Name: efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors
Uploaded: 2017-03-07T12:30:00
Description: Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

Dette håndskrift blev støttet af midler fra Toronto General og vestlige Foundation og Banting &amp; bedste Diabetes Center-University Health Network Graduate Award.

1. Udarbejdelse af løsninger og Medier BEMÆRK: Forberede alle medier til cellekultur i et sterilt miljø. Medier skal gøres og anvendes straks. Reagens oplysninger findes i Materials tabel. <ol><li> Differentiering medier <ol><li> Forbered Dag 0 Differentiation Medier: RPMI Medium med 1% glutamin, 2 uM CHIR 99.021, 100 ng / ml activin A, 10 4 M MTG. </li><li> Forbered Day 1-2 Differentiation Medie: RPMI Medium med 1% glutamin, 100 ng / ml act...

<ol>
	<li>Cogger, K., Nostro, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+cell+replacement+therapies+for+the+treatment+of+type+1+diabetes.">Recent advances in cell replacement therapies for the treatment of type 1 diabetes.</a> Endocrinology. 156 (1), 8-15 (2015).</li><li>Nostro, M. C., 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+NKX6-1++pancreatic+progenitors+from+multiple+human+pluripotent+stem+cell+lines.">Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines.</a> Stem Cell Reports. 4 (4), 591-604 (2015).</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> Nat Biotechnol. , (2014).</li><li>Kroon, 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=Pancreatic+endoderm+derived+from+human+embryonic+stem+cells+generates+glucose-responsive+insulin-secreting+cells+in+vivo.">Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.</a> Nat Biotechnol. 26 (4), 443-452 (2008).</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=Maturation+of+human+embryonic+stem+cell-derived+pancreatic+progenitors+into+functional+islets+capable+of+treating+pre-existing+diabetes+in+mice.">Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice.</a> Diabetes. 61 (8), 2016-2029 (2012).</li><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=Efficient+differentiation+of+human+embryonic+stem+cells+to+definitive+endoderm.">Efficient differentiation of human embryonic stem cells to definitive endoderm.</a> Nat Biotechnol. 23 (12), 1534-1541 (2005).</li><li>Gouon-Evans, V., 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=BMP-4+is+required+for+hepatic+specification+of+mouse+embryonic+stem+cell-derived+definitive+endoderm.">BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm.</a> Nat Biotechnol. 24 (11), 1402-1411 (2006).</li><li>Jonsson, J., Carlsson, L., Edlund, T., Edlund, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-promoter-factor+1+is+required+for+pancreas+development+in+mice.">Insulin-promoter-factor 1 is required for pancreas development in mice.</a> Nature. 371 (6498), 606-609 (1994).</li><li>Pan, F. C., Wright, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+organogenesis:+from+bud+to+plexus+to+gland.">Pancreas organogenesis: from bud to plexus to gland.</a> Dev Dyn. 240 (3), 530-565 (2011).</li><li>Schaffer, A. E., Freude, K. K., Nelson, S. B., Sander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nkx6+transcription+factors+and+Ptf1a+function+as+antagonistic+lineage+determinants+in+multipotent+pancreatic+progenitors.">Nkx6 transcription factors and Ptf1a function as antagonistic lineage determinants in multipotent pancreatic progenitors.</a> Dev Cell. 18 (6), 1022-1029 (2010).</li><li>Zhou, Q., 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+multipotent+progenitor+domain+guides+pancreatic+organogenesis.">A multipotent progenitor domain guides pancreatic organogenesis.</a> Dev Cell. 13 (1), 103-114 (2007).</li><li>Sander, 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=Homeobox+gene+Nkx6.1+lies+downstream+of+Nkx2.2+in+the+major+pathway+of+beta-cell+formation+in+the+pancreas.">Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas.</a> Development. 127 (24), 5533-5540 (2000).</li><li>Sharow, K. A., Temkin, B., Asson-Batres, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+stability+in+stem+cell+cultures.">Retinoic acid stability in stem cell cultures.</a> Int J Dev Biol. 56 (4), 273-278 (2012).</li><li>Korytnikov, R., Nostro, M. C. <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+polyhormonal+and+multipotent+pancreatic+progenitor+lineages+from+human+pluripotent+stem+cells.">Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells.</a> Methods. , 56-64 (2016).</li><li>Baumgarth, N., Roederer, 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+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> J Immunol Methods. 243 (1-2), 77-97 (2000).</li><li>Nostro, M. C., 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=Stage-specific+signaling+through+TGFbeta+family+members+and+WNT+regulates+patterning+and+pancreatic+specification+of+human+pluripotent+stem+cells.">Stage-specific signaling through TGFbeta family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells.</a> Development. 138 (5), 861-871 (2011).</li><li>Schulz, T. C., 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+scalable+system+for+production+of+functional+pancreatic+progenitors+from+human+embryonic+stem+cells.">A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.</a> PLoS One. 7 (5), e37004 (2012).</li></ol>

