Name: efficient generation of pancreas/duodenum homeobox protein 1+ posterior foregut/pancreatic progenitors from hpscs in adhesion cultures
Uploaded: 2019-03-27T12:30:00
Description: Watch this Scientific Journal Video about Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures at JoVE.com

Dette arbejde blev støttet i en del af finansiering fra Japan samfundet for fremme videnskab (JSP'ER) gennem Scientific Research (C) (JSP'ER KAKENHI Grant Number15K09385 og 18 K 08510) til T.T. og licensbetaling for JSP'ER forskningsstipendiater (JSP'ER KAKENHI Grant antallet 17J07622) til A.K. og Japan Agency for medicinalforskning og udvikling (AMED) gennem sin forskning give "Core Center for iPS Cell Research, Research Center netværk for realisering af regenerativ medicin" til K.O. Forfatterne takke Dr. Peter Karagiannis for læsning af manuskript.

Eksperimenter ved hjælp af hPSCs blev godkendt af den etiske komité i Institut for medicin og Graduate School of Medicine, Kyoto University.
1. forberedelse af materialer
Bemærk: Forberede alle medier og reagenser til cellekultur i et sterilt miljø. Varm op base dyrkningsmedier til stuetemperatur (RT) før brug. Medium for differentiering bruges inden for 6 h på RT. detaljer af reagenser er angivet i Tabel af materialer</...

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	<li>D'Amour, K. 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=Production+of+pancreatic+hormone-expressing+endocrine+cells+from+human+embryonic+stem+cells.">Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.</a> Nature Biotechnology. 24 (11), 1392-1401 (2006).</li><li>Pagliuca, F. W., 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=Generation+of+functional+human+pancreatic+beta+cells+in+vitro.">Generation of functional human pancreatic beta cells in vitro.</a> Cell. 159 (2), 428-439 (2014).</li><li>Rezania, 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=Reversal+of+diabetes+with+insulin-producing+cells+derived+in+vitro+from+human+pluripotent+stem+cells.">Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.</a> Nature Biotechnology. 32 (11), 1121-1133 (2014).</li><li>Kondo, Y., Toyoda, T., Inagaki, N., Osafune, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPSC+technology-based+regenerative+therapy+for+diabetes.">iPSC technology-based regenerative therapy for diabetes.</a> Journal of Diabetes Investigation. 9 (2), 234-243 (2017).</li><li>Millman, 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=Generation+of+stem+cell-derived+beta-cells+from+patients+with+type+1+diabetes.">Generation of stem cell-derived beta-cells from patients with type 1 diabetes.</a> Nature Communications. 7, 11463 (2016).</li><li>Zeng, H., 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=An+Isogenic+Human+ESC+Platform+for+Functional+Evaluation+of+Genome-wide-Association-Study-Identified+Diabetes+Genes+and+Drug+Discovery.">An Isogenic Human ESC Platform for Functional Evaluation of Genome-wide-Association-Study-Identified Diabetes Genes and Drug Discovery.</a> Cell Stem Cell. 19 (3), 326-340 (2016).</li><li>Hosokawa, 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=Insulin-producing+cells+derived+from+'induced+pluripotent+stem+cells'+of+patients+with+fulminant+type+1+diabetes:+Vulnerability+to+cytokine+insults+and+increased+expression+of+apoptosis-related+genes.">Insulin-producing cells derived from 'induced pluripotent stem cells' of patients with fulminant type 1 diabetes: Vulnerability to cytokine insults and increased expression of apoptosis-related genes.</a> Journal of Diabetes Investigation. , (2017).</li><li>Jennings, R. 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H., 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=Single-cell+RNA-seq+of+human+induced+pluripotent+stem+cells+reveals+cellular+heterogeneity+and+cell+state+transitions+between+subpopulations.">Single-cell RNA-seq of human induced pluripotent stem cells reveals cellular heterogeneity and cell state transitions between subpopulations.</a> Genome Research. 28 (7), 1053-1066 (2018).</li><li>Narsinh, K. H., 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=Single+cell+transcriptional+profiling+reveals+heterogeneity+of+human+induced+pluripotent+stem+cells.">Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells.</a> Journal of Clinical Investigation. 121 (3), 1217-1221 (2011).</li><li>Rosowski, K. A., Mertz, A. F., Norcross, S., Dufresne, E. 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Generation af PDX1+ celler består af flere trin; Derfor er det kritisk at behandle celler på et passende tidspunkt. Blandt skridt, endelige endoderm induktion effektivitet i høj grad påvirker den endelige induktion effektivitet, muligvis af interferens fra andre forurenende slægt celler (dvs. mesoderm og ectoderm), der kan formere sig og/eller udskiller faktorer, der forstyrre specifikke differentiering. Hvis andelen af SOX17+ celler er lavere end 80% på dag 4 (etape 1B), en effektiv induktion...

