Name: in vitro and in vivo models to study corneal endothelial-mesenchymal transition
Uploaded: 2016-08-20T14:00:00
Description: Watch this Scientific Journal Video about In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition at JoVE.com

We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

Alle procedurer, der følges i denne undersøgelse indrømmes med foreningen for Forskning i Vision og Oftalmologi Erklæring for anvendelse af dyr i Ophthalmic og Vision Research og blev godkendt af Institutional Animal Care og brug Udvalg for National Taiwan University Hospital. 1. Isolation, Primary Kultur Forberedelse og immunfarvning af Bovin CØE <ol><li> Anskaf friske bovine øjne fra et lokalt slagteri. </li><li> Desinficere øjnene i en 10% vægt / volumen povidon-iod-opløsning i 3 minutter. Vaske de...

<ol>
	<li>Maurice, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+location+of+the+fluid+pump+in+the+cornea.">The location of the fluid pump in the cornea.</a> J Physiol. 221 (1), 43-54 (1972).</li><li>Joyce, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferative+capacity+of+the+corneal+endothelium.">Proliferative capacity of the corneal endothelium.</a> Progress in Retinal and Eye Research. 22 (3), 359-389 (2003).</li><li>Morishige, N., Sonoda, K. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+and+its+receptor,+basic+fibroblast+growth+factor,+transforming+growth+factor+beta-1,+and+interleukin-1+alpha+messenger+RNA+production+in+human+corneal+endothelial+cells.">Epidermal growth factor and its receptor, basic fibroblast growth factor, transforming growth factor beta-1, and interleukin-1 alpha messenger RNA production in human corneal endothelial cells.</a> Invest Ophthalmol Vis Sci. 32 (10), 2747-2756 (1991).</li><li>Chen, K. H., Harris, D. L., Joyce, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta2+in+aqueous+humor+suppresses+S-phase+entry+in+cultured+corneal+endothelial+cells.">TGF-beta2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells.</a> Invest Ophthalmol Vis Sci. 40 (11), 2513-2519 (1999).</li><li>Senoo, T., Obara, Y., Joyce, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EDTA:+a+promoter+of+proliferation+in+human+corneal+endothelium.">EDTA: a promoter of proliferation in human corneal endothelium.</a> Invest Ophthalmol Vis Sci. 41 (10), 2930-2935 (2000).</li><li>Yue, B. Y., Sugar, J., Gilboy, J. E., Elvart, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+human+corneal+endothelial+cells+in+culture.">Growth of human corneal endothelial cells in culture.</a> Invest Ophthalmol Vis Sci. 30 (2), 248-253 (1989).</li><li>Peh, G. S., Beuerman, R. W., Colman, A., Tan, D. T., Mehta, 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=Human+corneal+endothelial+cell+expansion+for+corneal+endothelium+transplantation:+an+overview.">Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview.</a> Transplantation. 91 (8), 811-819 (2011).</li><li>Zhu, C., Rawe, I., Joyce, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+protein+expression+in+human+corneal+endothelial+cells+cultured+from+young+and+older+donors.">Differential protein expression in human corneal endothelial cells cultured from young and older donors.</a> Mol Vis. 14, 1805-1814 (2008).</li><li>Ko, M. K., Kay, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+role+of+FGF-2+on+type+I+collagen+expression+during+endothelial+mesenchymal+transformation.">Regulatory role of FGF-2 on type I collagen expression during endothelial mesenchymal transformation.</a> Invest Ophthalmol Vis Sci. 46 (12), 4495-4503 (2005).</li><li>Lee, H. T., Kay, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-2+induced+reorganization+and+disruption+of+actin+cytoskeleton+through+PI+3-kinase,+Rho,+and+Cdc42+in+corneal+endothelial+cells.">FGF-2 induced reorganization and disruption of actin cytoskeleton through PI 3-kinase, Rho, and Cdc42 in corneal endothelial cells.