Succesfuld generere NKX6-1 + bugspytkirtlen forfædre fra hPSCs in vitro bygger på anvendelsen af høj kvalitet kulturer af hPSCs og dirigeret differentiering involverer præcis regulering af specifikke signalveje, der styrer vigtige udviklingsstadier i bugspytkirtlen udvikling. Selv om denne protokol kan bruges til at fremkalde robust udtryk for NKX6-1 tværs af en række hPSC linjer som tidligere vist 3, at sikre en effektiv NKX6-1 generation følgende overvejelser bør gø...

Forekomsten af diabetes er stigende, og i henhold til den canadiske diabetesforening, anslås det, at mere end 11 millioner mennesker i Canada er diabetiker eller prædiabetisk, med 5-10% af disse personer, der har type 1-diabetes (T1D) 1. T1D er en autoimmun sygdom, der er forårsaget af ødelæggelse af de insulinproducerende p-celler, der er placeret inden for de langerhanske øer. I øjeblikket individer lever med T1D kræver udefra kommende kilder af insulin to. Trods fremskridt i insulinbehandling, T1D patienter fortsat have en vanskelig tid at regulere deres blodsukkerniveau og fortsat lider både hypo- og hyperglykæmi. En lovende form for behandling for at genoprette normoglykæmi i T1D er anvendelsen af humane embryonale stamceller (hESCs), som kunne anvendes til at generere en ubegrænset forsyning af insulinproducerende p-celler både in vivo og in vitro 3, 4, 5, 6, 7. Differentiering hESCs til p-lignende celler kunne gøre det muligt at studere diabetes in vitro, hvilket muliggør identifikationen af nye terapeutiske mål for type 2-diabetes og tilvejebringer celler til transplantation i T1D patienter. Den mest succesfulde forsøg på at generere insulinproducerende celler fra hESCs in vitro er at rekapitulere de embryonale begivenheder, der opstår under pancreasudviklingen 4, 5. Dette indebærer manipulation af distinkte signalveje til præcist at modellere vigtige stadier i udviklingslandene bugspytkirtlen. Pancreasudviklingen begynder med induktion af den endelige endoderm, som er kendetegnet ved ekspressionen af CXCR4 og CD117 (c-KIT) 8, 9. Præcis regulering af Definitive endoderm organisation er nødvendig for dannelsen af ​​tarmen rør, som så gennemgår anterior-til-posterior og ventral-dorsale mønsterdannelse. Den dorsale og ventrale pancreatiske knopper komme ud af området af den bageste fortarm der udtrykker pancreas og duodenal homeobox-genet (Pdx1), som er nødvendig for pancreasudviklingen 10. Den dorsale og ventrale knopper sikringen til at danne bugspytkirtlen, som derefter gennemgår omfattende epitelial remodeling og ekspansion 11. Engagement i endokrine og eksokrine afstamning er ledsaget af den generation af multipotente stamceller (MPCS), der udtrykker, blandt andre, transskription faktorer Pdx1, Nkx6.1 og Ptf1a 12, 13. Middelhavspartnerlandene, der bliver endokrine og duktale celler fortsætter med at udtrykke Nkx6-1 mens faldende Ptf1a udtryk. I modsætning til dette, vil exokrine afstamningsceller miste expression af Nkx6-1 og vedligeholde Ptf1a udtryk 12. Transkriptionsfaktoren Nkx6-1 har en central rolle i pancreas udvikling, især under differentieringen af endokrine progenitorceller til p-celler. Som tidligere beskrevet, sletning af Nkx6-1 resulterer i nedsat dannelse af p-celler under pancreasudvikling 14. Derfor genererer insulinproducerende p-celler både in vitro og in vivo kræver effektiv induktion af Nkx6-1. Vi har for nylig udviklet en protokol til effektivt at generere Pdx1 + / NKX6-1 + bugspytkirtlen forfædre fra hPSCs. Disse hPSC-afledte pancreas progenitorer generere modne p-celler ved transplantation i immundefekte mus 3. Differentieringen protokol kan opdeles i 4 faser karakteristisk for: 1) endelig endoderm induktion, 2) posterior fortarm mønsterdannelse, 3) pancreas specifikation og 4) NKX6-1 induktion. Her giver vi en detaljeret beskrivelse af hvert trin i rettet differentiering.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media and cytokines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-Thioglycerol (MTG)</td>
			<td>Sigma</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D</td>
			<td>338-AC/CF&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Life Technologies</td>
			<td>12587-010&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm Buffer</td>
			<td>BD Bioscience</td>
			<td>554722</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Perm/Wash buffer, 1x</td>
			<td>BD Bioscience</td>
			<td>554723</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR990210</td>
			<td>Tocris</td>
			<td>4423a</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>VWR</td>
			<td>80510-412&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Sigma</td>
			<td>P5499</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&#38;D</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Wisent</td>
			<td>88150</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Life Technologies</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>NO636</td>
			<td></td>
		</tr>
		<tr>
			<td>NOGGIN</td>
			<td>R&#38;D</td>
			<td>3344-NG</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>Life Technologies</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>SANT-1</td>
			<td>Tocris</td>
			<td>1974</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1x), phenol red</td>
			<td>Life Technologies</td>
			<td>12605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies for flow cytometry (working dilutions)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD117 PE (1:100)</td>
			<td>Life Technologies</td>
			<td>CD11705</td>
			<td></td>
		</tr>
		<tr>
			<td>CXCR4 APC (1:50)</td>
			<td>BD&#160; Bioscience</td>
			<td>551966</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 conjugate (1:400)</td>
			<td>Life Technologies</td>
			<td>A-31571</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Goat IgG (H+L), Alexa Fluor 488 (1:400)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td>705-546-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotype Control Mouse IgG&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.&#160;</td>
			<td>015-000-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotype Control Goat IgG</td>
			<td>R&#38;D&#160;&#160;</td>
			<td>AB-108-C</td>
			<td></td>
		</tr>
		<tr>
			<td>NKX6-1 (1:2000)</td>
			<td>DSHB</td>
			<td>F55A10</td>
			<td></td>
		</tr>
		<tr>
			<td>PDX1 (1:100)</td>
			<td>R&#38;D</td>
			<td>AF2419</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors

Pluripotente stamceller har evnen til at forny sig selv og differentiere til flere slægter, hvilket gør dem til et attraktivt kilde til generering af pancreasstamfaderceller, der kan anvendes til studiet af og fremtidige behandling af diabetes. Denne artikel beskriver en fire-trins differentiering protokol designet til at generere pancreasstamfaderceller fra humane embryonale stamceller (hESCs). Denne protokol kan anvendes på en række humane pluripotente stamcelle (hPSC) linjer. Den tilgang, til at generere pancreasstamfaderceller er at differentiere hESCs til præcist modellere vigtige stadier i bugspytkirtlen udvikling. Det begynder med induktion af den endelige endoderm, hvilket opnås ved dyrkning af cellerne i nærvær af activin A, basisk fibroblastvækstfaktor (bFGF) og CHIR990210. Yderligere differentiering og mønstret med fibroblast vækstfaktor 10 (FGF10) og Dorsomorphin genererer celler ligner den bageste fortarm. Tilsætningen af ​​Retinsyre, noggin, SANT-1 og FGF10 adskiller bageste fortarm celler i celler karakteristiske af pancreas endoderm. Endelig kombinationen af epidermal vækstfaktor (EGF), Nicotinamid og noggin fører til den effektive generering af PDX1 + / NKX6-1 + -celler. Flowcytometri er udført for at bekræfte ekspressionen af ​​specifikke markører på centrale stadier i bugspytkirtlen udvikling. De Pdx1 + / NKX6-1 + pancreas progenitorer i slutningen af trin 4 er i stand til at generere modne p-celler ved transplantation i immundefekte mus, og kan yderligere differentieres til frembringelse insulinproducerende celler in vitro. Således effektiv generering af PDX1 + / NKX6-1 + pancreas progenitorer, som påvist i denne protokol, er af stor betydning, da det giver en platform til at studere human pancreas udvikling in vitro og tilvejebringer en kilde til celler med potentialet til at differentiere til p-celler, der kunne eventually anvendes til behandling af diabetes.