Bugspytkirtlen består hovedsageligt af eksokrine og endokrine celler, og dens dysfunktion eller overbelastning forårsager adskillige sygdomme som betændelse i bugspytkirtlen, diabetes og kræft i bugspytkirtlen. For at belyse pathogeny af pancreatopathy, er det nødvendigt at analysere den udviklingsmæssige proces og funktion i bugspytkirtlen celler. Derudover kræves en stabil celle forsyning med robust kvalitet at etablere celler/væv tilskud terapi. Humane pluripotente stamceller (hPSC)-afledte pancreas celler er en lovende celle kilde til disse formål, og differentiering protokol mod pancreas celler har været intensivt undersøgt1,2,3, 4. Seneste fremskridt inden for den in vitro-generation celleområde, pancreas β efterligne generation af β-celler i voksen menneskelige, og disse celler Vis terapeutiske virkning efter implantation i diabetisk model mus2,3. Derudover viste analyse af β celler genereret fra den inducerede pluripotente stamceller (iPSCs) af sunde og type 1 diabetes patienten donorer ingen funktionelle forskelle, herunder når under stress5. Derudover er sygdom fænotyper blevet delvist gengivet i induceret i bugspytkirtlen celler med patienten-afledte iPSCs eller hPSCs husly genetiske mutationer i samme sted som patienter6,7.
Du kan generere i bugspytkirtlen celler fra hPSCs ved bruges trinvis styret differentiering, der sammenfatter udviklingsprocesser. Bugspytkirtlen er afledt fra endoderm lag af den tidlige embryon, som udtrykker sex bestemmelse af regionen Y-box 17 (SOX17) og forkhead boks A2 (FOXA2)8. Baseret på undersøgelser, mus, danner det endodermal lag primitive gut tube, som er præget af udtryk for hepatocyt nukleare faktor 1-beta (Hnf1β) og hepatocyt nukleare faktor 4-alpha (Hnf4α). Primitive gut tube elongates og udvikler sig til respiratorisk apparater, fordøjelseskanalen og organer. Efter strækforlængelse, posterior foregut område bliver regionen formodede i bugspytkirtlen som præget af udtryk for transcriptional faktor bugspytkirtlen/duodenum HOXD protein 1 (PDX1)8,9,10. De dorsale og ventrale dele af PDX1+ gut tube tykkere til form i bugspytkirtlen knopper, som er præget af fælles udtryk af bugspytkirtlen transskription faktor 1 subunit alpha (PTF1A) og NK6 HOXD 1 (NKX6.1)8,11. Dette udtryk markerer den morfologiske begyndelse i bugspytkirtlen organogenesis. Pancreas endoderm celler, som er komponenter af pancreas knopper, danner et forgrenet rørformede netværk af epitelial strukturer12 og i sidste ende udvikle sig til eksokrine og endokrine celler, herunder insulin-secernerende β-celler og Glucagon-secernerende α-cellerne. Udtryk for PDX1 registreres første ved den formodede pancreas region, som er så observeret gennem hele pancreas udvikling, og viser lokalisering til β-δ-cellerne og9,13,14. Selv om Pdx1+ celle-området, der ikke udtrykker Ptf1a eller Nkx6.1 differentierer i mavens antrum, duodenum, ekstrahepatisk galdegang og nogle tarmcellerne på midten til sent udviklingstrin i mus9, PDX1+ stamfaderen til bugspytkirtlen betragtes som celler den udviklingsmæssige tidligt i mennesker.
Vi præsenterer her, en detaljeret protokol for at differentiere hPSCs i PDX1+ celler for generation af pancreas lineages. Protokollen indviede differentiering af såning dissocieres enkelt celler15,16,17. Generelt, udifferentierede hPSCs vedligeholdes som kolonier eller celle aggregater i suspension eller vedhæftning. Som et resultat, indlede de fleste protokoller differentiering kort efter passaging. Dog i tilstanden af kolonier eller aggregater, celler er tilbøjelige til rumlige og transcriptional heterogeneities18,19,20,21,22, der kunne hæmme den første differentiering skridt mod endelige endoderm efterfulgt af ineffektive differentiering til PDX1+ celler. Denne protokol kan tilbyde nem håndtering for at forbedre og stabilisere differentiering effektivitet af nogle hPSC linjer, der fandtes tidligere for at differentiere ineffektivt at endodermal slægter og PDX1+ celler23, 24 , 25.

