Name: determining bile duct density in the mouse liver
Uploaded: 2019-04-30T14:45:00
Description: Watch this Scientific Journal Video about Determining Bile Duct Density in the Mouse Liver at JoVE.com

Forfatterne anerkender støtte fra National Institutes of Health (NIH) (R01 GM084135 og R01 DK109982), en pilot/gennemførligheds pris fra Texas Medical Center fordøjelses sygdoms Center under NIH P30 DK56338, og en Alagille syndrom Accelerator Award fra Det medicinske fundament.

Alle dyr blev anbragt i en barriere Animal facilitet på Baylor College of Medicine per institutionel dyrepasning og anvendelse udvalg retningslinjer og under godkendte dyre protokoller.
1. indsamling af mus lever væv
<ol><li>
Forberedelse af mus til leveren høst
	<ol>
<li>Euthanize musen ved hjælp af isoflurane.</li>
		<li>Udfør livmoderhals afplacering af musen for at sikre døden.</li>
		<li>Lav en tværgående indsnit ca. en tomme under ribburet.</li>
		<li>Udsætte ...

<ol>
	<li>Zong, 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=Notch+signaling+controls+liver+development+by+regulating+biliary+differentiation.">Notch signaling controls liver development by regulating biliary differentiation.</a> Development. 136 (10), 1727-1739 (2009).</li><li>Clotman, F., 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=Control+of+liver+cell+fate+decision+by+a+gradient+of+TGF&#946;+signaling+modulated+by+Onecut+transcription+factors.">Control of liver cell fate decision by a gradient of TGF&#946; signaling modulated by Onecut transcription factors.</a> Genes &amp; Development. 19 (16), 1849-1854 (2005).</li><li>Karpen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+the+etiologies+and+management+of+neonatal+cholestasis.">Update on the etiologies and management of neonatal cholestasis.</a> Clin Perinatol. 29 (1), 159-180 (2002).</li><li>Roskams, T. 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=Nomenclature+of+the+finer+branches+of+the+biliary+tree:+canals,+ductules,+and+ductular+reactions+in+human+livers.">Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.</a> Hepatology. 39 (6), 1739-1745 (2004).</li><li>Oda, 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=Mutations+in+the+human+Jagged1+gene+are+responsible+for+Alagille+syndrome.">Mutations in the human Jagged1 gene are responsible for Alagille syndrome.</a> Nature Genetics. 16 (3), 235 (1997).</li><li>Li, 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=Alagille+syndrome+is+caused+by+mutations+in+human+Jagged1,+which+encodes+a+ligand+for+Notch1.">Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.</a> Nature Genetics. 16 (3), 243 (1997).</li><li>Xue, 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=Embryonic+lethality+and+vascular+defects+in+mice+lacking+the+Notch+ligand+Jagged1.">Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1.</a> Human Molecular Genetics. 8 (5), 723-730 (1999).</li><li>Thakurdas, S. 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=Jagged1+heterozygosity+in+mice+results+in+a+congenital+cholangiopathy+which+is+reversed+by+concomitant+deletion+of+one+copy+of+Poglut1+(Rumi).">Jagged1 heterozygosity in mice results in a congenital cholangiopathy which is reversed by concomitant deletion of one copy of Poglut1 (Rumi).</a> Hepatology. 63 (2), 550-565 (2016).</li><li>Hadchouel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paucity+of+interlobular+bile+ducts.">Paucity of interlobular bile ducts.</a> Seminars in Diagnostic Pathology. 9 (1), 24-30 (1992).</li><li>Emerick, K. 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=Features+of+Alagille+syndrome+in+92+patients:+frequency+and+relation+to+prognosis.">Features of Alagille syndrome in 92 patients: frequency and relation to prognosis.</a> Hepatology. 29 (3), 822-829 (1999).</li><li>Poncy, 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=Transcription+factors+SOX4+and+SOX9+cooperatively+control+development+of+bile+ducts.">Transcription factors SOX4 and SOX9 cooperatively control development of bile ducts.</a> Dev Biol. 404 (2), 136-148 (2015).</li><li>Hofmann, J. 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=Jagged1+in+the+portal+vein+mesenchyme+regulates+intrahepatic+bile+duct+development:+insights+into+Alagille+syndrome.">Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development: insights into Alagille syndrome.</a> Development. 137 (23), 4061-4072 (2010).</li><li>McCright, B., Lozier, J., Gridley, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+Alagille+syndrome:+Notch2+as+a+genetic+modifier+of+Jag1+haploinsufficiency.">A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency.</a> Development. 129 (4), 1075-1082 (2002).