Effektiv generation af pancreas progenitorer er afhængig af korrekt vækst og vedligeholdelse af udifferentierede celler efterfulgt af den præcise tilsætning af specifikke signaleringsmolekyler under differentieringen protokol, som illustreret i diagrammet i figur 1A. På dag 0 bør udifferentierede celler være 80-95% sammenflydende og kolonier bør have definerede kanter (figur 1A). Under fase 1, vil medierne sandsynligvis vises overskyet da celled?...

Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

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

Her beskriver vi en 4-trins-protokol til at differentiere humane embryonale stamceller til NKX6-1 + bugspytkirtlen forfædre in vitro. Denne protokol kan anvendes på en række humane pluripotente stamcellelinjer.

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 ...

The overall goal of this procedure is to efficiently generate NKX6.1 expressing pancreatic progenitors from human pluripotent stem cells in vitro by differentiating the cells through four key stages of pancreatic development. By differentiating human pluripotent stem cells through four key stages of pancreatic devleopment, this protocol provides a model to study human pancreas development in vitro. The main advantage of this technique over existing protocols is that we are able to generate high percentages of NKX6.1 positive pancreatic progenitor cells using a variety of human pluripotent stem cell lines.Given that pancreatic progenitors have the potential to generate insulin producing beta like cells, this technique will give us the opportunity to develop cells for diabetes treatment, for drug discovery and for disease modeling. On day zero, begin differentiation by replacing the KnockOut serum replacement media with day zero differentiation media. Then one days one and two, gently shake the plate to remove any dead cells from the monolayer before aspirating the media.Afterward, replace it with day one to two differentiation media. On day three, harvest the cells for flow cytometry. If the cells express more than 90%of CXCR4 and CD117, the efficiency has been reached.Then gently shake the plate to remove any dead cells from the monolayer prior to aspiration and replace it with day three to five differentiation media. It is important to ensure efficient induction of the definitive under derm by day three by flow cytometry. If the percentage of cells expressing both CXCR4 and CD117 is lower than 90%differentiation to pancreatic progenitors will likely be compromised.On days six and seven, replace the medium with day six to seven differentiation media. On days eight, 10 and 12, replace the medium with day eight to 12 differentiation media. Then on day 13, harvest the cells for flow cytometry to determine PDX1 and NKX6.1 expression.To harvest the cells for flow cytometry analysis, disassociate the cells with commercial Trypsin solution according to the manufacturer's protocol and incubate the cells at 37 degrees Celsius for three minutes. After three minutes, remove the Trypsin solution and re-suspend the single cells in 1, 000 microliters of fax buffer with 30 microliters of DNA One. Then filter the cells using a 35 micrometer nylon mesh cell strainer and transfer them to a micro centrifuge tube.Centrifuge the cells at 455 times g for five minutes before preparing the cells for live or fixed staining. Now re-suspend the cells in 1, 000 microliters of fax buffer. Transfer 200 microliters into two wells of a 96 well plate for both unstained and stained samples.Afterward, centrifuge the plate at 931 times g for two minutes, afterward remove the supernatant by inverting the plate, stain the cells with primary conjugated antibody in a final volume of 100 microliters for 30 minutes at room temperature and protect them from light. Subsequently, centrifuge the plate at 931 times g for two minutes, after two minutes remove the supernatant by inverting the plate. Following that, re-suspend the samples in 100 microliters of fax buffer.Then centrifuge the plate 931 times g for two minutes. And remove the supernatant by inverting the plate. Next re-suspend the samples in 300 to 500 microliters of fax buffer, transfer them to the one milliliter micro test tubes or the five milliliter round bottom tubes and run the samples on flow cytometor.If the samples cannot be run immediately, store them at four degrees Celsius and protect it from light. In this procedure, re-suspend the cell pellet in commercial fixation and permeable Isation solution for 24 hours at four degrees Celsius. After 24 hours, centrifuge the sample at 455 times g for five minutes, then re-suspend it in 400 microliters of commercial perm wash solution.Next, transfer 200 microliters of the sample in two wells of a 96 well plate for IGG