td>
			<td>Dharmacon</td>
			<td>M-058489-01</td>
		</tr>
	</tbody>
</table>
Control siRNA: siRNA duplex targeting firefly luciferase (5'-CGTACGCGGAATACTTCGA) synthesized by Dharamcon.

oct4gip reporter assay to study genes that regulate mouse embryonic stem cell maintenance and self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

Watch this Scientific Journal Video about Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal at JoVE.com

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

oct4gip-reporter-assay-to-study-genes-that-regulate-mouse-embryonic

Research

JoVE Journal

Biology

10.4K Views.  National Institute of Environmental Health Sciences. We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Video: Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem 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 ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

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 ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...