This work was supported by Funda&ccedil;&atilde;o de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cient&iacute;fico e Tecnol&oacute;gico (CNPq) and Instituto Nacional de Ci&ecirc;ncia e Tecnologia (INCTC). We are grateful to Bruna S. Paulsen and Aline M. Fernandes for the EB images.

1. Fixation and Cryopreservation
Pluripotent stem cells were cultured onto mouse embryonic fibroblasts (MEF) inactivated with mitomycin C and maintained in DMEM/F12 supplemented with 20% knockout serum replacement (KSR) and 8ng/ mL of fibroblast growth factor (FGF-2). In order to induce EB formation H9 cells were transferred to non-adherent dishes and cultured for 7 days, maintained in DMEM/F12 supplemented with 15% KSR 8. 
Note (!): This technique can be used for EBs der...

<ol>
	<li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <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, 1145-117 (1998).</li><li>Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., Benvenisty, N. <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+human+embryonic+stem+cells+into+embryoid+bodies+compromising+the+three+embryonic+germ+layers.">Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.</a> Mol Med. 6, 88-95 (2000).</li><li>Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+inducing+embryoid+body+formation:+in+vitro+differentiation+system+of+embryonic+stem+cells.">Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells.</a> J Biosci Bioeng. 103, 389-398 (2007).</li><li>Conley, B. J., Young, J. C., Trounson, A. O., Mollard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+propagation+and+differentiation+of+human+embryonic+stem+cells.">Derivation propagation and differentiation of human embryonic stem cells.</a> Int J Biochem Cell Biol. 36, 555-567 (2004).</li><li>Moon, I. S., Cho, S. J., Jin, I., Walikonis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+combined+fluorescence+in+situ+hybridization+and+immunocytochemistry.">A simple method for combined fluorescence in situ hybridization and immunocytochemistry.</a> Mol Cells. 24, 76-82 (2007).</li><li>Daneshtalab, N., Dor&#233;, J. J., Smeda, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+tissue+specificity+and+antibody+selection:+Procedures+in+immunohistochemical+studies.">Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies.</a> J Pharmacol Toxicol Methods. 61, 127-135 (2009).</li><li>Krenacs, L., Krenacs, T., Stelkovics, E., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval+for+immunohistochemical+reactions+in+routinely+processed+paraffin+sections.">Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections.</a> Methods Mol Biol. , 588-5103 (2010).</li><li>Nones, J., Spohr, T. C., Furtado, D. R., Sartore, R. C., Paulsen, B. S., Guimar&#227;es, M. Z., Rehen, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+modulate+cell+survival+in+embryoid+bodies.">Cannabinoids modulate cell survival in embryoid bodies.</a> Cell Biol Int. 12, 399-408 (2010).</li><li>Huang, W. M., Gibson, S. J., Facer, P., Gu, J., Polak, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+section+adhesion+for+immunocytochemistry+using+high+molecular+weight+polymers+of+L-lysine+as+a+slide+coating.">Improved section adhesion for immunocytochemistry using high molecular weight polymers of L-lysine as a slide coating.</a> Histochemistry. 77, 275-279 (1983).</li><li>Kuenzi, M. J., Sherwood, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+specific+relaxin-binding+cells+in+thecervix,+mammary+glands,+and+nipples+of+pregnant+rats.">Immunohistochemical localization of specific relaxin-binding cells in thecervix, mammary glands, and nipples of pregnant rats.</a> Endocrinology. 136, 1367-1373 (1995).</li></ol>

No conflicts of interest declared.

The method described here provides an easy-to-follow protocol to obtain PFA fixed thin cryostat sections of embryoid bodies useful for immunofluorescence and in situ hybridization assays. The resulting cryosections permit the study of cellular and molecular aspects of human embryonic stem cells differentiation, while preserving their structure and organization as aggregates.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>D (+) Sucrose</td>
<td>Reagent</td>
<td>Vetec</td>
<td>228</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Rieden-de Ha&euml;n</td>
<td>16005</td>
<td>&nbsp;</td>
<tr>
<td>PBS solution</td>
<td>Reagent</td>
<td>LGC Biotecnology</td>
<td>13-30259.05</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P2636</td>
<td>200mg/mL in water</td>
</tr>
<tr>
<td>Tissue-Tek O.C.T. Compound</td>
<td>Reagent</td>
<td>Sakura</td>
<td>P2636</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose Solution</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10% Sucrose Solution in PBS w/v</td>
</tr>
<tr>
<td>Sucrose Solution</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>20% Sucrose Solution in PBS w/v</td>
</tr>
<tr>
<td>Sucrose Solution</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>30% Sucrose Solution in PBS w/v</td>
</tr>
<tr>
<td>Conic Tube</td>
<td>Tool</td>
<td>TPP</td>
<td>91015</td>
<td>15mL conic tube</td>
</tr>
<tr>
<td>Plate shaker</td>
<td>Tool</td>
<td>Biomixer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cryostat</td>
<td>Tool</td>
<td>Leica</td>
<td>CM 1850</td>
<td>&nbsp;</td>
<tr>
<td>Mold for OCT platform</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Plastic mold plataform</td>
</tr>		</tbody>
		</table>
		</div>

analysis of pluripotent stem cells by using cryosections of embryoid bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Watch this Scientific Journal Video about Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies at JoVE.com

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

analysis-pluripotent-stem-cells-using-cryosections-embryoid

Research

JoVE Journal

Biology

13.8K Views.  Universidade Federal do Rio de Janeiro (UFRJ), Brazil. Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates. 