<table border="0" cellpadding="0" cellspacing="0" style="width:617px;" width="616">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">3-Keto-N-aminoethyl-N&#8242;-aminocaproyldihydrocinnamoyl cyclopamine</td>
			<td style="width:100px;">Toronto Research Chemicals</td>
			<td style="width:123px;">K171000</td>
			<td style="width:172px;">CYC</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid</td>
			<td style="width:100px;">Santa Cruz Biotechnology</td>
			<td style="width:123px;">SC-203303</td>
			<td style="width:172px;">TTNPB</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">50 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">339652</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Anti-CDX2 antibody [EPR2764Y]</td>
			<td style="width:100px;">Abcam</td>
			<td style="width:123px;">Ab76541</td>
			<td style="width:172px;">Anti-CDX2, &#215; 1/1000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">B-27 Supplement (50 &#215;)</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">17504-044</td>
			<td style="width:172px;">Serum-free supplement</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">BD FACSAria II Cell Sorter</td>
			<td style="width:100px;">BD Biosciences</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For flow cytometry</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Biomedical freezer</td>
			<td style="width:100px;">SANYO</td>
			<td style="width:123px;">MDF-U538</td>
			<td>For -30 &#176;C storing</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Cell Counting Slides for TC10/TC20 Cell Counter, Dual-Chamber</td>
			<td style="width:100px;">BIO-RAD</td>
			<td style="width:123px;">1450011</td>
			<td style="width:172px;">Counting slide glass</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CELL CULTURE MULTIWELL PLATE, 6 WELL, PS, CLEAR</td>
			<td style="width:100px;">Greiner bio-one</td>
			<td style="width:123px;">657165</td>
			<td style="width:172px;">For differentiation culture/6-well plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Centrifuge</td>
			<td style="width:100px;">TOMY</td>
			<td style="width:123px;">AX-310</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Centrifuge</td>
			<td style="width:100px;">TOMY</td>
			<td style="width:123px;">MX-305</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CHIR99021</td>
			<td style="width:100px;">Axon Medchem</td>
			<td style="width:123px;">Axon 1386</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CLEAN BENCH</td>
			<td style="width:100px;">SHOWA KAGAKU&#160;</td>
			<td style="width:123px;">S-1601PRV</td>
			<td style="width:172px;">Clean bench</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Corning CellBIND 6-well plate</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">3335</td>
			<td style="width:172px;">For feeder-free culture of hPSCs/6-well plate</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Corning Matrigel Basement Membrane Matrix Growth Factor Reduced</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">354230</td>
			<td style="width:172px;">Basement membrane matrix</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Corning Synthemax II-SC Substrate</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">3535</td>
			<td style="width:172px;">For feeder-free culture of hPSCs/synthetic surface material for hPSCs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Cryostat</td>
			<td style="width:100px;">Leica</td>
			<td style="width:123px;">Leica CM1510 S</td>
			<td style="width:172px;">For immunostaining of aggregates.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:223px;">Cytofix/Cytoperm Kit</td>
			<td style="width:100px;">Becton Dickinson</td>
			<td style="width:123px;">554714</td>
			<td style="width:172px;">Perm/Wash buffer is&#160; Permeabilization/Wash buffer. Cytofix/Cytoperm buffer is fixation and permeabilization buffer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Dako pen</td>
			<td style="width:100px;">Dako</td>
			<td style="width:123px;">S2002</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">dNTP mix (10 mM)</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td>18427-088</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A11055</td>
			<td style="width:172px;">Secondary antibody, &#215; 1/500 dilution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A10036</td>
			<td style="width:172px;">Secondary antibody, &#215; 1/500 dilution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A10040</td>
			<td style="width:172px;">Secondary antibody, &#215; 1/500 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Donkey Serum</td>
			<td style="width:100px;">Merck Millipore</td>
			<td style="width:123px;">S30</td>
			<td style="width:172px;">Donkey serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">D-PBS(-) without Ca or Mg</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">14249-95</td>
			<td style="width:172px;">DPBS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Essential 8 Medium</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A1517001</td>
			<td style="width:172px;">For feeder-free culture of hPSCs/hPSC maintenance medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Falcon 5mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">352235</td>
			<td style="width:172px;">5 mL round bottom polystyrene tube with cell strainer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter Tip, 1000 &#181;L</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">124-1000S</td>
			<td style="width:172px;">Use together with pipettes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter Tip, 20 &#181;L</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">124-P20S</td>
			<td style="width:172px;">Use together with pipettes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter Tip, 200 &#181;L</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">124-P200S</td>
			<td style="width:172px;">Use together with pipettes</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Fluorescence Microscope</td>
			<td style="width:100px;">Keyence</td>
			<td style="width:123px;">BZ-X700</td>
			<td style="width:172px;">For immunostaining</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Forma Steri-Cycle CO2 incubator</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">370A</td>
			<td style="width:172px;">Incubator</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">HNF-1&#946; Antibody (C-20)</td>
			<td style="width:100px;">Santa Cruz Biotechnology</td>
			<td style="width:123px;">sc-7411</td>
			<td style="width:172px;">Anti-HNF1&#946;, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">HNF-4&#945; Antibody (H-171)</td>
			<td style="width:100px;">Santa Cruz Biotechnology</td>
			<td style="width:123px;">sc-8987</td>
			<td style="width:172px;">Anti-HNF4&#945;, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Hoechst 33342</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">H3570</td>
