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 ønsker å takke dr Rossant og Dr. Bilodeau for Nkx2-1 mcherry ESC brukt i eksperimenter vist i figur 1-3. FACS ble utført i Den SickKids-uhn flowcytometrisystemer Facility. Dette arbeidet ble støttet av driftstilskudd fra den kanadiske Institutes for Health Research og en infrastruktur stipend (CSCCD) fra kanadiske Foundation of Innovation.

Dyreforsøk ble utført i samsvar med Animal Care komiteens retningslinjer Hospital for Sick Children Research Institute. 1. Stillas Forberedelse <ol><li> Decellularization av lungene <ol><li> Avlive voksne Wistar rotter ved hjelp av CO 2 kammer. Plasser dyr i kammeret og begynne 100% CO2 eksponering ved en fyllgrad på 10-30% av kammervolumet per minutt. <ol><li> Observere dyr for bevisstløshet; Dette vil skje etter ca 2-3 min. Hvis bevisstløshet ikke forekommer i denne tidsperioden, s...

<ol>
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Protokollen er beskrevet her genererer modne ESC-avledet luftveis epitel med kun naturlig lunge stillasene til direkte differensiering med ingen andre tilskudd. Denne kulturen oppsettet er definert, serum-fri, billig, og reproduserbar. Ingen vekstfaktor tilskudd av basen differensiering media er nødvendig. Tidligere publiserte metoder for å generere stamcelle-avledet lunge epitelceller har brukt to-dimensjonale strategier med vekstfaktor tilskudd for å fremme avstamning begrensning 3,4,8,18,19. Teknikken s...

Regissert differensiering av pluripotente celler til lungene avstamning er avhengig av presise signal hendelser i mikromiljøet 1,2. På grunn av dynamikken i denne prosessen har det vært utfordrende å etterligne de nøyaktige hendelsene i lunge organogeneseperioden in vitro. Nyere rapporter har brukt trinnvise avstamning restriksjons strategier med løselig vekstfaktor tilskudd av to-dimensjonale kulturer for å oppnå lunge differensiering 3-8. I trinn-messig differensiering protokoller, pluripotente celler, enten embryonale stamceller (ESC) eller induserte pluripotente stamceller, ble først differensiert til den definitive endoderm bakterie lag. Endodermal celler ble deretter presset til en fremre endoderm skjebne, og deretter til lunge progenitor-celler, som er identifisert ved ekspresjon av homeodomain-inneholdende transkripsjonsfaktor NKX2-1. Disse lungestamfedre ble ytterligere differensiert i proksimale (luftveier) eller distal (alveolære) lunge epitelceller with fortsatt vekst faktor tilskudd. Slike to-dimensjonale strategier har hatt en viss suksess i å generere lunge epitelceller, men det er flere begrensninger, inkludert uklare effektivitet, mulig forurensning fra andre endodermal slektsnavn, mangel på en tre-dimensjonal (3D) struktur, og i noen tilfeller bruk av udefinerte kulturer med serum tilskudd. Kultur av pluripotent eller differensierte celler på decellularized lungestillasene blir stadig mer brukt som en analyse for å vurdere den regenerative potensialet til utsådd celler til å danne tette epiteliale strukturer 3,5,6,8,9. Slike rapporter kultur seeded celler på stillasene med fortsatt vekstfaktor eller serum tilskudd. Lunge utvikling involverer divisjonen, migrasjon, genekspresjon og differensiering av individuelle celler i respons til miljømessige signaler. Den ekstra matriks (ECM) er et rammeverk av glykoproteiner som i tillegg til å gi strukturell støtte, dirigerer tissue morphogenesis ved å integrere og regulere disse prosessene 10,11. Ved hjelp av lunge ECM stillaset som en naturlig plattform for endoderm kultur for å bedre etterligne in vivo lunge utviklingsmiljøet, har vi generert stamcelle-avledet luftveis epitelceller i et definert 3D-kultur innstilling med høy effektivitet og reproduserbarhet. Rat lunge ECM stillasene ble generert av decellularization samt mus ESC-avledet endodermal celler ble generert og deretter sådd ut på disse stillasene. Dual uttrykk for CXCR4 &amp; c-KIT proteiner indikerer en definitiv endoderm celle identitet og positiv for både SOX2 &amp; NKX2-1 uttrykk er identifisert som luftveis (proksimale lunge) stamceller celler. Definitive endoderm celler ble dyrket i luften væske-grensesnittet (ALI) for opptil tre uker å lage funksjonelle Airway epitelceller in vitro. Denne protokollen fremmer lunge avstamning differensiering av Definitiontive endoderm så tidlig som 7 dager, observert med fremveksten av NKX2-1 + / SOX2 + tidlige proksimale lunge stamfedre. Ved dag 14 og 21 av kultur modne luftveier epiteliale cellepopulasjoner dukker inkludert Cilierte (TUBB4A +), klubb (SCGB1A1 +), og basal (TRP63 +, KRT5 +) celler med morfologisk og funksjonell likhet med innfødte mus luftveiene. Denne protokollen demonstrerer viktigheten av 3D-matrix mikromiljøet for å oppnå robust differensiering til luftveis epitelceller.

