Name: in vitro pancreas organogenesis from dispersed mouse embryonic progenitors
Uploaded: 2014-07-19T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

Detta arbete har finansierats sekventiellt av en NCCR Frontiers i genetik pilot utmärkelse, Juvenile Diabetes Research Foundation Grant 41-2009-775 och Grant 12-126875 från Det Frie Forskningsråd / Sundhed og Sygdom. Författarna tackar Spagnoli labbet för värd för videoinspelning.

Detta protokoll ska växa pankreas organoids härrör från mus-E10.5 dissocierade epithelial celler i bukspottkörteln. Protokollet kräver etiskt godkännande för djurförsök. 1. Dissektion av Dorsal bukspottskörteln Bud från E10.5 musembryon <ol><li> Offra tidsinställd-dräktiga möss på embryonala dag (E) 10.5, öppna buken med en sax, ta bort de två livmoderhornen och placera dem i en 10 cm petriskål fylld med kall fosfatbuffert saltlösning (PBS) ...

<ol>
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Storskalig produktion av funktionella betaceller in vitro är fortfarande ineffektivt 1. I detta utmanande sammanhang, kan utvecklingsbiologiska studier hjälpa dechiffrera de exakta signaler som krävs för differentiering av funktionella betaceller. Detta protokoll möjliggör för underhåll, utbyggnad och differentiering av embryonala pankreas stamceller in vitro. Detta omfattar bildandet av insulinproducerande betaceller som inte samar uttrycka andra endokrina hormoner, har höga nivåe...

Organkultur ger en användbar modell som överbryggar klyftorna mellan den komplexa men mycket relevant in vivo-undersökningar och bekvämt men ungefärlig simulering av cellinje modeller. I fallet med bukspottkörteln, det inte finns någon cell linje perfekt motsvarar pankreas progenitorer även om det är transformerade cellinjer som simulerar endokrina och exokrina celler. Den vuxna Hela bukspottkörteln kan inte odlas; isolerade endokrina cellöar kan upprätthållas under några få veckor, utan att celltillväxt och vävnadsskivor kan hållas in vitro under några timmar 5. Embryonala bukspottkörteln kultur har använts i stor utsträckning inte bara för att studera dess utveckling, men också för att undersöka epitelceller-mesenkymala interaktioner 4,6,7, att bilden processer 8 eller kemiskt störa dem 9. Två organodlingsmetoder används främst: den första består i att odla pankreas knoppar på fibronektin belagda plattor 2, vilket är convenient för avbildningsändamål; det andra alternativet är att odla organ på filter på luft-vätske-gränssnittet 3,4 som bäst bevarar morfogenes. Även mycket användbara, är dessa metoder leder till en viss grad av tillplattning; expansionen av progenitorceller är mycket begränsad i jämförelse med den normala utvecklingen och utgångspopulationen är komplex innefattande alla typer av pankreatiska celler och mesenkymala celler. Förmågan att kulturen och expandera spridda primära celler är värdefullt att studera härstamning relationer och avslöja de inneboende egenskaper isolerade celltyper 10. Sugiyama m.fl. 11. Skulle kunna behålla pankreas stamceller och endokrina stamfäder som behållit vissa funktionella tecken i 3-5 dagar i kultur på matarskikt. Pancreatospheres, besläktad med neurosfärer 12 och mammospheres 13, har utökats från vuxna cellöar och duktala celler även om vilken typ av progenitorer / stamcellersom genererar dessa sfärer är inte klart. Dessutom, i motsats till fysiologisk utveckling innehöll pancreatospheres vissa nervceller 14,15. Klot har också nyligen producerat från embryonala pankreasgångare 16,17 och regenere pancreata 18 med god stamfader expansion och efterföljande differentiering men misslyckades med att rekapitulera morfogenes. 3D-modeller från spridda och ofta definierade celler som själv organiserar i miniatyriserade organ har nyligen blomstrade och simulera utvecklingen eller en vuxen omsättning på flera organ såsom tarm 19,20, magen 21, lever 22, prostata 23 och luftstrupen 24. I vissa fall har utvecklings morfogenes och differentiering valts rekapituleras i 3D från ES-celler, såsom är fallet med optiska kopparna 25, tarmen 26 eller hjärn 27. Här, vi desCribe en metod att expandera dissocierade multi pankreas stamceller i en 3D Matrigel byggnadsställning där de kan differentiera och självorganisation.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>3148-25ML</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Calbiotech</td>
			<td>524400-1MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma Aldrich</td>
			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-spondin 1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock keept at -80&#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td>Stock keept at -80&#176;C- do not include to increase beta cell production</td>
		</tr>
		<tr>
			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
			<td></td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>B27 x50 (supplement)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>R&#38;D</td>
			<td>233-FB-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>RNAlater - RNA stabilizing reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4818-2MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>BSA for reconstitution</td>
			<td>Milipore</td>
			<td><a href="http://www.millipore.com/catalogue/item/81-068-3">81-068</a></td>
			<td>For reconstituition of cytokines&#160; - Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td>16141079</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>60 well MicroWell trays</td>
			<td>Sigma Aldrich</td>
			<td>M0815-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>95-well plates F bottom</td>
			<td>Greiner Bio</td>
			<td>6555180</td>
			<td></td>
		</tr>
		<tr>
			<td>Glas bottom plates</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal micropittes</td>
			<td>Blaubrand</td>
			<td>708745</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

E10.5 dorsala bukspottskörteln stamceller dissocierade och heat i 3D Matrigel rekapitulera bukspottkörteln utveckling. Stamceller kan enklast följas med fluorescerande reportrar. I vårt fall använde vi en transgen mus som uttrycker en nukleär GFP protein styrs av Pdx1 promotor (Pdx1-Ngn3-ER TM-nGFP) (film 1) i avsaknad av tamoxifen och därmed utan att aktivera Neurog3 4 (figur 2). Med organoid medium, en första packning av små...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

Den tredimensionella kultur metod som beskrivs i detta protokoll rekapitulerar bukspottkörteln utveckling från spridda embryonala mus bukspottkörteln progenitorceller, inklusive deras betydande expansion, differentiering och morfogenes i ett grenat orgel. Denna metod är mottaglig för bildbehandling, funktionella störningar och manipulation av nisch.

In vitro Bukspottkörteln Organogenesis från spridda Mouse Embryonala stamfäder

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

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Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

In vitro-differentiering av Mouse embryonala stamceller (MES) celler med hängande släpp-metoden

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

In vitro-märkning av mänskliga embryonala stamceller för magnetisk resonanstomografi

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Encelliga Sug Inspelningar från fotoreceptorer Mus Cone

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging och terapeutiska behandlingar i en Orthotopic musmodell av äggstockscancer

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Purification of Progenitors from Skeletal Muscle

Rening av stamceller från skelettmuskulatur

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

A System for ex vivo Culturing of Embryonic Pancreas

A System for ex vivo Culturing of Embryonic Pancreas

Ett system för<em&gt; Ex vivo</em&gt; Odling av embryonala Pancreas

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

Generering av möss härledda från inducerade pluripotenta stamceller

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Cell Märkning och Injektion i utvecklings Embryonala Mus Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

RNA Isolering från mus Pankreas: En Ribonuclease rika Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Protein Isolering från utvecklings Embryonala Mus hjärtklaff Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...