			<td style="width:172px;">For nucleus staining, &#215; 1/200 dilution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Human Pancreas Total RNA</td>
			<td style="width:100px;">Ambion</td>
			<td style="width:123px;">AM7954</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Human PDX-1/IPF1 Antibody</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">AF2419</td>
			<td style="width:172px;">Anti-PDX1, goat IgG, &#215; 1/200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Human SOX17 Antibody</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">AF1924</td>
			<td style="width:172px;">Anti-SOX17, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Improved MEM Zinc Option medium</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">10373-017</td>
			<td style="width:172px;">iMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Incubation chamber</td>
			<td style="width:100px;">Cosmo Bio</td>
			<td style="width:123px;">10DO</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Latex Examination Gloves</td>
			<td style="width:100px;">Adachi</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">MAS coated slide glass</td>
			<td style="width:100px;">Matsunami Glass</td>
			<td>83-1881</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">MicroAmp Fast 96-well Reaction Plate</td>
			<td style="width:100px;">Applied Biosystems/Thermo Fisher Scientific</td>
			<td style="width:123px;">4346907</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Microscope</td>
			<td style="width:100px;">Olympus</td>
			<td style="width:123px;">CKX41N-31PHP</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Microtube</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">131-515CS</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Monoclonal Anti-&#945;-Fetoprotein</td>
			<td style="width:100px;">SIGMA</td>
			<td style="width:123px;">A8452</td>
			<td style="width:172px;">Anti-AFP, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Nanodeop 8000</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Oligo dT</td>
			<td style="width:100px;">FASMAC</td>
			<td style="width:123px;">Custom made Oligo&#160;</td>
			<td style="width:172px;">For RT-qPCR of sequence is &#34;TTTTTTTTTTTTTTTTTTTT&#34;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Paraformaldehyde, powder</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">26126-54</td>
			<td style="width:172px;">PFA, fixative, diluted in DPBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Pharmaceutical refrigerator</td>
			<td style="width:100px;">SANYO</td>
			<td style="width:123px;">MPR-514</td>
			<td>For 4 &#176;C storing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">PIPETMAN P&#160;</td>
			<td style="width:100px;">GILSON</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">Pipette</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Recombinant Human KGF/FGF-7</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">251-KG</td>
			<td style="width:172px;">KGF</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Recombinant Human Noggin</td>
			<td style="width:100px;">PeproTech</td>
			<td style="width:123px;">120-10C</td>
			<td style="width:172px;">NOGGIN</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Recombinant Human/Mouse/Rat Activin A</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">338-AC</td>
			<td style="width:172px;">Activin A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">ReverTra Ace (100 U/&#956;L)</td>
			<td style="width:100px;">TOYOBO</td>
			<td style="width:123px;">TRT-101</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Rnase-Free Dnase Set (50)</td>
			<td style="width:100px;">QIAGEN</td>
			<td style="width:123px;">79254</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Rneasy Mini Kit</td>
			<td style="width:100px;">QIAGEN</td>
			<td style="width:123px;">74104</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">RPMI 1640 with L-Gln</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">30264-85</td>
			<td style="width:172px;">RPMI 1640</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sealing Film for Real Time</td>
			<td style="width:100px;">Takara</td>
			<td style="width:123px;">NJ500</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Serological pipettes 10 mL</td>
			<td style="width:100px;">Costar/Corning</td>
			<td style="width:123px;">4488</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Serological pipettes 25 mL</td>
			<td style="width:100px;">Costar/Corning</td>
			<td>4489</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Serological pipettes 5 mL</td>
			<td style="width:100px;">Costar/Corning</td>
			<td style="width:123px;">4487</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sox2 (D6D9) XP Rabbit mAb</td>
			<td style="width:100px;">Cell signaling</td>
			<td style="width:123px;">3579S</td>
			<td style="width:172px;">Anti-SOX2, &#215; 1/200 dilution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">StepOnePlus</td>
			<td style="width:100px;">Applied Biosystems/Thermo Fisher Scientific</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sucrose</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td>30406-25</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">TB Green 
			Premix Ex Taq II&#160;</td>
			<td style="width:100px;">Takara</td>
			<td>RR820B</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">TC20 Automated Cell Counter</td>
			<td style="width:100px;">BIO-RAD</td>
			<td style="width:123px;">1450101J1</td>
			<td style="width:172px;">Automatic cell counter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Tissue-Tek OCT compound 4583&#160;</td>
			<td style="width:100px;">Sakura Finetechnical</td>
			<td>4583</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Tissue-Tek Cryomold Molds/Adapters</td>
			<td style="width:100px;">Sakura Finetechnical</td>
			<td>4566</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Triton X-100</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">35501-15</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Trypan Blue</td>
			<td style="width:100px;">BIO-RAD</td>
			<td style="width:123px;">1450021</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Ultracold freezer</td>
			<td style="width:100px;">SANYO</td>
			<td style="width:123px;">MDF-U33V</td>
			<td>For -80 &#176;C storing</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">15575-038</td>
			<td style="width:172px;">Dilute with DPBS to prepare 0.5 mM EDTA</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Veriti Thermal Cycler</td>
			<td style="width:100px;">Applied Biosystems/Thermo Fisher Scientific</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Y-27632</td>
			<td style="width:100px;">Wako</td>
			<td style="width:123px;">251-00514</td>
			<td style="width:172px;"></td>
		</tr>
	</tbody>
</table>