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="22">
			<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 avstamning differensiering krever integrering av komplekse miljø signaler som omfatter vekstfaktor-signalering, celle-celle interaksjoner og celle-matriks-interaksjoner. På grunn av denne kompleksiteten, til gjentagelse av lunge utvikling in vitro fremme differensiering av stamceller til lunge epitelceller har vært utfordrende. I denne protokollen, decellularized lunge stillasene benyttes for å etterligne den tre-dimensjonale miljøet i lunge og generere stamcelle-avledede luftveis epitelceller. Mus embryonale stamceller blir først differensiert til endoderm linjen ved hjelp av en embryoid legeme (EB) dyrkningsmetode med aktivin A. endoderm Cellene blir deretter sådd ut på decellularized stillaser og dyrket ved luft-væske-grenseflate i opp til 21 dager. Denne teknikken fremmer differensiering av seeded celler til funksjonell Airway epitelceller (Cilierte celler, klubb celler, og basal celler) uten ytterligere vekstfaktor tilskudd. Denne kulturen oppsettet er definert, serum-fri, billig, og reproduserbar. Selv om det er begrenset forurensning fra ikke-lunge endoderm linjene i kultur, denne protokollen genererer bare luftveis epitel populasjoner og ikke gir opphav til alveolære epitelceller. Airway epithelia generert med denne protokollen kan brukes til å studere celle-matriks interaksjoner under lunge organogeneseperioden og for sykdom modellering eller narkotika-discovery plattformer av luftveisrelaterte sykdommer som cystisk fibrose.

Som beskrevet i denne protokollen, til robuste differensiering av definitive endoderm modne airway epitelceller kan oppnås ved hjelp av utvidet kultur seeded celler på decellularized lunge stillas seksjoner. Det anbefales at decellularized stillasene kjennetegnes for å sikre (1) vertsceller kan fjernes helt, og (2) ekstracellulære matrix proteiner er bevart før du bruker stillaser for differensiering. Decellularization kan vurderes ved hjelp vev farging med hematoksylin og eosin (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

Generering av MGP-avledet Mouse Airway Epitelceller Bruke Decellularized Lung Stillas

Denne protokollen dirigerer effektivt mus embryonale stamcelle-avledet definitive endoderm til modne luftveier epitelceller. Denne differensieringen teknikken bruker tre-dimensjonale decellularized lunge stillasene for å dirigere lunge avstamning spesifikasjon, i et definert, serumfritt kultur innstilling.

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 Stillas for Ledd chondrocyttransplantasjon 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

Epitelcelle repopulation og klargjøring av gnagere Ekstracellulær Matrix Stillas for Nedsatt Tissue Development

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

Fabrikasjon av Decellularized brusk-avledet Matrix stillaser

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

Konstruert lungevev tilberedt av decellulariserte 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 

Disseksjon og isolering av regionspesifikt decellularisert lungevev

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 ...