Special thanks to Dr. Boris Greber for setting up the Activin A ELISA protocol as published in Greber et al. 2007. We are particularly grateful to Mrs. Monica Shevack for preparing the graphical overview. We are very grateful to Dr Heiko Fuchs for all help and valuable suggestions before and during the filming. We would like to thank all members of the Adjaye laboratory, especially Elisabeth Socha for maintaining a constant supply of MEFs and CM. We also acknowledge our colleagues at the animal facility of the MPIMG for their permanent support. This work was partly funded by The Max Planck Society and the [BMBF; grant number 0315717A], partners of the ERASysBio+ initiative supported under the EU ERA-NET Plus scheme in FP7.

1. Isolation of Mouse Embryonic Fibroblasts (MEFs)
The following two steps are performed under non-aseptic conditions.
<ol>
	<li>Sacrifice a pregnant mouse (CF1, Harlan, USA) at 13 or 14 d.p.c. (day post-coitum) by cervical dislocation.</li>
	<li>Dissect out the uterine horns, briefly rinse in 70% (v/v) ethanol and place into a falcon tube containing PBS without Ca2+Mg2+ (Gibco, Invitrogen).</li>
</ol>
The following steps are carried out in a tissue culture hood under aseptic conditions and using sterile instruments.
<ol start="3">
	<li>Place uterine horns into a Petri dish and separate each embryo from its placenta and embryonic sac.</li>
	<li>Dissect head and red organs, wash in PBS and place all embryos in a clean Petri dish. Finely mince the tissue using a sterile razor blade until it becomes possible to pipette.</li>
	<li>Add 1 ml of 0.05% trypsin/EDTA (Gibco, Invitrogen), including 100 Kunitz units of DNase I (USB), per embryo.</li>
	<li>Transfer the tissue into a 50 ml falcon tube and incubate for 15 min at 37 &deg;C. After each 5 min of incubation, dissociate cells by pipetting up and down thoroughly.</li>
	<li>Inactivate the trypsin by adding about 1 volume of freshly prepared MEF medium. 
	MEF culture medium (components to make 500 ml of media, mix all components and filter): 
	450 ml of DMEM, 50 ml of FBS (10% (v/v)), 5 ml of 200 mM L-glutamine (1/100 (v/v)), 5 ml of Penicillin-streptomycin (1/100 (v/v)).</li>
	<li>Centrifuge the cells with low-speed (300 x g), 5 min, carefully remove the supernatant and resuspend cell pellet in warm MEF medium.</li>
	<li>Plate approximately a number of cells which is equivalent to 3-4 embryos in each T150 (TPP) flask coated with 0.2% gelatine (Gelatine from bovine skin, Type B, Sigma) for 2 hr. The fibroblasts (P0, passage 0) are the only cells that have the ability to attach to the gelatine-coated flasks.</li>
	<li>Ideally, cells are 80-90% confluent after 24 hr and at this stage a major part of P0 cells is frozen for future usage.</li>
	<li>Expand the remaining T150 flask(s) of P0 cells till P3 or P4, then inactivate and use as feeders to replate hESCs or to produce conditioned medium (CM).</li>
</ol>
2. Inactivation and Plating MEFs (Feeder Cells Preparation)
All steps are carried out in a tissue culture hood under aseptic conditions.
<ol>
	<li>Coat T150 flasks with 0.2% gelatine and incubate at RT for at least 2 hr.</li>
	<li>Dilute mitomycin C in PBS (1 mg/ml) and filter.</li>
	<li>Aspirate media from MEFs and wash with PBS without Ca2+Mg2+.</li>
	<li>Place 20 ml of medium containing 10 &mu;g/ml of mitomycin C on MEFs.</li>
	<li>Incubate for 2 hr at 37 &deg;C with mitomycin C containing medium then wash twice with PBS, trypsinize, centrifuge (for 5 min at 300 x g) and resuspend cells in warm medium.</li>
	<li>Count cells and plate at a density of 56.000 cells/cm2 in T150 flasks and use for CM production for the following 6 days.</li>
</ol>
3. Conditioned Medium (CM) Preparation
All steps are carried out in a tissue culture hood under aseptic conditions.
<ol>
	<li>The day after plating inactivated MEFs at a density of 56.000 cells/cm2 replace the MEF medium with hESC medium (UM, unconditioned medium) (0.5 ml/cm2) supplemented freshly with 4 ng/ml of FGF2.</li>
	<li>Collect CM from feeder flasks after 24 h incubation and add fresh hESC medium containing 4 ng/ml of FGF2 to the feeders.</li>