efficient generation of pancreas/duodenum homeobox protein 1+ posterior foregut/pancreatic progenitors from hpscs in adhesion cultures

Humane pluripotente stamceller (hPSC)-afledte pancreas celler er en lovende celle kilde for regenerativ medicin og en platform til at studere menneskelige udviklingsprocesser. Trinvis instrueret differentiering, der sammenfatter udviklingsprocesser er en af de vigtigste måder at generere pancreas celler herunder bugspytkirtlen/duodenum HOXD protein 1+ (PDX1+) i bugspytkirtlen stamceller. Konventionelle protokoller indlede differentiering med små kolonier kort efter passage. Men i tilstanden af kolonier eller aggregater, celler er tilbøjelige til at heterogeneities, som kan hæmme differentiering til PDX1+ celler. Vi præsenterer her, en detaljeret protokol for at differentiere hPSCs i PDX1+ celler. Protokollen består af fire trin og initierer differentiering af seeding dissocierede enkelt celler. Induktion af SOX17+ endelige endoderm celler blev efterfulgt af udtryk for to primitive gut tube markører, HNF1β og HNF4α og eventuel differentiering i PDX1+ celler. Denne protokol giver nem håndtering og kan forbedre og stabilisere differentiering effektiviteten af nogle hPSC linjer, der fandtes tidligere for at differentiere ineffektivt i endodermal lineages eller PDX1+ celler.

Formeringsmaterialets hiPSCs (585A129,30) er kondenseret og danner en homogen éncellelag (figur 1B), der er velegnet til differentiering. Udifferentierede hiPSCs (fase 0) er adskilt og re seedet som enkelt celler ved lav celle tætheder (1-1,5 x 105 celler/cm2). Inden for 1 time, cellerne er knyttet til pladen og begynde at vise fremspring. På dag 1, er cellerne tilvækst og godt fordelt til at dække 80-90% a...

