Our research was supported by grants from Instituto Nacional de Perinatolog&#237;a de M&#233;xico (21041 and 21081) and CONACYT (A1-S-8450 and&#160;252756). We thank Jessica Gonz&#225;lez Norris and Lidia Yuriria Paredes Vivas for the technical support.

This protocol was approved by the ethical committee of Instituto Nacional de Perinatolog&#237;a in Mexico City (Registry number 212250-21041). All procedures performed in these studies were in accordance with the ethical standards of the Instituto Nacional de Perinatolog&#237;a, the Helsinki Declaration, and the guidelines set forth in the Ministry of Health&#8217;s Official Mexican Standard.
1. Preparation
<ol>
	<li>Prepare a solution of 1x PBS with EDTA. To do so, add 500 &#956;L of 0.5 M ...

<ol>
	<li>Shahbazi, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+the+human+embryo+in+the+absence+of+maternal+tissues.">Self-organization of the human embryo in the absence of maternal tissues.</a> Nature Cell Biology. 18 (6), 700-708 (2016).</li><li>Garcia-Lopez, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amniotic+epithelium+(HAE)+as+a+possible+source+of+stem+cells+(SC).">Human amniotic epithelium (HAE) as a possible source of stem cells (SC).</a> Gaceta Medica de Mexico. 151 (1), 66-74 (2015).</li><li>Gramignoli, R., Srinivasan, R. C., Kannisto, K., Strom, S. C. <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+Human+Amnion+Epithelial+Cells+According+to+Current+Good+Manufacturing+Procedures.">Isolation of Human Amnion Epithelial Cells According to Current Good Manufacturing Procedures.</a> Current Protocols in Stem Cell Biology. 37, (2016).</li><li>Murphy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amnion+epithelial+cell+isolation+and+characterization+for+clinical+use.">Amnion epithelial cell isolation and characterization for clinical use.</a> Current Protocols in Stem Cell Biology. , (2010).</li><li>Garcia-Castro, I. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+of+Pluripotency+in+Human+Amniotic+Epithelial+Cells+and+Their+Differentiation+to+Progenitor+of+Cortical+Neurons.">Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons.</a> PLoS One. 10 (12), 0146082 (2015).</li><li>Garcia-Lopez, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotency+markers+in+tissue+and+cultivated+cells+in+vitro+of+different+regions+of+human+amniotic+epithelium.">Pluripotency markers in tissue and cultivated cells in vitro of different regions of human amniotic epithelium.</a> Experimental Cell Research. 375 (1), 31-41 (2019).</li><li>Yang, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+characterization+of+human+amniotic+epithelial+cells+in+a+serum-free+system+and+their+safety+evaluation.">Biological characterization of human amniotic epithelial cells in a serum-free system and their safety evaluation.</a> Acta Pharmacological Sinica. 39 (8), 1305-1316 (2018).</li><li>Zou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA32+silences+WWP2+expression+to+maintain+the+pluripotency+of+human+amniotic+epithelial+stem+cells+and+beta+isletlike+cell+differentiation.">MicroRNA32 silences WWP2 expression to maintain the pluripotency of human amniotic epithelial stem cells and beta isletlike cell differentiation.</a> International Journal of Molecular Medicine. 41 (4), 1983-1991 (2018).</li><li>Avila-Gonzalez, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+the+ephemeral+human+pluripotent+state.">Capturing the ephemeral human pluripotent state.</a> Developmental Dynamics. 245 (7), 762-773 (2016).</li><li>Niknejad, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+amniotic+membrane+for+potential+use+in+tissue+engineering.">Properties of the amniotic membrane for potential use in tissue engineering.</a> European Cells &amp; Materials. 15, 88-99 (2008).</li><li>Miki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+characteristics+and+the+therapeutic+potential+of+amniotic+epithelial+cells.">Stem cell characteristics and the therapeutic potential of amniotic epithelial cells.</a> American Journal of Reproductive Immunology. 80 (4), 13003 (2018).