</li><li>Andersson, E. R., 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=Mouse+Model+of+Alagille+Syndrome+and+Mechanisms+of+Jagged1+Missense+Mutations.">Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations.</a> Gastroenterology. 154 (4), 1080-1095 (2018).</li><li>Loomes, K. 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=Bile+duct+proliferation+in+liver-specific+Jag1+conditional+knockout+mice:+effects+of+gene+dosage.">Bile duct proliferation in liver-specific Jag1 conditional knockout mice: effects of gene dosage.</a> Hepatology. 45 (2), 323-330 (2007).</li><li>Brulet, P., Babinet, C., Kemler, R., Jacob, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+against+trophectoderm-specific+markers+during+mouse+blastocyst+formation.">Monoclonal antibodies against trophectoderm-specific markers during mouse blastocyst formation.</a> Proc Natl Acad Sci U S A. 77 (7), 4113-4117 (1980).</li><li>Skalli, O., 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=A+monoclonal+antibody+against+alpha-smooth+muscle+actin:+a+new+probe+for+smooth+muscle+differentiation.">A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.</a> J Cell Biol. 103 (6 Pt 2), 2787-2796 (1986).</li><li>Kamath, B. 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=A+longitudinal+study+to+identify+laboratory+predictors+of+liver+disease+outcome+in+Alagille+syndrome.">A longitudinal study to identify laboratory predictors of liver disease outcome in Alagille syndrome.</a> Journal of Pediatric Gastroenterology and Nutrition. 50 (5), 526 (2010).</li><li>Mouzaki, 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=Early+life+predictive+markers+of+liver+disease+outcome+in+an+International,+Multicentre+Cohort+of+children+with+Alagille+syndrome.">Early life predictive markers of liver disease outcome in an International, Multicentre Cohort of children with Alagille syndrome.</a> Liver International. 36 (5), 755-760 (2016).</li><li>Shiojiri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+differentiation+of+bile+ducts+in+the+mammalian+liver.">Development and differentiation of bile ducts in the mammalian liver.</a> Microsc Res Tech. 39 (4), 328-335 (1997).</li><li>Crawford, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+intrahepatic+biliary+tree.">Development of the intrahepatic biliary tree.</a> Semin Liver Dis. 22 (3), 213-226 (2002).</li><li>Kaneko, K., Kamimoto, K., Miyajima, A., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+remodeling+of+the+biliary+architecture+underlies+liver+homeostasis.">Adaptive remodeling of the biliary architecture underlies liver homeostasis.</a> Hepatology. 61 (6), 2056-2066 (2015).</li><li>Schaub, J. R., 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=De+novo+formation+of+the+biliary+system+by+TGFbeta-mediated+hepatocyte+transdifferentiation.">De novo formation of the biliary system by TGFbeta-mediated hepatocyte transdifferentiation.</a> Nature. 557 (7704), 247-251 (2018).</li><li>Sparks, E. E., Huppert, K. A., Brown, M. A., Washington, M. K., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+regulates+formation+of+the+three-dimensional+architecture+of+intrahepatic+bile+ducts+in+mice.">Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.</a> Hepatology. 51 (4), 1391-1400 (2010).</li><li>Tanimizu, 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=Intrahepatic+bile+ducts+are+developed+through+formation+of+homogeneous+continuous+luminal+network+and+its+dynamic+rearrangement+in+mice.">Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.</a> Hepatology. 64 (1), 175-188 (2016).</li><li>Walter, T. J., Sparks, E. E., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-dimensional+resin+casting+and+imaging+of+mouse+portal+vein+or+intrahepatic+bile+duct+system.">3-dimensional resin casting and imaging of mouse portal vein or intrahepatic bile duct system.</a> J Vis Exp. (68), e4272 (2012).</li><li>Popper, H., Kent, G., Stein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductular+cell+reaction+in+the+liver+in+hepatic+injury.">Ductular cell reaction in the liver in hepatic injury.</a> J Mt Sinai Hosp N Y. 24 (5), 551-556 (1957).</li><li>Yimlamai, 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=Hippo+pathway+activity+influences+liver+cell+fate.">Hippo pathway activity influences liver cell fate.</a> Cell. 157 (6), 1324-1338 (2014).</li></ol>

Analyse af BD udvikling og reparation i mus er et vigtigt redskab til at studere patogenesen og mekanismen af kolestatiske lidelser. Desuden er udviklingen af nye terapier delvis afhængig af, at der etableres en reproducerbar og fortrinsvis kvantificerbare fænotype. Nuværende fænotypebestemmelse i musemodeller involverer normalt serum kemi, lever histologi og immun farvning for celle-typespecifikke markører. Selv om disse teknikker genererer værdifulde oplysninger om strukturen og funktionen af biliær systemet, de...

