Name: generation of esc-derived mouse airway epithelial cells using decellularized lung scaffolds
Uploaded: 2016-05-05T15:00:00
Description: Watch this Scientific Journal Video about Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds at JoVE.com

Vi vil gerne takke Dr. Rossant og Dr. Bilodeau for Nkx2-1 mcherry ESC anvendes til forsøg er afbildet i figur 1-3. FACS blev udført i The SickKids-UHN flowcytometri Facility. Dette arbejde blev støttet af driftstilskud fra de canadiske Institutes for Health Research og en infrastruktur tilskud (CSCCD) fra den canadiske Foundation of Innovation.

Dyreforsøg blev udført i overensstemmelse med Animal Care udvalgets retningslinjer Hospital for Sick Children Research Institute. 1. Stillads Forberedelse <ol><li> Decellularization af lunger <ol><li> Euthanize voksne Wistar-rotter under anvendelse af CO 2 kammer. Placer dyr i kammeret og start 100% CO2 eksponering ved et fyld på 10-30% af kammerets volumen per minut. <ol><li> Overhold dyr til bevidstløshed; dette vil forekomme efter ca. 2-3 min. Hvis bevidstløshed ikke forekommer i ...

<ol>
	<li>Discher, D. E., Mooney, D. J., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+Factors,+Matrices,+and+Forces+Combine+and+Control+Stem+Cells.">Growth Factors, Matrices, and Forces Combine and Control Stem Cells.</a> Science. 324 (5935), 1673-1677 (2009).</li><li>Daley, W. P., Peters, S. B., Larsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+dynamics+in+development+and+regenerative+medicine.">Extracellular matrix dynamics in development and regenerative medicine.</a> J. Cell Sci. 121 (3), 255-264 (2008).</li><li>Ghaedi, 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=Human+iPS+cell-derived+alveolar+epithelium+repopulates+lung+extracellular+matrix.">Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix.</a> J. Clin. Invest. 123 (11), 4950-4962 (2013).</li><li>Huang, S. X. 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=Efficient+generation+of+lung+and+airway+epithelial+cells+from+human+pluripotent+stem+cells.">Efficient generation of lung and airway epithelial cells from human pluripotent stem cells.</a> Nat. Biotechnol. 32 (1), 84-91 (2014).</li><li>Jensen, 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=A+rapid+lung+de-cellularization+protocol+supports+embryonic+stem+cell+differentiation+in+vitro+and+following+implantation.">A rapid lung de-cellularization protocol supports embryonic stem cell differentiation in vitro and following implantation.</a> Tissue Eng. Part C: Methods. 18 (8), 632-646 (2012).</li><li>Longmire, 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=Efficient+derivation+of+purified+lung+and+thyroid+progenitors+from+embryonic+stem+cells.">Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells.</a> Cell stem cell. 10 (4), 398-411 (2012).</li><li>Wong, A. P., 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=Directed+differentiation+of+human+pluripotent+stem+cells+into+mature+airway+epithelia+expressing+functional+CFTR+protein.">Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein.</a> Nat. Biotechnol. 30 (9), 876-882 (2012).</li><li>Gilpin, S. E., 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=Enhanced+Lung+Epithelial+Specification+of+Human+Induced+Pluripotent+Stem+Cells+on+Decellularized+Lung+Matrix.">Enhanced Lung Epithelial Specification of Human Induced Pluripotent Stem Cells on Decellularized Lung Matrix.