Name: in vitro culture of epithelial cells from different anatomical regions of the human amniotic membrane
Uploaded: 2019-11-28T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane at JoVE.com

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 ...

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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 ...

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 ...

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

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. ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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 ...

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 ...

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 ...

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

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 ...