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.

כל הנהלים המיושמים במחקר זה עולה בקנה אחד עם האגודה לחקר משפט חזון עיניים עבור שימוש בבעלי חי עיניים וחזון המחקר אושרו על ידי ועדת הטיפול בבעלי החיים המוסדיים השתמש בבית החולים האוניברסיטאי הלאומיים בטייוואן. 1. ניתוק, הכנת התרבות ראשית ולאח?...

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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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CECs ידוע נטייתן לעבור EnMT במהלך התפשטות תאים. כדי לפתח אסטרטגיות דיכוי תהליך EnMT למטרות טיפוליות, הבנה מעמיקה של מנגנון EnMT הכרחית. תיארנו 2 דגמים לחקור EnMT, כלומר CEC שור במודל תרבות במבחנה דגם cryoinjury האנדותל עכברוש הקרנית. התוצאות שלנו הוכיחו את תהליך EnMT בשני המוד?...

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.

לאחר בידוד של CECs שור, התאים היו בתרבית במבחנה. איור 1 מציג את התמונות בניגוד שלב של CECs שור. צורת המשושה של התאים בנקודת המפגש ציינה כי התאים לא היו מזוהמים על ידי פיברובלסטים סטרומה הקרניים במהלך בידוד תא. איור 2 מתארים את immunostaini...

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

תרבות עיקרית של תאי אנדותל קרניים שור שמשה לחקור את המנגנון של מעבר אנדותל-mesenchymal הקרני. יתר על כן, מודל cryoinjury האנדותל עכברוש הקרני שמש להפגין מעבר אנדותל-mesenchymal קרני in vivo.

במבחנה מודלים Vivo ללמוד קרנית האנדותל-mesenchymal מעבר

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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Live Imaging of Mouse Secondary Palate Fusion

הדמיה חיה של עכבר משניים פיוז &#39;ן

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

ניתוח Immunofluorescent מכתים של פטריות הגוף והנפש ומה קולט אור נוירונים במוח למבוגרים דרוזופילה melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

הנדסת רקמות הקרנית: במבחנה מודל של אינטראקציות סטרומה-עצב של הקרנית האנושית

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

היישום של RNAi חום-הלם-induced בביטוי פקטור שעתוק כדי לתכנת בתאי הנבט את הנוירונים 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

להמחיש את צומת ואת צלחת Notochordal Gastrulating עוברי העכבר באמצעות מיקרוסקופ אלקטרונים סריקה כל הר Immunofluorescence

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

דג זברה בוגרת מודלים פציעה ללמוד את השפעות Prednisolone רגנרציה רקמת העצם

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

שיטת In Vivo ללמוד העכבר דם Testis מכשול שלמות

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