Galde træet er en kritisk del af pattedyr leveren, tillader passage af galde fra hepatocytter i tarmen. Intrahepatiske galdekanaler (BDS) dannes af cholangiocytter, som adskiller fra bipotential hepatoblaster gennem hak og tgfβ signalering1,2. Korrekt specifikation og engagement af cholangiocytes og deres samling i modne BDs er afgørende for udviklingen af det intrahepatiske Biliære træ. Som leveren vokser under udvikling eller ved organ regenerering, galdesystemet skal udvikle sig langs leveren for at sikre korrekt galde dræning. Desuden, en række syndromer og ikke-syndromiske sygdomme resultere i Brandt af intrahepatiske BDS3. Desuden giver en række akutte og kroniske leversygdomme anledning til såkaldte ductular reaktioner i leveren, som defineres som tilstedeværelsen af et betydeligt antal celler, der udtrykker Biliære markører, men ikke nødvendigvis skyldes galde celler eller form patent BDs4. I multi-system Disorder Alagille syndrom (ALGS), haploinsufficiens af notch ligand jagged1 (JAG1) resulterer i dårlig BD dannelse og kolestase5,6. Vores laboratorium har for nylig påvist, at en tidligere genereret Jag1 heterozygot muse linje7 er en dyremodel af BD Brandt i ALGS8. I denne mus model af ALGS, cholangiocytter er stadig til stede. Men de undlader at forpligte sig til inkorporering i moden, patent BDs8. Derfor, analyse af leveren i en model af BD Brandt kræver mere end den tilsyneladende tilstedeværelse eller fravær af cholangiocytter. Det er vigtigt præcist at vurdere, i hvilken grad modne BD'er er til stede i leveren.
I anatomiske patologi er der accepterede kvantitative metoder til at vurdere, om BD Brandt findes9. For eksempel, undersøgelser på ALGS hos mennesker patienter ofte kvantificere BD til Portal vene (PV) ratio ved at analysere mindst 10 Portal fartøjer pr leveren biopsi9,10. Analyse af form og generel tilstedeværelse eller fravær af patent BDS, kombineret med serum kemi, kan give værdifulde oplysninger om BD udvikling i mus11,12,13. Men, mus kan miste et betydeligt antal BDs med kun en beskeden stigning i serum bilirubinniveau8. Derfor kan en kvantitativ metode, der evaluerer antallet af BDS til stede pr. PV, give en mere direkte måling af graden af BD Brandt i mus. I en nylig rapport kvantificerede vi antallet af BDs pr. PV på tværs af alle leverlobes og rapporterede et signifikant fald i BD til PV-forholdet i Jag1 +/– dyr8. I løbet af vores analyse, bemærkede vi, at på trods af den betydelige variation i graden af inflammatorisk respons og ductular reaktioner, BD til PV ratio viser ikke meget variation8. Desuden, kvantificering af BD til PV ratio tillod os at påvise, at fjerne en kopi af glykosyltransferase genet Poglut1 i Jag1 +/– dyr kan væsentligt forbedre deres BD Brandt8. I en Jag1 +/+ baggrund resulterer betinget tab af Poglut1 i vaskulære glatte muskelceller i en progressiv stigning i BD-numre, hvilket er beskedent (20-30%) på P7, men bliver fremtrædende i voksne8. Igen, denne teknik tillod os at vise, at selv på P7, stigningen i BD tæthed i disse dyr er statistisk signifikant. Bemærk, at den øgede BD-tæthed i denne genotype ved fire måneders alderen også blev valideret ved hjælp af harpiks støbe analyse. 8 disse observationer og andre rapporter, som målte BD-tæthed i forskellige ALGS-musemodeller14,15 fik os til at indarbejde denne metode i vores overordnede strategi om at analysere Biliære defekter i forskellige mutante og Transgene mus.
Her, vi detaljer en ligetil teknik, som kan bruges til at undersøge graden af BD Brandt i musemodeller af leversygdom (figur 1). I denne metode, samtidig farvning med cholangiocyte markører cytokeratin (CK) 8 og CK19 (herefter bredspektret CK, wsCK) bruges til at visualisere BDs og uindarbejdet cholangiocytes i muse leveren. Et antistof mod Alpha-glat muskel actin (αSMA) tilsættes til farvning til etiketten fartøjer. Systematisk analyse af BD-til-PV-forholdet i et afsnit, der dækker alle leverlobes, sikrer, at et stort antal PVs analyseres for hver genotype. Da vores metode er afhængig af kvantificering af BDs og PVs i 2D-billeder, er det ikke egnet til at studere virkningerne af en given mutation på 3D-strukturen i galde træet eller integriteten af de små Biliære ledninger. Ikke desto mindre, det giver en enkel og objektiv strategi for efterforskere til at vurdere biliær udvikling i musen.