</a> Annal. Thorac. Surg. 98, 1721-1729 (2014).</li><li>Cortiella, 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=Influence+of+Acellular+Natural+Lung+Matrix+on+Murine+Embryonic+Stem+Cell+Differentiation+and+Tissue+Formation.">Influence of Acellular Natural Lung Matrix on Murine Embryonic Stem Cell Differentiation and Tissue Formation.</a> Tissue Eng. Part A. 16 (8), 2565-2580 (2010).</li><li>Princivalle, M., De Agostini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+roles+of+heparan+sulfate+proteoglycans:+a+comparative+review+in+Drosophila,+mouse+and+human.">Developmental roles of heparan sulfate proteoglycans: a comparative review in Drosophila, mouse and human.</a> Int. J. Dev. Biol. 46, 267-278 (2002).</li><li>Thompson, S. M., Jesudason, E. C., Turnbull, J. E., Fernig, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+in+lung+morphogenesis:+The+elephant+in+the+room.">Heparan sulfate in lung morphogenesis: The elephant in the room.</a> Birth Defects Res. Part C, Embryo Today. 90 (1), 32-44 (2010).</li><li>Zimmermann, 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=Improved+reproducibility+in+preparing+precision-cut+liver+tissue+slices.">Improved reproducibility in preparing precision-cut liver tissue slices.</a> Cytotechnology. 61 (3), 145-152 (2009).</li><li>Ying, Q. -. 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=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453 (7194), 519-523 (2008).</li><li>Fox, E., 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=Three-Dimensional+Culture+and+FGF+Signaling+Drive+Differentiation+of+Murine+Pluripotent+Cells+to+Distal+Lung+Epithelial+Cells.">Three-Dimensional Culture and FGF Signaling Drive Differentiation of Murine Pluripotent Cells to Distal Lung Epithelial Cells.</a> Stem Cells Dev. 24 (1), 21-35 (2014).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Specific+Cell+Population+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS).</a> J. Vis. Exp. (41), e1546 (2010).</li><li>Shojaie, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acellular+lung+scaffolds+direct+differentiation+of+endoderm+to+functional+airway+epithelial+cells:+requirement+of+matrix-bound+HS+proteoglycans.">Acellular lung scaffolds direct differentiation of endoderm to functional airway epithelial cells: requirement of matrix-bound HS proteoglycans.</a> Stem Cell Reports. 4, 1-12 (2015).</li><li>Kubo, 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=Development+of+definitive+endoderm+from+embryonic+stem+cells+in+culture.">Development of definitive endoderm from embryonic stem cells in culture.</a> Development. 131 (7), 1651-1662 (2004).</li><li>Longmire, 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=Efficient+Derivation+of+Purified+Lung+and+Thyroid+Progenitors+from+Embryonic+Stem+Cells.">Efficient Derivation of Purified Lung and Thyroid Progenitors from Embryonic Stem Cells.</a> Cell stem cell. 10 (4), 398-411 (2012).</li><li>Wong, M. D., Dorr, A. E., Walls, J. R., Lerch, J. P., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+mouse+embryo+atlas+based+on+micro-CT.">A novel 3D mouse embryo atlas based on micro-CT.</a> Development. 139 (17), 3248-3256 (2012).</li></ol>

