Name: Forfatter Spotlight: Fremskridt og udfordringer i β-cellers differentiering fra pluripotente stamceller
Uploaded: 2024-02-02T13:00:00
Description: Watch this Scientific Journal Video about Author Spotlight: Advancements and Challenges in β-Cells Differentiation from Pluripotent Stem Cells at JoVE.com

Ines Cherkaoui blev støttet af et Diabetes UK-studieophold (BDA 18/0005934) til GAR, som også takker Wellcome Trust for en Investigator Award (212625/Z/18/Z), UKRI MRC for et programtilskud (MR/R022259/1), Diabetes UK for Project-tilskud (BDA16/0005485), CRCHUM for opstartsfonde, Innovation Canada for en John R. Evans Leader Award (CFI 42649), NIH-NIDDK (R01DK135268) for et projekttilskud og CIHR, JDRF for et teamtilskud (CIHR-IRSC:0682002550; JDRF 4-SRA-2023-1182-S-N). Camille Dion og Dr. Harry Leitch for deres hjælp med human hiPSC'er generation og kultur, NIHR Imperial BRC (Biomedical Research Centre) Organoid facilitet, London.

Før differentiering påbegyndes, anbefales det at bestemme det krævede antal ølignende organoider til eksperimentelle formål. I en 6-brøndsplade består en enkelt brønd med over 80% sammenløb typisk af 2-2,3 millioner hPSC'er. Mens en nøjagtig forudsigelse er udfordrende på grund af variationer i hPSC-linjer og differentieringseffektivitet, er et groft skøn 1,5 gange antallet af indledende brønde. En effektivt rettet differentiering giver normalt 1,6 til 2 millioner celler pr. brønd i seks-brøndplader, der o...

Differentiering af humane pluripotente stamceller til β-celler i bugspytkirtlen

<ol>
	<li>Kolios, G., Moodley, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+stem+cells+and+regenerative+medicine.">Introduction to stem cells and regenerative medicine.</a> Respiration. 85 (1), 3-10 (2013).</li><li>Thomson, J. 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li><li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+pluripotent+cell+line+from+early+mouse+embryos+cultured+in+medium+conditioned+by+teratocarcinoma+stem+cells.">Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.</a> Proceedings of the National Academy of Sciences. 78 (12), 7634-7638 (1981).</li><li>Nair, G. G., Tzanakakis, E. S., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+routes+to+the+generation+of+functional+&#946;-cells+for+diabetes+mellitus+cell+therapy.">Emerging routes to the generation of functional &#946;-cells for diabetes mellitus cell therapy.</a> Nature Reviews Endocrinology. 16 (9), 506-518 (2020).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+classification+of+diabetes+mellitus.">Diagnosis and classification of diabetes mellitus.</a> American Diabetes Association. Diabetes Care. 32 (Supplement_1), S62-S67 (2009).</li><li>Sui, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Cell+Replacement+in+mice+using+human+Type+1+diabetes+nuclear+transfer+embryonic+stem+cells.">&#946;-Cell Replacement in mice using human Type 1 diabetes nuclear transfer embryonic stem cells.</a> Diabetes. 67 (1), 26-35 (2018).</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>Hogrebe, N. J., Maxwell, K. G., Augsornworawat, P., Millman, J. R. <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+insulin-producing+pancreatic+&#946;-cells+from+multiple+human+stem+cell+lines.">Generation of insulin-producing pancreatic &#946;-cells from multiple human stem cell lines.</a> Nature Protocols. 16 (9), 4109-4143 (2021).</li><li>Jiang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+insulin-producing+islet-like+clusters+from+human+embryonic+stem+cells.">Generation of insulin-producing islet-like clusters from human embryonic stem cells.</a> Stem Cells. 25 (8), 1940-1953 (2007).</li><li>Sui, L., Leibel, R. L., Egli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+&#946;-cell+differentiation+from+human+pluripotent+stem+cells.">Pancreatic &#946;-cell differentiation from human pluripotent stem cells.</a> Current Protocols in Human Genetics. 99 (1), e68 (2018).</li><li>Sui, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+replication+fork+speed+promotes+pancreatic+endocrine+differentiation+and+controls+graft+size.">Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size.</a> JCI Insight. 6 (5), 141553 (2021).</li><li>Veres, 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=Charting+cellular+identity+during+human+in+vitro+&#946;-cell+differentiation.">Charting cellular identity during human in vitro &#946;-cell differentiation.</a> Nature. 569 (7756), 368-373 (2019).</li><li>Rutter, G. A., Georgiadou, E., Martinez-Sanchez, A., Pullen, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+and+functional+specialisations+of+the+pancreatic+&#946;-cell:+gene+disallowance,+mitochondrial+metabolism+and+intercellular+connectivity.">Metabolic and functional specialisations of the pancreatic &#946;-cell: gene disallowance, mitochondrial metabolism and intercellular connectivity.</a> Diabetologia. 63 (10), 1990-1998 (2020).</li><li>Micallef, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=INS(GFP/w)+human+embryonic+stem+cells+facilitate+isolation+of+in+vitro+derived+insulin-producing+cells.">INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.</a> Diabetologia. 55 (3), 694-706 (2012).</li><li>Finch, P. W., Rubin, J. S., Miki, T., Ron, D., Aaronson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+KGF+is+FGF-related+with+properties+of+a+paracrine+effector+of+epithelial+cell+growth.">Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth.</a> Science. 245 (4919), 752-755 (1989).</li></ol>