</li><li>Wu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+proliferation,+migration+and+angiogenic+properties+of+human+amniotic+epithelial+and+mesenchymal+stem+cells+and+their+effects+on+endothelial+cells.">Comparison of the proliferation, migration and angiogenic properties of human amniotic epithelial and mesenchymal stem cells and their effects on endothelial cells.</a> International Journal of Molecular Medicine. 39 (4), 918-926 (2017).</li><li>Hammer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amnion+epithelial+cells,+in+contrast+to+trophoblast+cells,+express+all+classical+HLA+class+I+molecules+together+with+HLA-G.">Amnion epithelial cells, in contrast to trophoblast cells, express all classical HLA class I molecules together with HLA-G.</a> American Journal of Reproductive Immunology. 37 (2), 161-171 (1997).</li><li>Benirschke, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+Pathology+of+the+Placental+Membranes.">Anatomy and Pathology of the Placental Membranes.</a> Pathology of the Human Placenta. , 268-318 (1995).</li><li>Banerjee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+metabolic+activity+in+placental+and+reflected+regions+of+the+human+amniotic+membrane.">Different metabolic activity in placental and reflected regions of the human amniotic membrane.</a> Placenta. 36 (11), 1329-1332 (2015).</li><li>Kim, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-143+regulation+of+prostaglandin-endoperoxidase+synthase+2+in+the+amnion:+implications+for+human+parturition+at+term.">miR-143 regulation of prostaglandin-endoperoxidase synthase 2 in the amnion: implications for human parturition at term.</a> PLoS One. 6 (9), 24131 (2011).</li><li>Han, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Region-specific+gene+expression+profiling:+novel+evidence+for+biological+heterogeneity+of+the+human+amnion.">Region-specific gene expression profiling: novel evidence for biological heterogeneity of the human amnion.</a> Biology of Reproduction. 79 (5), 954-961 (2008).</li><li>Alcaraz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+TGF-beta+induces+epithelial+to+mesenchymal+transition+in+human+amniotic+epithelial+cells.">Autocrine TGF-beta induces epithelial to mesenchymal transition in human amniotic epithelial cells.</a> Cell Transplantation. 22 (8), 1351-1367 (2013).</li><li>Canciello, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+prevents+epithelial-mesenchymal+transition+of+ovine+amniotic+epithelial+cells+and+enhances+their+immunomodulatory+properties.">Progesterone prevents epithelial-mesenchymal transition of ovine amniotic epithelial cells and enhances their immunomodulatory properties.</a> Scientific Reports. 7 (1), 3761 (2017).</li><li>Canciello, A., Greco, L., Russo, V., Barboni, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+Epithelial+Cell+Culture.">Amniotic Epithelial Cell Culture.</a> Methods in Molecular Biology. 1817, 67-78 (2018).</li><li>Singh, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+network+crosstalk+in+human+pluripotent+cells:+a+Smad2/3-regulated+switch+that+controls+the+balance+between+self-renewal+and+differentiation.">Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation.</a> Cell Stem Cell. 10 (3), 312-326 (2012).</li><li>Villa-Diaz, L. G., Kim, J. K., Laperle, A., Palecek, S. P., Krebsbach, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Focal+Adhesion+Kinase+Signaling+by+Integrin+alpha6beta1+Supports+Human+Pluripotent+Stem+Cell+Self-Renewal.">Inhibition of Focal Adhesion Kinase Signaling by Integrin alpha6beta1 Supports Human Pluripotent Stem Cell Self-Renewal.</a> Stem Cells. 34 (7), 1753-1764 (2016).</li><li>Bednar, A. D., Beardall, M. K., Brace, R. A., Cheung, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+and+regional+distribution+of+aquaporins+in+amnion+of+normal+and+gestational+diabetic+pregnancies.">Differential expression and regional distribution of aquaporins in amnion of normal and gestational diabetic pregnancies.</a> Physiological Reports. 3 (3), 12320 (2015).</li><li>Avila-Gonzalez, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amniotic+epithelial+cells+as+feeder+layer+to+derive+and+maintain+human+embryonic+stem+cells+from+poor-quality+embryos.">Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.</a> Stem Cell Research. 15 (2), 322-324 (2015).</li></ol>