<table>
	<tbody>
		<tr>
			<td>Isothesia (Isoflurane)</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td></td>
		</tr>
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			<td>Desiccator</td>
			<td>Bel-Art</td>
			<td>16-800-552</td>
			<td></td>
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		<tr>
			<td>10% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15712</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL tube</td>
			<td>ThermoScientific</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2401</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2801</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoChoice</td>
			<td>VWR Life Sciences</td>
			<td>H103-4L</td>
			<td>clearing agent</td>
		</tr>
		<tr>
			<td>Omnisette Tissue Cassette</td>
			<td>Fisher HealthCare</td>
			<td>15-197-710E</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosette</td>
			<td>Simport</td>
			<td>M512</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast X-TRA</td>
			<td>McCormick Scientific</td>
			<td>39503002</td>
			<td>Parrafin</td>
		</tr>
		<tr>
			<td>Tissue Mold</td>
			<td>Fisher Scientific</td>
			<td>62528-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Microm</td>
			<td>HM 325</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>C8H10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Based Antigen Retrieval</td>
			<td>Vector Laboratories</td>
			<td>H-3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Cooker</td>
			<td>Instant Pot</td>
			<td>Lux Mini</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Pap Pen</td>
			<td>Life Technologies</td>
			<td>8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td>
			<td>J.T. Baker</td>
			<td>X251-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Octyl Phenol Ethoxylate (Triton-X-100)</td>
			<td>J.T. Baker</td>
			<td>X198-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Jackson Immunoresearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CK8</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I</td>
			<td>Antibody Registry ID AB531826</td>
		</tr>
		<tr>
			<td>anti-CK19</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-III</td>
			<td>Antibody Registry ID AB2133570</td>
		</tr>
		<tr>
			<td>anti-&#945;SMA</td>
			<td>Sigma Aldrich</td>
			<td>A2547, Clone 1A4</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat-Alexa488</td>
			<td>ThermoFisher</td>
			<td>A21208</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse-Cy5</td>
			<td>Jackson Immunoresearch</td>
			<td>715-175-151</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>22x50 micro cover glass</td>
			<td>VWR Life Sciences</td>
			<td>48393 059</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Leica</td>
			<td>DMI6000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech Science</td>
			<td>05511</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Antifade Mounting Medium</td>
		</tr>
	</tbody>
</table>

determining bile duct density in the mouse liver

Mus er bredt anvendt som en model organisme til at studere galde sygdomme. For at evaluere udviklingen og funktionen af galdesystemet anvendes der forskellige teknikker, herunder serum kemi, histologisk analyse og immun farvning for specifikke markører. Selv om disse teknikker kan give vigtige oplysninger om galde system, de ofte ikke præsenterer et fuldt billede af galdegang (BD) udviklingsmæssige defekter på tværs af hele leveren. Dette er til dels på grund af den robuste evne af musen leveren til at dræne galde selv hos dyr med signifikant svækkelse i galde udvikling. Her præsenterer vi en simpel metode til at beregne det gennemsnitlige antal BD'er, der er forbundet med hver Portal vene (PV) i sektioner, der dækker alle lapper af mutant/Transgene mus. I denne metode monteres lever og sektioneres på en stereotypisk måde for at lette sammenligningen mellem forskellige genotyper og eksperimentelle forhold. BDs identificeres via let mikroskopi af cytokeratin-farvede cholangiocytter, og derefter tælles og divideret med det samlede antal PVs til stede i leveren afsnit. Som et eksempel viser vi, hvordan denne metode klart kan skelne mellem vild-type mus og en musemodel af Alagille syndrom. Den metode, der præsenteres her, kan ikke erstatte teknikker, som visualiserer den tredimensionale struktur i biliær træet. Men, det giver en nem og direkte måde at kvantitativt vurdere BD udvikling og graden af ductular reaktion dannelse i mus.