Den vellykkede differentiering af hPSC'er i bugspytkirtlen β-celler afhænger af optimering af alle aspekter af rutinemæssig dyrkning og passage af de valgte hPSC'er. Dette inkluderer at sikre, at cellelinjen har en normal karyotype, er negativ for mycoplasmainfektion og er fri for plasmid eller virale vektorgenomer. Når der anvendes hiPSC'er, er det desuden vigtigt at undgå at anvende den tidligste passage, som stadig er under omprogrammering, til pilotforsøg. Disse eksperimenter bør udføres i lille skala for at ...

Humane pluripotente stamceller (hPSC'er) har den unikke evne til at differentiere sig til forskellige celletyper, hvilket gør dem til et levedygtigt alternativ til humane bugspytkirtel-β-celler1. Disse hPSC'er er kategoriseret i to typer: embryonale stamceller (hESC'er), afledt af blastocyst2, og inducerede pluripotente celler (hiPSC'er), genereret ved omprogrammering af somatiske celler direkte3. Udviklingen af teknikker til differentiering af hPSC'er i β-celler har vigtige konsekvenser for både grundforskning og klinisk praksis 1,4. Diabetes mellitus er en kronisk sygdom, der rammer >400 millioner mennesker verden over og skyldes kroppens manglende evne til at regulere glykæmi på grund af funktionsfejl eller tab af β-celler i bugspytkirtlen5. Den begrænsede tilgængelighed af bugspytkirteløceller til transplantation har hindret udviklingen af celleerstatningsterapier til diabetes 2,4,6,7. Evnen til at generere glukoseresponsive insulinudskillende celler ved hjælp af hPSC'er tjener som en nyttig cellulær model til undersøgelse af menneskelig øudvikling og funktion. Det kan også bruges til at teste potentielle terapeutiske kandidater til diabetesbehandling i et kontrolleret miljø. Desuden har hPSC'er potentialet til at producere bugspytkirteløceller, der er genetisk identiske med patienten, hvilket reducerer risikoen for immunafstødning efter transplantation 2,4,7.
I de senere år har der været betydelige fremskridt i forfiningen af hPSC-kultur og differentieringsprotokoller, hvilket resulterer i øget effektivitet og reproducerbarhed af differentieringsprocessen mod generering af bugspytkirtel-β-celler 8,9.
Følgende protokol skitserer de væsentlige stadier af rettet differentiering af β-celler i bugspytkirtlen. Det indebærer regulering af specifikke cellesignalveje på forskellige tidspunkter. Den er baseret på protokollen udviklet af Sui L. et al.10 (2018) til generering af hPSC'er i β-celler i bugspytkirtlen. Protokollen blev justeret til nylige opdateringer fra Sui L. et al.11 (2021), da den seneste forskning understreger betydningen af at bruge aphidicolin (APH) behandling for at forbedre differentieringen af β-celler. Den nuværende protokol omfatter tilsætning af APH til mediet i de senere faser af processen. Desuden er der foretaget ændringer i substratets sammensætning i de tidlige stadier af differentiering sammenlignet med den oprindelige protokol. En bemærkelsesværdig ændring er tilføjelsen af keratinocytvækstfaktor (KGF) på dag 6 og fortsætter indtil dag 8. Keratinocytvækstfaktoren (KGF) introduceres fra dag 6 til dag 8, hvilket adskiller sig lidt fra den oprindelige protokol10, hvor KGF ikke var inkluderet i trin 4-mediet.
Det første og væsentlige trin i dannelsen af β-cellelignende celler er den rettede differentiering af hPSC'er til endelig endoderm (DE), et primitivt kimlag, der giver anledning til epitelforing af forskellige organer, herunder bugspytkirtlen. Efter dannelsen af DE gennemgår cellerne differentiering i det primitive tarmrør, som efterfølges af specifikationen af den bageste foregutskæbne. Den bageste fortarm udvikler sig derefter til pancreas stamceller, som har potentiale til at differentiere sig til alle celletyper i bugspytkirtlen, herunder de endokrine og eksokrine celler. Den efterfølgende fase i processen involverer pancreas endokrine forfædre, hvilket giver anledning til de hormonudskillende celler, der findes i øerne Langerhans. I sidste ende når differentieringsprocessen sit sidste stadium ved at producere fuldt funktionelle bugspytkirtel- β-cellelignende celler 9,10. Det er vigtigt at bemærke, at denne proces er kompleks og ofte kræver optimering af dyrkningsbetingelserne, såsom specifikke vækstfaktorer og ekstracellulære matrixkomponenter, for at forbedre effektiviteten og specificiteten af differentiering 9,10. Desuden er generering af funktionelle β-cellelignende celler fra hPSC'er in vitro stadig en stor udfordring. Igangværende forskning fokuserer på at forbedre differentieringsprotokoller og forbedre modningen og funktionen af de resulterende β-celler9.
I denne protokol er brugen af blid celledissociation under dyrkning og passage af hPSC'er afgørende for at opretholde cellelevedygtighed og pluripotens, hvilket signifikant forbedrer effektiviteten af differentiering i bugspytkirtlen β-celler. Derudover er hvert trinspecifikt medium blevet omhyggeligt optimeret efter protokollen udviklet af Sui L. et al.10 for at fremme et højt udbytte af insulinudskillende celler i klynger, der ligner den menneskelige ø.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL TubeOne Microcentrifuge Tube</td>
			<td>Starlabs</td>
			<td>S1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Cell culture plate</td>