Watch this Scientific Journal Video about Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures at JoVE.com

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Vi præsenterer her, en detaljeret protokol for at differentiere humane pluripotente stamceller (hPSCs) i bugspytkirtlen/duodenum HOXD protein 1+ (PDX1+) celler for generation af pancreas lineages baseret på ikke-koloni type éncellelag vækst af adskilles enkelt celler. Denne metode er velegnet til producerer homogene hPSC-afledte celler, genmanipulation og screening.

Effektiv Generation af bugspytkirtlen/Duodenum HOXD Protein 1+ Posterior Foregut/pancreas ophav fra hPSCs i vedhæftning kulturer

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Den største fordel ved denne teknik er, at før stimulation, celler spredes jævnt og derefter stimuleres jævnt i tandem celle vedhæftning. Demonstration af proceduren vil være Azuma Kimura, en kandidat studerende fra vores laboratorium. For at begynde denne procedure, overføre seks milliliter RPMI 1640 i et rør afkølet til fire grader Celsius på is.Ved hjælp afkølet en milliliter pipette tips, tilføje to milligram kælder membran matrix. Bland godt ved blid pipettering at gøre det til en kælder membran matrix med en koncentration på 0,33 milligram pr milliliter. Dernæst skal du bruge en pipette til at overføre to milliliter af den fortyndede kældermembranmatrix i hver brønd af en seks-brøndcellekulturplade.Inkuber pladen ved 37 grader Celsius i 60 til 90 minutter. Derefter opbevares pladen ved stuetemperatur i op til tre timer, indtil den er klar til brug. Først aspirere det brugte medium og bruge en pipette til at tilføje to milliliter 0,5 millimolar EDTA til hver brønd til at vaske hPCSs dyrket i seks-brønd plade.Derefter aspirere EDTA fra brøndene og tilsæt to milliliter frisk 0,5 millimolar EDTA til hver brønd. Inkuber pladen ved 37 grader Celsius i fem minutter. Efter dette, aspirere EDTA fra hver brønd.Tilføj en milliliter hPSC vedligeholdelse medium ved stuetemperatur, suppleret med 10 mikromolar Y27632 til hver brønd. Pipette forsigtigt, men hurtigt at blæse de vedhæftede celler på pladen og til at adskille eventuelle klumpede celler i enkelte celler. Cellesuspension overføres i et 50 milliliter centrifugerør indeholdende fire milliliter hPSC vedligeholdelsesmedium suppleret med 10 mikromolar Y27632 pr. brønd og blandes ved pipettering.Dernæst blandes 15 mikroliter celleaffjedring med 15 mikroliter trypanblå i røret. Brug en 10 mikroliterpipette til at overføre 10 mikroliter af den fortyndede cellesuspension til celletællingsdias i dublet. Ved hjælp af en automatiseret celletæller skal du tælle cellenummeret og beregne celletætheden i cellesuspensionen.Cellesuspensionen fordeles i et 50 milliliterrør med en massetæthed på en til halvanden million celler for hver brønd, der skal anvendes. Centrifuge røret ved 200 gange G og ved stuetemperatur i fem minutter. Derefter skal du bruge en pipette til at aspirere supernatanten og re-suspendere cellerne ved hjælp af en milliliter fase 1A medium pr brønd.Forsigtigt pipette celle suspension og derefter tilføje en anden milliliter af fase 1A medium pr brønd. Ved hjælp af en 1000 mikroliterpipette, aspirere den fortyndede kælder membran matrix fra brønde af den tidligere forberedte celle kultur plade. Straks pipette suspensionen i røret forsigtigt og overføre to milliliter i hver brønd af en seks-brønd plade.Dæk pladen med aluminiumsfolie for at beskytte den mod lys og læg pladen på en ren bænk ved stuetemperatur i 10 til 15 minutter. Derefter overføres pladen forsigtigt til en inkubator ved 37 grader Celsius med 5 % kuldioxid og en befugtet atmosfære i 24 timer. Ryst forsigtigt pladen, og brug en pipette til at aspirere det anvendte medium.Derefter tilsættes to milliliter DPBS til hver brønd. Ryst forsigtigt pladen igen og aspirere den brugte DPBS. Tilføj fire milliliter af fase 1B medium, forvarmt til 37 grader Celsius, til hver brønd.Overfør forsigtigt pladen til en inkubator ved 37 grader Celsius med 5% kuldioxid og en befugtet atmosfære til kultur i 48 timer. Derefter rystes pladen forsigtigt, og indsugning af det brugte medium. Tilføj to milliliter DPBS til hver brønd.Gentag