</a> Mol Vis. 9, 624-634 (2003).</li><li>Pipparelli, 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=ROCK+Inhibitor+Enhances+Adhesion+and+Wound+Healing+of+Human+Corneal+Endothelial+Cells.">ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells.</a> PloS one. 8 (4), e62095 (2013).</li><li>Roy, O., Leclerc, V. B., Bourget, J. M., Theriault, M., Proulx, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+process+of+corneal+endothelial+morphological+change+in+vitro.">Understanding the process of corneal endothelial morphological change in vitro.</a> Invest Ophthalmol Vis Sci. 56 (2), 1228-1237 (2015).</li><li>Jakobiec, F. A., Bhat, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrocorneal+membranes:+a+comparative+immunohistochemical+analysis+of+keratocytic,+endothelial,+and+epithelial+origins.">Retrocorneal membranes: a comparative immunohistochemical analysis of keratocytic, endothelial, and epithelial origins.</a> Am J Ophthalmol. 150 (2), 230-242 (2010).</li><li>Okumura, 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=Involvement+of+ZEB1+and+Snail1+in+excessive+production+of+extracellular+matrix+in+Fuchs+endothelial+corneal+dystrophy.">Involvement of ZEB1 and Snail1 in excessive production of extracellular matrix in Fuchs endothelial corneal dystrophy.</a> Lab Invest. 95 (11), 1291-1304 (2015).</li><li>Okumura, 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=Enhancement+on+primate+corneal+endothelial+cell+survival+in+vitro+by+a+ROCK+inhibitor.">Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor.</a> Invest Ophthalmol Vis Sci. 50 (8), 3680-3687 (2009).</li><li>Zhu, Y. T., Chen, H. C., Chen, S. Y., Tseng, S. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+p120+catenin+unlocks+mitotic+block+of+contact-inhibited+human+corneal+endothelial+monolayers+without+disrupting+adherent+junctions.">Nuclear p120 catenin unlocks mitotic block of contact-inhibited human corneal endothelial monolayers without disrupting adherent junctions.</a> J Cell Sci. 125 (15), 3636-3648 (2012).</li><li>Ho, W. T., 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=Inhibition+of+Matrix+Metalloproteinase+Activity+Reverses+Corneal+Endothelial-Mesenchymal+Transition.">Inhibition of Matrix Metalloproteinase Activity Reverses Corneal Endothelial-Mesenchymal Transition.</a> Am J Pathol. 185 (8), 2158-2167 (2015).</li><li>Kay, E. D., Cheung, C. C., Jester, J. V., Nimni, M. E., Smith, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+collagen+and+fibronectin+synthesis+by+retrocorneal+fibrous+membrane.">Type I collagen and fibronectin synthesis by retrocorneal fibrous membrane.</a> Invest Ophthalmol Vis Sci. 22 (2), 200-212 (1982).</li><li>Li, C., 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=Notch+Signal+Regulates+Corneal+Endothelial-to-Mesenchymal+Transition.">Notch Signal Regulates Corneal Endothelial-to-Mesenchymal Transition.</a> Am J Pathol. 183 (3), 786-795 (2013).</li><li>Okumura, 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=The+ROCK+Inhibitor+Eye+Drop+Accelerates+Corneal+Endothelium+Wound+Healing.">The ROCK Inhibitor Eye Drop Accelerates Corneal Endothelium Wound Healing.</a> Invest Ophthalmol Vis Sci. 54 (4), 2493-2502 (2013).</li><li>Mannermaa, E., Vellonen, K. 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CØE er kendt for deres tilbøjelighed til at undergå EnMT under celleproliferation. At udvikle strategier for at undertrykke EnMT processen til terapeutiske formål, en grundig forståelse af EnMT mekanisme er nødvendig. Vi beskrev 2 modeller til at undersøge EnMT, nemlig den bovine CEC in vitro kultur model og rotte hornhindeendothel kryobeskadigelse model. Vores resultater viste den EnMT processen i begge modeller. Endvidere blev EnMT-undertrykkende virkning af marimastat gengivet i begge modeller, antyde...