Den her beskrevne protokol genererer modne ESC-afledte luftvejsepitel kun bruger naturlige lunge stilladser til direkte differentiering uden anden tilskud. Denne opsætning kultur er defineret, serumfrit, billig og reproducerbar. Ingen vækst faktor tilskud af basen differentiering medier er påkrævet. Tidligere offentliggjorte metoder til at generere stamcelle-afledte lungeepitelceller har anvendt 2-dimensionelle strategier med vækstfaktor tilskud til fremme af afstamning begrænsning 3,4,8,18,19. Den her ...

Rettet differentiering af pluripotente celler til lungen afstamning er afhængig af præcise signaleringsbegivenheder i mikromiljøet 1,2. På grund af den dynamiske natur af denne proces har det været en udfordring at efterligne de nøjagtige begivenheder af lunge organogenese in vitro. Nylige rapporter har brugt trinvise afstamning restriktionssteder strategier med opløselig vækstfaktor tilskud af todimensionale kulturer for at opnå lunge differentiering 3-8. I trinvise differentiering protokoller, pluripotente celler, uanset om embryonale stamceller (ESC) eller inducerede pluripotente stamceller, blev først differentieret til den endelige endoderm kim lag. Endodermale celler blev efterfølgende presset til en anterior endoderm skæbne og derefter til lunge progenitorceller, som identificeret ved ekspressionen af ​​homeodomænet-holdige transcriptionsfaktor NKX2-1. Disse lunge forfædre blev yderligere differentieret til proksimal (luftvejene) eller distale (alveolære) lunge epitelceller with fortsat vækst faktor tilskud. Sådanne 2-dimensionelle strategier har haft en vis succes med at skabe lungeepitelceller, men der er flere begrænsninger, herunder uklare effektivitet, mulig forurening fra andre endodermale afstamninger, manglen på en 3-dimensional (3D) struktur, og i nogle tilfælde anvendelse af udefinerede kulturer med serum tilskud. Kultur af pluripotente eller differentierede celler på decellulariseret lunge stilladser stigende grad anvendes som et assay til at vurdere den regenerative potentiale sederede celler ved dannelse lungeepitelceller strukturer 3,5,6,8,9. Sådanne rapporter kultur seedede celler på stilladser med fortsat vækst faktor eller serum tilskud. Lunge udvikling indebærer opdelingen, migration, genekspression og differentiering af individuelle celler som respons på miljømæssige signaler. Den ekstracellulær matrix (ECM) er et gitterværk af glycoproteiner, der ud over at tilvejebringe strukturel støtte, dirigerer tissue morfogenese ved at integrere og regulere disse processer 10,11. Ved at bruge lunge ECM stillads som en naturlig platform for endoderm kultur bedre efterligne in vivo lunge udviklingsmæssige miljø, har vi frembragt stamcelle-afledte luftvejs-epitelceller i en defineret 3D-kultur omgivelser med høj effektivitet og reproducerbarhed. Rottelunge ECM scaffolds blev dannet ved decellularization samt muse ESC-afledte endodermale celler blev dannet og efterfølgende podet på disse scaffolds. Dobbelt ekspression af CXCR4 &amp; c-kit-proteiner indikerer en definitiv endoderm celleidentitet og celler positive for både SOX2 &amp; NKX2-1 ekspression er identificeret som luftvej (proksimal lunge) progenitorceller. Endelige endoderm celler blev dyrket ved luft væske-grænsefladen (ALI) i op til tre uger til at generere funktionelle luftvejsepitelceller in vitro. Denne protokol fremmer lunge afstamning differentiering af definitive endoderm så tidligt som 7 dage, observeret med fremkomsten af NKX2-1 + / SOX2 + tidlige proksimale lunge stamfædre. Ved dag 14 og 21 i kultur modne luftvejsepitelceller cellepopulationer emerge herunder cilierede (TUBB4A +), klub (SCGB1A1 +), og basal (TRP63 +, KRT5 +) celler med morfologisk og funktionel lighed med native mus luftveje. Denne protokol viser betydningen af ​​3D-matrix mikromiljø for at opnå robust differentiering for luftvejsobstruktion epitelceller.