denne aspiration og tilføjelse af DPBS en ekstra gang. Ryst forsigtigt pladen og opsugning af den brugte DPBS. Tilføj fire milliliter af fase 1B medium, forvarmt til 37 grader Celsius, til hver brønd.Overfør forsigtigt pladen til en inkubator, ved 37 grader Celsius med 5% kuldioxid og en befugtet atmosfære til kultur i 24 timer. Ryst forsigtigt pladen og opsugning af det brugte medium. Tilføj to milliliter DPBS til hver brønd af seks-brønd plade.Ryst derefter forsigtigt pladen og opsugning af den brugte DPBS. Tilføj fire milliliter af fase to medium, forvarmt til 37 grader Celsius, til hver brønd. Overfør forsigtigt pladen til en inkubator ved 37 grader Celsius med 5% kuldioxid og en befugtet atmosfære til kultur i fire dage.Ryst forsigtigt pladen og aspirere det brugte medium. Tilføj to milliliter DPBS til hver brønd. Gentag denne proces med at aspirere og tilføje DPPS igen.Derefter rystes pladen forsigtigt, og indsugning af den brugte DPBS. Tilføj fire milliliter af fase tre medium, forvarmt til 37 grader Celsius, til hver brønd. Overfør forsigtigt pladen til en inkubator ved 37 grader Celsius med 5% kuldioxid og en befugtet atmosfære til kultur i tre dage.I denne undersøgelse kondenseres formerings-hIPSEs og danner et homogent monolag, der er egnet til differentiering. Udifferentierede hIPSEs adskilles og genfrøes som enkelte celler ved lav celletæthed. Inden for en time er cellerne fastgjort til pladen og begynder at vise fremspring.På dag ét, cellerne er spredt og godt fordelt til at dække 80 til 90% af overfladearealet. På dag tre og fire danner cellerne et homogent monolagsark, der kan beskrives som et brostensbelagt udseende. På dette tidspunkt holder de fleste celler op med at udtrykke SOX2, som er en markør for udifferentierede celler, og udtrykker i stedet en endelig endodermmarkør, SOX17, ved mere end 90% Mest SOX17-positive celler udtrykker FOXA2.Så cellerne begynder at udtrykke den primitive goptupe markører, HNF1-beta og HNF4-alpha, og i sidste ende udtrykke en posterior foregrate pancreas progenitor markør, PDX1, på mere end 90% PDX1-positive celle induktion er reproducerbare i en anden HIPSE linje, 1231A3, og en hESE linje, KhES-3. QRTPCR-resultaterne af MRNA-ekspressionen af scenemarkører er i overensstemmelse med immunstaining, og MRNA-udtrykket af PDX1 er tydeligt i trin tre og stiger betydeligt bagefter. Da udifferentierede ESIPS-celler dyrkes som kolonier, er de enkelte celler lokalt i en anden tilstand.Denne protokol giver mulighed for at stimulere cellerne jævnt for den valgte differentiering.

Research

JoVE Journal

Developmental Biology

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Effektiv Udledning af retinale pigmentepitel Celler fra stamceller

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Generation af menneskelige fedt-stamceller gennem dedifferentiering af Ældre Adipocytter i Loft Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Generering af genetisk modificerede Organotypiske Skin Cultures Brug devitaliseret Menneskelig Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Instrueret differentiering af Primitive og endelige hæmatopoietisk ophav fra humane pluripotente stamceller

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Effektiv Differentiering af pluripotente stamceller til NKX6-1<sup&gt; +</sup&gt; Pankreatisk progenitorer

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Isolation, kultur og differentiering af knoglemarven stromale celler og osteoklast ophav fra mus

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation af stillads-fri, tre-dimensionelle Insulin udtrykker Pancreatoids fra mus i bugspytkirtlen stamfaderen In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

Generation af første hjertet felt-lignende hjerte ophav og ventrikel-lignende Cardiomyocytes fra humane pluripotente stamceller

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

I Vitro Generation af Somite derivater fra menneskelige induceret celler pluripotente stamceller

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

Differentiering og karakterisering af neurale forfædre og neuroner fra museembryst stamceller

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...