We implemented a new protocol to isolate HAEC from term membranes. It differs from previous reports in that each membrane was divided into its three anatomical regions prior to isolation to analyze cells from each one.
One of the most critical steps in the protocol is the washing of the membrane to remove all blood clots, because they can interfere with the activity of trypsin when separating the epithelial cells. Failure to carry out this step properly can lead to obtaining a primary culture ...

Human amniotic epithelium&#160;cells&#160;(HAEC) originate during the early stages of embryonic development, at around eight days postfertilization. They arise from a population of squamous epithelial cells of the epiblast that derive from the innermost layer of the amniotic membrane1. Thus, HAEC are considered&#160;remnants of pluripotent cells from the epiblast that have the potential to differentiate into the three germ layers of the embryo2. In the last decade, diverse&#160;research groups have developed methods to isolate&#160;these cells from the amniotic membrane at the term of gestation to characterize their presumptive pluripotency-related properties in a culture model&#160;in vitro3,4.&#160;
Accordingly, it&#160;has been found that HAEC feature traits characteristic of human pluripotent stem cells (HPSC), such as the surface antigens SSEA-3, SSEA-4, TRA 1-60, TRA 1-81; the core of pluripotency transcription factors OCT4, SOX2, and NANOG; and the proliferation marker KI67, suggesting that they are self-renewing5,6,7. Moreover, these cells have been challenged using differentiation protocols to obtain cells positive for lineage-specific markers of the three germ layers (ectoderm, mesoderm, and endoderm)4,5,8, as well as in animal models of human diseases. Finally, HAEC express E-cadherin, which demonstrate that they retain an epithelial nature much like the HPSC5,9.
Apart from their embryonic origin, HAEC have other intrinsic properties that make them suitable for different clinical applications, such as the secretion of anti-inflammatory and antibacterial molecules10,11, growth factors and cytokine release12, no formation of teratomas when they are transplanted into immunodeficient mice in contrast with HPSC2, and immunological tolerance because they express HLA-G, which decreases the risk of rejection after transplantation13.
However, previous reports have assumed that the human amnion is a homogenous membrane, without considering that it can be anatomically and physiologically divided into three regions: placental (the amnion that covers the decidua basalis), umbilical (the part that envelops the umbilical cord), and reflected (the rest of the membrane not attached to the placenta)14. It has been shown that the placental and reflected regions of the amnion display differences in morphology, mitochondrial activity, detection of reactive oxygen species15, miRNA expression16, and activation of signaling pathways17. These results suggest that the human amnion is integrated by a heterogeneous population with different functionality that should be considered for further studies carried out in either in situ or in vitro models. While other laboratories have designed protocols for the isolation of HAEC from the whole membrane, our laboratory has established a protocol to isolate, culture, and characterize cells from different anatomical regions.