control and PDX1, NKX6.1 staining, centrifuge the plate at 931 times g for two minutes. Then re-suspend the cell pellets in 100 microliters of commercial perm wash solution containing anti-PDX1 and anti-NKX61 primary or isotope control antibodies. Incubate them overnight at four degrees Celsius.The next day, centrifuge the plate at 931 times g for two minutes, remove the supernatant by inverting the plate and re-suspend the samples in 100 microliters of the commercial perm wash solution. Next, centrifuge the plate at 931 times g for two minutes. Remove the supernatant by inverting the plate.Then re-suspend the samples in 100 microliters of perm wash solution containing secondary antibody at room temperature for one hour and protect it from light. After an house, centrifuge the plate 931 times g for two minutes, then remove the supernatant by inverting the plate, following that, re-suspend the samples in 100 microliters of commercial perm wash solution, centrifuge the plate at 931 times g for two minutes, remove the supernatant by inverting the plate. Then, re-suspend the sample in 100 microliters of commercial perm wash solution and centrifuge the plate at 931 times g for two minutes again before removing the supernatant.Next, re-suspend the samples in 300 to 500 microliters of fax buffer and transfer them into the separate one milliliter test tubes or five milliliter round bottom tubes for running flow cytometry. Shown here is a sematic of the induction of pancreatic progenitor from human embryonic stem cells which indicates day of differentiation, develop mental stage, base media and the cytokines added at every stage. And here are the phase contrast images at the key stages of pancreatic differentiation.H1 cells were differentiated according to the four stage differentiation protocol depicted. With undifferentiated human embryonic stem cells at day zero, definitive endo derm at stage one, posterior four gut at stage two, pancreatic endo derm at stage three and NKX6.1 positive endo derm pancreatic progenitors at stage four. This figure shows the day three representative flow cytometry plots for CXCR4 PE and CD117 APC antibody staining.Here is the unstained sample and here is the stained sample. And this figure shows day 13 representative flow cytometry plots for stage four derived pancreatic progenitor cells expressing NKX61 and PDX1. This is the IGG control and this is the stained sample.Once mastered, this technique can be done in 13 days. However this may vary depending on the cell line used. While attempting this procedure, it is important to test all agents due to batch to batch variability.After following the procedure, the NKX6.1 positive pancreatic progenitor cells can be further differentiated to insulin producing beta like cells using other published protocols. This provides a platform for studying beta cell maturation and development in vitro. After watching this video, you should have a good understanding of how to efficiently differentiate human pluripotent stem cells to pancreatic progenitors by taking the cells through four key stages of development.

Research

JoVE Journal

Developmental Biology

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

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 ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In vitro differentiering af humane pluripotente stamceller til trofoblastceller

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Hurtig, instrueret differentiering af retinale Pigment epitel celler fra menneskelige embryonale eller induceret pluripotente stamceller

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Effektiv og skalerbar styret differentiering af klinisk kompatibel hornhinde Limbal epitelial stamceller fra humane pluripotente stamceller

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Effektiv differentiering af humane pluripotente stamceller i leverceller

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

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 ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Forbigående behandling af humane pluripotente stamceller med DMSO for at fremme differentiering

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

Vurdering af oxidative skader i den primære mus okulære overfladeceller / stamceller som reaktion på Ultraviolet-C (UV-C) Skader

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Effektiv neurale differentiering ved hjælp af enkelt cellekultur af humane embryonale stamceller

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Effektiv differentiering af postganglioniske sympatiske neuroner ved hjælp af humane pluripotente stamceller under Feeder-fri og kemisk definerede kulturbetingelser

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...