	<li>Repeat this procedure for the next 6 days. Each day store collected CM at -20 &deg;C.</li>
	<li>After 6 days mix all aliquots of medium and filter (Corning, 0.22 &mu;m, PAS). Make 50 ml aliquots and store at -80 &deg;C.</li>
	<li>Supplement CM with additional 4 ng/ml of FGF2 before adding to hESCs grown on Matrigel.</li>
</ol>
4. Measurement of Activin A in Conditioned Media (ELISA)15
<ol>
	<li>Bring all samples and reagents to room temperature.</li>
	<li>Dilute capture antibody (Human\Mouse\Rat Activin A MAb, R&amp;D Systems) in PBS with 1% BSA, add to microplate (100 &mu;l/well) and incubate overnight at RT.</li>
	<li>After 24 hr wash the wells three times with PBST (PBS with 0.05% Tween 20) (300 &mu;l/well) then block (1% BSA/PBS, 300 &mu;l/well) for 1 hr at RT.</li>
	<li>In this time prepare an Activin A (R&amp;D Systems) standard curve, including 7 dilutions (concentration less than 30 ng/ml) and blank sample. The linear working range of the Activin A is between 0.25 and 32 ng/ml.</li>
	<li>Add duplicates of standards and samples to the wells (100 &mu;l/well) and incubate for 2 hr at RT.</li>
	<li>Wash wells three times (300 &mu;l of PBST).</li>
	<li>Add the secondary (biotinylated) antibody (Human/Mouse/Rat Biotinylated Activin A MAb, R&amp;D Systems) (0.25 &mu;g/ml in 1% BSA/PBS) and incubate for 2 hr at RT.</li>
	<li>Wash wells three times (300 &mu;l of PBST), add Streptavidin-HRP (diluted in 1%BSA/PBS, R&amp;D Systems) and incubate for 20 min at RT.</li>
	<li>Wash wells three times (300 &mu;l of PBST), add 100 &mu;l of substrate solution (Quantikine, R&amp;D Systems) and incubate for 30 min at RT in the dark.</li>
	<li>Add 100 &mu;l of stop solution (Quantikine, R&amp;D Systems) to each well and mix gently.</li>
	<li>Set Microplate reader (Molecular Devices Spectra Max 250, Global Medical Instrumentation, Inc., Minnesota) to 450 nm, with wavelength correction at 540 or 570 nm, to determine the optical density of each well.</li>
</ol>
5. Representative Results
The overall scheme of the isolation procedure is presented in Figure 1. The typical morphology of hESCs and hiPSCs cultured under different conditions is presented in Figure 2. The morphology of MEFs and inactivated feeder cells used to prepare CM is presented in Figure 3. In general, cells should be confluent 24 hours post-isolation and ready to be frozen or expanded. However, sometimes it might take 2-3 days before obtaining confluent cultures. The CM should be prepared from cells at passage 4 and not later. This is crucial because primary cells can only be expanded for 4-5 passages before the onset of senescence.
The cytokine, Activin A, is considered as the most critical factor secreted by feeder cells for the support of undifferentiated growth of pluripotent cells14. The measurement of the level of Activin A in CM (Figure 4) is a very convenient quantitative assay to monitor the quality of MEFs.
<img alt="Figure 1" src="/files/ftp_upload/3854/3854fig1.jpg" /> 
Figure 1. A schematic representation of the MEFs isolation procedure.
<img alt="Figure 2" src="/files/ftp_upload/3854/3854fig2.jpg" /> 
Figure 2. The typical morphology of undifferentiated hESCs cultured in the presence of (A) feeder cells, (B) conditioned medium and (C) defined medium. The typical morphology of hiPSCs cultured in the presence of (D, E) feeder cells.
<img alt="Figure 3" src="/files/ftp_upload/3854/3854fig3.jpg" /> 
Figure 3. The typical morphology of mouse embryonic fibroblasts (MEFs). (A) Passage 0 (P0) two days after plating/isolation, (B) inactivated feeder layer at a density of 56.000 cells/cm.2
<img alt="Figure 4" src="/files/ftp_upload/3854/3854fig4.jpg" /> 
Figure 4. Enzyme-linked immunosorbent assay (ELISA)-based measurements of the concentration of Activin A in conditioned medium (CM) prepared with mouse embryonic fibroblasts derived from the CF1 mouse strain. CM was collected for 6 days and then pooled. CM"1" and CM"2" refer to different batches of media and UM to unconditioned media. As the function of the conditioning process is Activin A secretion into the medium by MEFs, Activin A is almost undetectable in UM.