<table>
	<tbody>
		<tr>
			<td>Culture reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>21985023</td>
			<td>55 mM</td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>15240062</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>12430054</td>
			<td>Supplemented with high glucose and HEPES</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo Fisher Scientific/Ambion</td>
			<td>AM9260G</td>
			<td>0.5 M</td>
		</tr>
		<tr>
			<td>Embryonic stem-cell FBS, qualified</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>10439024</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>11140050</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>10010023</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Saline solution (sodium chloride 0.9%)</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>11360070</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA 0.05%</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m Cell Strainer</td>
			<td>Corning/Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Clear TC-treated Multiple Well Plates</td>
			<td>Corning/Costar</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Clear TC-treated Multiple Well Plates</td>
			<td>Corning/Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Pyrogenic Sterile Centrifuge Tube</td>
			<td>any brand</td>
			<td></td>
			<td>with conical bottom</td>
		</tr>
		<tr>
			<td>Non-Pyrogenic sterile tips of 1,000 &#181;l, 200 &#181;l and 10 &#181;l.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cotton gauzes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile serological pipettes of 5, 10 and 25 mL</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical gloves</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized Pipet Filler/Dispenser</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile beakers of 500 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic cutting board</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpels, scissors, forceps, clamps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile stainless steel container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Rotator</td>
			<td>MaCSmix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies and Kits</td>
			<td></td>
			<td></td>
			<td>Antibody ID</td>
		</tr>
		<tr>
			<td>Anti-E-cadherin</td>
			<td>BD Biosciences</td>
			<td>610181</td>
			<td>RRID:AB_3975</td>
		</tr>
		<tr>
			<td>Anti-KI67</td>
			<td>Santa Cruz</td>
			<td>23900</td>
			<td>RRID:AB_627859)</td>
		</tr>
		<tr>
			<td>Anti-NANOG</td>
			<td>Peprotech</td>
			<td>500-P236</td>
			<td>RRID:AB_1268274</td>
		</tr>
		<tr>
			<td>Anti-OCT4</td>
			<td>Abcam</td>
			<td>ab19857</td>
			<td>RRID:AB_44517</td>
		</tr>
		<tr>
			<td>Anti-SOX2</td>
			<td>Millipore</td>
			<td>AB5603</td>
			<td>RRID:AB_2286686</td>
		</tr>
		<tr>
			<td>Anti-SSEA-4</td>
			<td>Cell Signaling</td>
			<td>4755</td>
			<td>RRID:AB_1264259</td>
		</tr>
		<tr>
			<td>Anti-TRA-1-60</td>
			<td>Cell Signaling</td>
			<td>4746</td>
			<td>RRID:AB_2119059</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11029</td>
			<td>RRID:AB_2534088</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11036</td>
			<td>RRID:AB_10563566</td>
		</tr>
		<tr>
			<td>Tunel Assay Kit</td>
			<td>Abcam</td>
			<td>66110</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro culture of epithelial cells from different anatomical regions of the human amniotic membrane

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

HAEC were isolated from each of the three anatomical regions of the amniotic membrane and individually cultured in vitro. After 48 h of culture, cells with an epithelial phenotype adhered to the surface of the plate, although the media also contained cell debris and floating cells, which were removed once the medium was changed (Figure 3).
During the processing of primary culture (passage zero, P0), some complications could arise that can interfere with the experi...

Watch this Scientific Journal Video about In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane at JoVE.com