			<td>ThermoFisher Scientific</td>
			<td>165218</td>
			<td></td>
		</tr>
		<tr>
			<td>AggreWell 400 6-well plate&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>34425</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Glucagon&#160;</td>
			<td>Sigma-aldrich</td>
			<td>G2654-100UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Insulin&#160;</td>
			<td>Dako</td>
			<td>A0564</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NKX6.1</td>
			<td>Novus Biologicals</td>
			<td>NBP1-49672SS</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PDX1&#160;</td>
			<td>Abcam</td>
			<td>ab84987</td>
			<td></td>
		</tr>
		<tr>
			<td>Aphidicolin</td>
			<td>Sigma-Aldrich</td>
			<td>A4487</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, fatty acid free</td>
			<td>Sigma-Aldrich</td>
			<td>A3803-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium/Magnesium free D-PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclopamine-KAAD</td>
			<td>Calbiochem</td>
			<td>239804</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose,BioXtra</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>94046-100ML-</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM plus GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>10566016</td>
			<td>For Washing Medium 2: DMEM plus GlutaMAX 1% PS.&#160;</td>
		</tr>
		<tr>
			<td>DMEM/F-12 (Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredi SuperFrost Plus Adhesion slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>20821.33</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270098</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamma-Secretase Inhibitor XX</td>
			<td>Thermo Fisher Scientific</td>
			<td>J64904</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free Reduced Growth Factor Basement</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1413302</td>
			<td>Geltrex 1:1 into cold DMEM/F-12 medium to provide a final dilution of 1:100.</td>
		</tr>
		<tr>
			<td>Goat Anti-Guinea pig, Alexa Fluor 555</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21435</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Guinea pig, Alexa Fluor 647</td>
			<td>Abcam</td>
			<td>ab150187</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse Secondary Antibody, Alexa Fluor 633</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21052</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11011</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF-7 (KGF) Recombinant Protein</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0094</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen chloride</td>
			<td>Sigma-Aldrich</td>
			<td>295426</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge Hydrophobic Barrier PAP Pen</td>
			<td>Agar Scientific</td>
			<td>AGG4582</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Sigma-Aldrich</td>
			<td>SML0559-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M9272-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound 118 mL</td>
			<td>Agar Scientific</td>
			<td>AGR1180</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Tablets, Phosphate Buffered Saline, Fisher BioReagents</td>
			<td>Thermo Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (PS)</td>
			<td>Thermo Fisher Scientific,</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human EGF Protein</td>
			<td>R&#38;D Systems</td>
			<td>236-EG-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular cover glasses, 22&#215;50 mm</td>
			<td>VWR</td>
			<td>631-0137</td>
			<td></td>
		</tr>
		<tr>
			<td>RepSox (Hydrochloride)</td>
			<td>STEMCELL Technologies</td>
			<td>72394</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, GlutaMAX Supplement&#160;&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>61870036</td>
			<td>For Washing Medium 1: RPMI 1640 plus GlutaMAX 1% PS.</td>
		</tr>
		<tr>
			<td>Shandon Immu-mount</td>
			<td>Thermo Fisher Scientific</td>
			<td>9990402</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>7558-80-7</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Endoderm&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>5110</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFlex Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>A3349401</td>
			<td>Thaw the StemFlex Supplement overnight at 4&#176;C, transfer any residual liquid of the supplement bottle to StemFlex Basal Medium.</td>
		</tr>
		<tr>
			<td>Stemolecule All-Trans Retinoic Acid</td>
			<td>Reprocell</td>
			<td>04-0021&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Thyroid Tormone 3 (T3)</td>
			<td>Sigma-Aldrich</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>ThermoFisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypL Express Enzyme (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Adherent Plate for Suspension Culture</td>
			<td>Thermo Fisher Scientific</td>
			<td>38071</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 (Dihydrochloride)&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Sulfate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Z4750</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized protocol for generating functional pancreatic insulin-secreting cells from human pluripotent stem cells