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	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Thomson, J. A. <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+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Amit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonally+derived+human+embryonic+stem+cell+lines+maintain+pluripotency+and+proliferative+potential+for+prolonged+periods+of+culture.">Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.</a> Dev. Biol. 227, 271-278 (2000).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat. Biotechnol. 19, 971-974 (2001).</li><li>Ludwig, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+human+embryonic+stem+cells+in+defined+conditions.">Derivation of human embryonic stem cells in defined conditions.</a> Nat. Biotechnol. 24, 185-187 (2006).</li><li>Yao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+self-renewal+and+directed+differentiation+of+human+embryonic+stem+cells+in+chemically+defined+conditions.">Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 6907-6912 (2006).</li><li>Lu, J., Hou, R., Booth, C. J., Yang, S. H., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+culture+conditions+of+human+embryonic+stem+cells.">Defined culture conditions of human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 5688-5693 (2006).</li><li>Skottman, H., Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+conditions+for+human+embryonic+stem+cells.">Culture conditions for human embryonic stem cells.</a> Reproduction. 132, 691-698 (2006).</li><li>Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., Bongso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeders+support+prolonged+undifferentiated+growth+of+human+inner+cell+masses+and+embryonic+stem+cells.">Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.</a> Nat. Biotechnol. 20, 933-936 (2002).</li><li>Richards, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+various+human+feeders+for+prolonged+undifferentiated+growth+of+human+embryonic+stem+cells.">Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.</a> Stem Cells. 21, 546-556 (2003).</li><li>Amit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeder+layers+for+human+embryonic+stem+cells.">Human feeder layers for human embryonic stem cells.</a> Biol. 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J. Dev. Biol. 52, 353-363 (2008).</li><li>Greber, B., Lehrach, H., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+2+modulates+transforming+growth+factor+beta+signaling+in+mouse+embryonic+fibroblasts+and+human+ESCs+(hESCs)+to+support+hESC+self-renewal.">Fibroblast growth factor 2 modulates transforming growth factor beta signaling in mouse embryonic fibroblasts and human ESCs (hESCs) to support hESC self-renewal.</a> Stem Cells. 25, 455-464 (2007).</li><li>Vallier, L., Alexander, M., Pedersen, 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=Activin/Nodal+and+FGF+pathways+cooperate+to+maintain+pluripotency+of+human+embryonic+stem+cells.">Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells.</a> J. 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No conflicts of interest declared.

The MEFs isolation procedure presented here enables the establishment of a standardized culture condition for hESCs and hiPSCs. Moreover the ELISA-based system used to evaluate cytokine production by feeder cells is a useful indicator of the quality of the MEF-derived conditioned media. The routine maintenance of the mouse strain (CF1) providing supporting fibroblasts is necessary to avoid batch-to-batch variation of culture media. Moreover, it is recommended to isolate embryos from several mice simultaneously to obtain consistent quality of cells. Certainly freshly isolated MEFs can be kept frozen at P0 and P1. Also inactivated MEFs can be kept frozen in aliquots of appropriate amounts depending on the requirements for cell culture. Usually approximately 250.000 cells should be plated in a single well of a 6-well plate to culture hESCs and iPS cells. The established and optimized method can be routinely used to minimize the variability between different experiments.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM (High Glucose)</td>
			<td>Gibco, Invitrogen</td>
			<td>41966-052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biochrom</td>
			<td>S0115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco, Invitrogen</td>
			<td>25030-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco, Invitrogen</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vacuum filter system</td>
			<td>Corning</td>
			<td>431097</td>
			<td>500 ml, 0.22 &mu;m, PAS</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco, Invitrogen</td>
			<td>10829-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Gibco, Invitrogen</td>
			<td>10828-028</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Gibco, Invitrogen</td>
			<td>11140-035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>beta-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco, Invitrogen</td>
			<td>14190-169</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>D4527</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Roche</td>
			<td>10107409001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Basic Fibroblast Growth Factor</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biotinylated Anti-human/mouse/rat Activin A Antibody</td>
			<td>R&amp;D Systems</td>
			<td>BAM3381</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human/Mouse/Rat Activin A MAb</td>
			<td>R&amp;D Systems</td>
			<td>MAB3381</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco, Invitrogen</td>
			<td>25300-054</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Extra Thin Iris Scissors</td>
			<td>FST</td>
			<td>14088-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps</td>
			<td>FST</td>
			<td>11150-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pierse Fixation Forceps</td>
			<td>FST</td>
			<td>18155-13</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

Watch this Scientific Journal Video about Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells at JoVE.com

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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90.4K Views.  Max Planck Institute for Molecular Genetics. The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgfβ1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Video: Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Rozprzestrzenianie się człowieka zarodkowych komórek macierzystych (ES) Cells

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...