Vi har tidligere dokumenteret Biliære defekter i Jag1 +/– dyr, en musemodel på ALGS8. For at bestemme BD til PV ratio, vi sektionerede P30 mus lever og co-plettet dem for CK8 og CK19 (wsCK) sammen med den vaskulære markør αSMA. Vi derefter afbildet alle PVS i hver af leveren lobes. Som vist i figur 2adefinerede vi PVS som αsma-farvede fartøjer, der har tilstødende wsCK-farvning (arrowheads). De αSMA-farvede struktur...

Watch this Scientific Journal Video about Determining Bile Duct Density in the Mouse Liver at JoVE.com

Determining Bile Duct Density in the Mouse Liver

Vi præsenterer en temmelig enkel og følsom metode til nøjagtig kvantificering af galdegang tæthed i muse leveren. Denne metode kan hjælpe med at bestemme virkningerne af genetiske og miljømæssige modifikatorer og effektiviteten af potentielle terapier i musemodeller af Biliære sygdomme.

Bestemmelse galdegang tæthed i muse leveren

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Denne protokol gør det muligt for investigator at kvantificere galdegang udvikling i musemodeller af leversygdom. Det giver også mulighed for at evaluere virkningerne af genetiske modifikatorer på galdegang udvikling. Fordelen ved denne teknik er, at det er en enkel, følsom og kvantitativ metode til direkte vurdering af udviklingen af galdegang.Til at begynde, med huden holdt stramt af hændebånd, bruge små saks til at gøre en tværgående snit cirka en tomme under brystkassen af en aflivet mus. Eksponere hele ventrale overflade af leveren. Brug små saks til omhyggeligt at skære gennem ledbånd forbinder leveren til de andre organer i maven.Derefter skæres gennem den fælles galdegang til at frigøre leveren fra tarmen. Hold fast i galdeblæren og fjern forsigtigt leveren. Umiddelbart placere den i en 50 milliliter rør fyldt tre fjerdedele med 4% paraformaldehyd.For at fastsætte levervæv, inkubere det i 4% PFA ved fire grader Celsius i 48 timer. Efter en række ethanol vasker, vaskes leveren med clearing agent tre gange i 30 minutter hver ved stuetemperatur. At begynde indlejring, vaske en vævskassette i et væv skimmel tre gange i 30 minutter i paraffin voks forvarmet til 60 grader Celsius.Derefter fylde vævsform med paraffin til tre fjerdedele højde og holde det på en varmeblok ved 60 grader Celsius. Placer leveren i formen med ventrale side opad. Efter omhyggeligt at fjerne formen fra varmeblokken, skal du placere toppen af kassetten på formen.Top off med varm flydende paraffin og lad det køle af til stuetemperatur natten over. Efter fjernelse af leveren blok fra formen, bruge en mikrotom til at begynde at sektionere gennem den overfladiske dorsale side af leveren, hvilket gør fem mikrometer sektioner. Kontroller de overfladiske sektioner under et dissektionsmikroskop for at sikre, at sektionerne ikke er skåret eller foldet.For at behandle dias til immunhisistokkemi skal du vælge et dias pr. genotype, der skal analyseres, og vaske det i 15 minutter i xylen, 100% ethanol, 95% ethanol og endelig 70% ethanol. Efter vask af diaset i det ioniserede vand, nedsænkes det i Tris-baserede høj pH antigen hentningsopløsning. Derefter, varme det under pres i en trykkoger i tre minutter på 10 pounds per kvadrattomme.Når du har ladet diaset køle af til stuetemperatur, skal du bruge en PAP-pen til at skitsere sektionerne på diaset. Efter vask med PBS Tween blokeres vævssektionerne ved at tilsætte 100 mikroliter blokerende opløsning pr. sektion og inkuberes dækket ved fire grader Celsius i en time. Der påføres 100 mikroliter af den fortyndede antistofopløsning, der indeholder alle tre primære antistoffer på hver sektion, og de inkuberes ved fire grader Celsius natten over.Efter vask af objektglassene tilsættes 100 mikroliter af den sekundære antistofopløsning, der indeholder både sekundære antistoffer, og inkuberes i en time ved stuetemperatur. Endelig gælder montering medium og en coverslip til dias. Brug et fluorescerende mikroskop til at tage 20x billeder ved 1x zoom i hver sektion.Sørg for, at hver portal vene på tværs af leveren er afbildet. Opret et regneark med følgende kolonner:Dyre-/eksempelnummer, Billednummer, antal portalvener og antal galdegange. Identificer og optag antallet af portalvener pr. billede for hvert billede.Derefter identificere