<table border="0" cellpadding="0" cellspacing="0" width="901">
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			<td height="22" style="height:22px;width:291px;">Reagents</td>
			<td style="width:163px;"></td>
			<td style="width:168px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Perfusion solution</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">H0777</td>
			<td style="width:281px;">10U/mL heparin</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:291px;">Perfusion solution</td>
			<td style="width:163px;">Gibco</td>
			<td align="right" style="width:168px;">14170112</td>
			<td style="width:281px;">dissolved in Hank&#39;s balanced salt solution (HBSS-)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">BioShop</td>
			<td style="width:168px;">CHA003</td>
			<td style="width:281px;">8mM CHAPS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">E9884</td>
			<td style="width:281px;">25mM EDTA&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">BioShop</td>
			<td style="width:168px;">SOD002</td>
			<td style="width:281px;">1M NaCl</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">14190-144</td>
			<td style="width:281px;">dissolved in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Benzonase nuclease</td>
			<td style="width:163px;">Novagen</td>
			<td style="width:168px;">70664-3</td>
			<td style="width:281px;">90U/mL Benzonase nuclease&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Benzonase nuclease</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">14190-144</td>
			<td style="width:281px;">diluted in PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antimicrobial solution&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td align="right" style="width:168px;">15140</td>
			<td style="width:281px;">200U/mL penicillin streptomycin&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Antimicrobial solution&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td align="right" style="width:168px;">15290</td>
			<td style="width:281px;">25&#956;g/mL amphotericin B&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antimicrobial solution&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">14190-144</td>
			<td style="width:281px;">diluted in PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Trypsinization&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">12605-028</td>
			<td style="width:281px;">TrypLE</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">IMDM 2440-053, F12 11765-054</td>
			<td style="width:281px;">3:1 ratio of IMDM and Ham&#8217;s modified F12 medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">12587-010</td>
			<td style="width:281px;">B27 supplement (50x dilution)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">17502-048</td>
			<td style="width:281px;">N2 supplement (100x dilution)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">15260-037</td>
			<td style="width:281px;">0.05% (Fraction V) bovine serum albumin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">35050-061</td>
			<td style="width:281px;">200mM Glutamax&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">M6145</td>
			<td style="width:281px;">4&#956;M monothioglycerol</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">A4403&#160;</td>
			<td style="width:281px;">0.05mg/mL ascobic acid</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Endoderm induction</td>
			<td style="width:163px;">R&#38;D</td>
			<td style="width:168px;">338-AC/CF</td>
			<td style="width:281px;">Activin A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antibodies</td>
			<td style="width:163px;"></td>
			<td style="width:168px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">CDH1</td>
			<td style="width:163px;">BD Biosciences</td>
			<td align="right" style="width:168px;">610181</td>
			<td style="width:281px;">Mouse, non-conjugated, 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">C-KIT</td>
			<td style="width:163px;">BD Biosciences</td>
			<td align="right" style="width:168px;">558163</td>
			<td style="width:281px;">Rat, PE-Cy7, 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">CXCR4</td>
			<td style="width:163px;">BD Biosciences</td>
			<td align="right" style="width:168px;">558644</td>
			<td style="width:281px;">Rat, APC, 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">KRT5</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:168px;">ab24647</td>
			<td style="width:281px;">Rabbit, non-conjugated, 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">NKX2-1</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:168px;">ab76013</td>
			<td style="width:281px;">Rabbit, non-conjugated, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Laminin</td>
			<td style="width:163px;">Novus Biologicals</td>
			<td style="width:168px;">NB300-144</td>
			<td style="width:281px;">Rabbit, non-conjugated, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">SCGB1A1</td>
			<td style="width:163px;">Santa Cruz</td>
			<td style="width:168px;">sc-9772&#160;</td>
			<td style="width:281px;">Goat, non-conjugated, 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">SOX2</td>
			<td style="width:163px;">R&#38;D Systems</td>
			<td style="width:168px;">AF2018&#160;</td>
			<td style="width:281px;">Goat, non-conjugated, 1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">TRP63</td>
			<td style="width:163px;">Santa Cruz</td>
			<td style="width:168px;">sc-8431</td>
			<td style="width:281px;">Mouse, non-conjugated, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">TUBB4A</td>
			<td style="width:163px;">BioGenex</td>
			<td style="width:168px;">MU178-UC</td>
			<td style="width:281px;">Mouse, non-conjugated, 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Goat IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-11055</td>
			<td style="width:281px;">Donkey, Alexa Fluor 488, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mouse IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-21202</td>
			<td style="width:281px;">Donkey, Alexa Fluor 488, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mouse IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-31571</td>
			<td style="width:281px;">Donkey, Alexa Fluor 647, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rabbit IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-21206</td>
			<td style="width:281px;">Donkey, Alexa Fluor 488, 1:200</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Rabbit IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-31573</td>
			<td style="width:281px;">Donkey, Alexa Fluor 647, 1:200</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Other Materials</td>
			<td style="width:163px;"></td>
			<td style="width:168px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low adherent plates</td>
			<td style="width:163px;">Nunc</td>
			<td style="width:168px;">Z721050&#160;</td>
			<td style="width:281px;">Low cell binding plates,&#160; 6 wells&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Air-liquid interface membranes&#160;</td>
			<td>Whatman</td>
			<td align="right">110614</td>
			<td>Hydrophobic Nucleopore membrane, 8&#956;m pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1200S&#160;</td>
			<td>Leica Vibratome</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tissue Adhesive</td>
			<td>Ted Pella</td>
			<td align="right">10033</td>
			<td>Pelco tissue adhesive</td>
		</tr>
	</tbody>
</table>