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.
The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGF&#946;2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function. 
In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

<table border="0" cellpadding="0" cellspacing="0" width="1447">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">trypsin</td>
			<td style="width:269px;">ThermoFisher Scientific</td>
			<td style="width:143px;">12604-013</td>
			<td style="width:340px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle medium and Ham&#39;s F12 medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human epidermal growth factor</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">insulin, transferrin, selenium&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cholera toxin</td>
			<td>Sigma</td>
			<td>C8052-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentamicin</td>
			<td>ThermoFisher Scientific</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">amphotericin B</td>
			<td>ThermoFisher Scientific</td>
			<td>15290-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>111219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">marimastat</td>
			<td>Sigma</td>
			<td>M2699-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-active beta-catenin antibody</td>
			<td>Millpore</td>
			<td>05-665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-snail antibody</td>
			<td>Santa cruz</td>
			<td>sc28199</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-slug antibody</td>
			<td>Santa cruz</td>
			<td>sc15391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11001</td>
			<td>for staining of ABC of bovine CECs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11003</td>
			<td>for staining of ABC of rat corneal endothelium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-rabbit IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11008</td>
			<td>for staining of snail and slug of bovine CECs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibody diluent</td>
			<td>Genemed Biotechnologies</td>
			<td>10-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">laser scanning confocal microscope</td>
			<td>ZEISS</td>
			<td>LSM510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">xylazine&#160;</td>
			<td>Bayer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tiletamine plus zolazepam</td>
			<td>Virbac</td>
			<td>N/A</td>
			<td>veterinary drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">proparacaine hydrochloride ophthalmic solution</td>
			<td>Alcon</td>
			<td>N/A</td>
			<td>veterinary drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% atropine</td>
			<td>Wu-Fu Laboratories Co., Ltd</td>
			<td>N/A</td>
			<td>clinical drug&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.3% gentamicin sulfate</td>
			<td>Sinphar Group</td>
			<td>N/A</td>
			<td>clinical drug&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">basic fibroblast growth factor</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0024</td>
			<td>clinical drug&#160;</td>
		</tr>
	</tbody>
</table>

in vitro and in vivo models to study corneal endothelial-mesenchymal transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

Efter isoleringen af bovine CØE, blev cellerne dyrket in vitro. Figur 1 viser fasekontrast billeder af bovine CØE. Den sekskantede form af cellerne på konfluens indikerede, at cellerne ikke var forurenet af corneal stromal fibroblast under celleisolering. Figur 2 viser immunfarvning, der blev udført under anvendelse af antistoffer mod ABC, sneglen, og slug på en angivet tidspunkt. Bortset fra fænotypiske ændringer i in vitro kult...

Watch this Scientific Journal Video about In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition at JoVE.com

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

En primær kultur af bovine hornhindeendothelceller blev anvendt til undersøgelse af mekanismen af ​​hornhindeendothel-mesenkymal overgang. Endvidere blev en rotte hornhindeendothel kryobeskadigelse model, der anvendes til at demonstrere hornhindeendothel-mesenchymal overgang in vivo.

In vitro og in vivo modeller til at studere hornhindeendothel-mesenkymale Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

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In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

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Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Anvendelse af RNAi og Heat-shock-induceret transskription faktor udtryk til at omprogrammere kønsceller til neuroner i C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

Visualisere Node og Notochordal plade i Gastrulating mus embryoner ved hjælp af Scanning elektronmikroskopi og hele Mount immunfluorescens

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Voksen zebrafisk skade modeller at studere virkningerne af Prednisolon i regenererende knoglevæv

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

En In Vivo -metode til at studere mus blod-Testis barriere integritet

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...