patent galdegangene i hvert billede, ved tilstedeværelsen af cholangiocytter omkring en definerbar lumen. Disse strukturer bør være adskilt og adskilt af mesenchyme fra andre bredspektrum cytokeratin-positive celler. En af de største udfordringer ved denne teknik er at identificere patentgaldegange.Bestemmelse af, hvad der er et patent kanal kræver en analyse af formen af kanalen og cellerne omkring lumen. Tæl hver patent galdegang og sted i samme kolonne som billednummeret og gentag for hver portal vene billede. Endelig beregne summen af alle portal vener og alle galdegange i leveren prøven og beregne galdegang til portal vene forholdet for leveren prøven.P30 mus lever blev opdelt og co-plettet for CK8 og CK19 sammen med den vaskulære markør, alpha-SMA, at bestemme BD til PV forholdet. Når alle portal vener i hver leverlap er afbildet, portal vener blev defineret som alfa-SMA farvede fartøjer, der har tilstødende bredt spektrum cytokeratin farvning. Alfa-SMA-pletstrukturerne uden bredspektret cytokeratin var centrale vener, som blev udelukket fra analysen.Patentkanaler har en klart definerbar lumen, der er omgivet af bredspektret cytokeratin-positive cholangioctyes og er normalt adskilt fra nærliggende kanaler eller cholangiocytter af mesenchyme. Wide-spektrum cytokeratin-positive celler, der ikke har en definerbar lumen, tælles ikke med i det samlede antal galdegange. Lever sektion fra en vild-type dyr viser en portal vene forbundet med en fuldt patent kanal sammen med flere personlige celler.Repræsentativ lever sektion fra en JAG1 heterozygot dyr viser ingen patent galdegange er til stede omkring de tre portal vener. Alle bredspektrede cytokeratin-positive celler her er ikke-inkorporerede og bør derfor ikke tælles med. Analyse af BD til PV-forholdet for tre vilde dyr og tre JAG1 heterozygotdyr viser, hvordan de to genotyper let kan skelnes baseret på galdegangstællinger.Det er vigtigt at vurdere alle portalfartøjer til galdegange. Det er især afgørende for bestemmelse galdegang tæthed, hvis der er fænotopypisk variation på tværs af leveren. Denne teknik blev brugt til at identificere en genetisk suppressor af galdehæotype i en musemodel af Helgason.Derfor kan det være nyttigt ved vurderingen af behandlingsmodealiteter for dyremodeller for galdesygdom.

determining-bile-duct-density-in-the-mouse-liver

Research

JoVE Journal

Developmental Biology

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Stimulering af Notch signalering i Mouse osteoklastpræcursorer

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Histologiske analyser af akut alkoholisk leverskader i sebrafisk

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

In Vitro Vækst af mus Preantral follikler Under simulerede vægtløshed

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

Kvantificering af forgreningsdensitet i rotte mammary Gland Whole-mounts ved hjælp af Sholl analysemetoden

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Live Imaging of Mouse Secondary Palate Fusion

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 cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

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

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

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

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissektion og immunfluorescent farvning af champignon krop og fotoreceptor neuroner i voksen Drosophila melanogaster hjerner

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

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

En roman brug af tre-dimensionelle høj frekvens ultralyd for tidlig graviditet karakterisering i mus

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

En familiær hyperkolesterolæmi menneskelige lever kimære musen Model ved hjælp af induceret pluripotente stamceller-afledt hepatocytter

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

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

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

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 ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Generation af Organoids fra mus ekstrahepatisk galdeveje

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...