generation of esc-derived mouse airway epithelial cells using decellularized lung scaffolds

Lunge afstamning differentiering kræver integration af komplekse miljømæssige påvirkninger, der omfatter vækstfaktorsignalering, celle-celle-interaktioner og celle-matrix-interaktioner. På grund af denne kompleksitet, at sammenfatning af lunge udvikling in vitro fremme differentiering af stamceller til lungeepitelceller har været en udfordring. I denne protokol, er decellulariseret lunge stilladser anvendt til at efterligne den 3-dimensionelle miljø af lungen og generere stamcelle-afledte luftvejs-epitelceller. Muse embryonale stamceller først differentieres til endoderm afstamning under anvendelse af en embryoid legeme (EB) dyrkningsmetode med activin A. endoderm celler podes derefter på decellulariserede scaffolds og dyrket ved luft-væske-grænseflade i op til 21 dage. Denne teknik fremmer differentiering af sederede celler til funktionelt luftvejsepitelceller (cilierede celler, klub celler og basale celler) uden yderligere vækstfaktor tilskud. Denne opsætning kultur er defineret, Serum-fri, billig og reproducerbar. Selv om der er begrænset forurening fra ikke-lunge endoderm slægter i kultur, denne protokol kun genererer luftvejsepitelceller befolkninger og ikke giver anledning til alveolære epitelceller. Luftvejsepitel genereret med denne protokol kan bruges til at studere celle-matrix interaktioner under lunge organogenese og sygdom modellering eller lægemiddel-discovery platforme af luftvejs-relaterede patologier såsom cystisk fibrose.

Som skitseret i denne protokol, til robust differentiering af definitiv endoderm modne luftvejs epitelceller kan opnås ved hjælp af udvidet kultur af seedede celler på decellulariseret lunge stillads sektioner. Det anbefales, at decellulariserede stilladser karakteriseres for at sikre (1) værtsceller er helt fjernet, og (2) ekstracellulære matrixproteiner er bevaret inden anvendelse stilladser til differentiering. Decellularization kan vurderes ved anvendelse af væv farvning med h?...

Watch this Scientific Journal Video about Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds at JoVE.com

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Generation af ESC-afledt Mouse Airway epitelceller Brug decellulariserede Lung Stilladser

Denne protokol effektivt dirigerer muse embryonale stamceller afledt endelig endoderm til modne luftvejs-epitelceller. Denne differentiering teknik anvender 3-dimensionelle decellulariseret lunge stilladser til direkte lunge afstamning specifikation, i en defineret, serumfrit indstilling kultur.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Research

JoVE Journal

Bioengineering

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

3D Hydrogel stillads til Artikulære chondrocyt Kultur og brusk Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

Epithelial cellerepopulationskilde og Udarbejdelse af Gnaver ekstracellulær matrix Stilladser for Renal Tissue Udvikling

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Fabrikation af Decellularized brusk-afledte Matrix stilladser

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

Konstrueret lungevæv fremstillet af decellulariserede lungeskiver

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Regional Decellularized Lung Tissue Isolation to Study Cell-Cell and Cell-ECM Interactions

Dissection and Isolation of Region-Specific Decellularized Lung Tissue

Dissection and Isolation of Region-Specific Decellularized Lung Tissue (Video) | JoVE 

Dissektion og isolering af regionsspecifikt decellulariseret lungevæv

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of ...

Comparative Study of Decellularization Techniques for Bovine Lung ECM Hydrogels

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...