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

This protocol can help answer key questions about heterogeneity and functional properties of human amniotic epithelial cells isolated from amniotic membrane. While previous protocols isolated cells from co-amniotic membrane without discerning between regions, our protocol allows the culture of cell population isolated from three distinct anatomical regions. To separate the amniotic membrane by region, in a biosafety cabinet under sterile conditions, identify the umbilical amnion enveloping the umbilical cord, the placental amnion covering the decidua basalis, and the reflected region, which is considered the rest of the amnion that is not attached to the placenta.To dissect the umbilical amnion region, use dissecting forceps to hold the portion of the amnion membrane that covers the junction of the placenta and the umbilical cord, and use a scalpel to dissect the region that surrounds the cord, while stretching to separate the region from the chorion. Then, deposit the separated tissue in a labeled beaker with 100 milliliters of saline solution. To dissect the placental amnion region, use a sterile cotton gauze to remove the blood clots from the surface of the chorion/amnion that covers the placenta.And use dissecting forceps to grasp the membrane on the border between the placenta and the reflected region. Use a scalpel to cut along the circumference of the placenta and separate the placental amnion from the chorion, being careful not to cut any vessels from the placenta. Place the separated tissue in another labeled beaker with 300 milliliters of saline solution.And separate the rest of the amniotic membrane that is not attached to the placenta from the chorion. Then, place the reflected region in another labeled beaker with 300 milliliters of saline solution. To wash the membranes, use tweezers to place them on a sterile surface to be able to discard the saline solution from each container of tissue.Place the membranes back in the container and add 100 milliliters of fresh saline solution to the umbilical region and 300 milliliters of fresh saline solution to the placental and reflected regions. Use dissecting forceps to stir the membranes to remove any blood residue and place the membranes on the sterile surface to be able to discard the saline solutions. Then, wash the membranes at least two more times, as just demonstrated, until the tissues are translucent.For enzymatic digestion of the different amniotic membrane regions, cut the reflected and placental regions into two or three fragments. And place the fragments and the umbilical region into individual 50-milliliter centrifuge tubes. Add 20 milliliters of 0.5%trypsin-EDTA to each tube of reflected and placental region tissue fragments and five milliliters of 0.5%trypsin-EDTA to the tube containing the umbilical region tissue.Shake the tubes gently for 30 seconds. Then place the membranes on a sterile surface, discarding the trypsin-EDTA solution and replace it with 30 milliliters of fresh 0.5%trypsin-EDTA for the reflected and placental region tissue fragments and 15 milliliters of fresh 0.5%Trypsin-EDTA for the umbilical region tissue. Then place the tubes in a rotator inside an incubator for a 40-minute rotation at 20 rotations per minute at 37 degrees Celsius.At the end of the incubation, transfer the entire volume of cell solution into new conical tubes. And add two times the volume of 37-degree-Celsius human AE cells medium to each tube on ice. Digest any leftover tissue fragments in the original tubes with fresh 0.5%trypsin-EDTA, as just demonstrated.At the end of the second digestion, use a pair of dissecting forceps to hold one end of the amnion portion and a second pair of dissecting forceps to squeeze along the remaining tissue to remove any rows of epithelial cells that did not completely peel off during the previous incubation periods. Then, remove the membranes to be able to transfer the second digestion solution to a second set of centrifuge tubes. And inactivate the digestion enzymes with two times the volume of fresh 37-degree-Celsius human AE cell medium on ice.For human AE cell isolation, sediment the cells by centrifugation. And resuspend the pellet in each tube with 10 milliliters of fresh 37-degree-Celsius human AE cell medium. Pull each pair of digestions into a single tube and filter the suspensions through 100-micrometer strainers to remove any extracellular matrix debris.After counting, seed the human AE cells from each of the three regions into individual 100-millimeter plates at a three times 10 to the fourth cells per centimeters squared concentration in 37-degree-Celsius human AE cell medium, supplemented with 10 nanograms per milliliter of human epidermal growth factor. Then, incubate the cultures at 37 degrees Celsius under normoxic conditions in a humidified incubator until the downstream analysis of interest. After 48 hours of culture, human AE cells with an epithelial phenotype adhere to the surface of the plate, while the supernatant contains cell debris and floating cells that can be removed once the medium is changed.It is advisable to discard the cultures and process another membrane upon identifying the presence of bacterial contamination, excessive erythrocytes due to insufficient washing of the membranes, deficiently or nonadherent cells, or cells with a fibroblast morphology. Human AE cell morphology depends on the origin of the cells, as cells from the reflected zone demonstrate a cuboidal morphology and grow in a cobbled monolayer, and cells from the placental and umbilical regions are flatter and squamous. Immunofluorescence against E-cadherin reveals that the primary cultures and subcultures maintain their epithelial phenotype, suggesting that there is no contamination of another cell type.In addition, the cells are viable, as evidenced by their expression of the Ki-67 proliferation marker. Although the subpopulations from the amnion differ in their morphology and function, the expression and presence of the core of the pluripotency factors does not change in human AE cells derived from the placental and reflected regions. Since human amniotic epithelial cells are a possible source of pluripotent stem cells, we can challenge them through a line of specific definition protocols to elucidate whether these cells from each region have different potentials.Previous to a started protocol, review the medical history of the patient to be sure that the tissue exhibits no microbiological characteristics of infection.

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Research

JoVE Journal

Developmental Biology

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch&#039;s Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...