Humane pluripotente stamceller (hPSC'er) kan differentiere sig til enhver form for celle, hvilket gør dem til en fremragende alternativ kilde til humane bugspytkirtel-β-celler. hPSC'er kan enten være embryonale stamceller (hESC'er) afledt af blastocysten eller inducerede pluripotente celler (hiPSC'er), der genereres direkte fra somatiske celler ved hjælp af en omprogrammeringsproces. Her præsenteres en videobaseret protokol for at skitsere de optimale dyrknings- og passagebetingelser for hPSC'er forud for deres differentiering og efterfølgende generering af insulinproducerende bugspytkirtelceller. Denne metode følger seks-trins processen for β-celle rettet differentiering, hvor hPSC'er differentierer sig til endelig endoderm (DE), primitivt tarmrør, bageste foregut skæbne, pancreas forfædre, pancreas endokrine forfædre og i sidste ende pancreas β-celler. Det er bemærkelsesværdigt, at denne differentieringsmetode tager en periode på 27 dage at generere humane β-celler i bugspytkirtlen. Potentialet for insulinsekretion blev evalueret gennem to forsøg, som omfattede immunfarvning og glucosestimuleret insulinsekretion.

Human pluripotent stem cells (hPSCs) have the unique ability to differentiate into various cell types, making them a viable alternative to human pancreatic &#946;-cells1. These hPSCs are categorized into two types: embryonic stem cells (hESCs), derived from the blastocyst2, and induced pluripotent cells (hiPSCs), generated by reprogramming somatic cells directly3. The development of techniques to differentiate hPSCs into &#946;-cells, has important implications for both fundamental research and clinical practice1,4. Diabetes mellitus is a chronic disease affecting &#62;400 million people worldwide and results from the inability of the body to regulate glycemia due to malfunction or loss of pancreatic &#946;-cells5. The limited availability of pancreatic islet cells for transplantation has hindered the development of cell replacement therapies for diabetes2,4,6,7. The ability to generate glucose-responsive insulin-secreting cells using hPSCs serves as a useful cellular model for studying human islet development and function. It can also be used to test potential therapeutic candidates for diabetes treatment in a controlled environment. Moreover, hPSCs have the potential to produce pancreatic islet cells that are genetically identical to the patient, reducing the risk of immune rejection after transplantation2,4,7.
In recent years, there have been significant advancements in the refinement of hPSC culture and differentiation protocols, resulting in increased efficiency and reproducibility of the differentiation process toward generating pancreatic &#946;-cells8,9.
The following protocol outlines the essential stages of directed differentiation of pancreatic &#946;-cells. It involves the regulation of specific cell signaling pathways at distinct time points. It is based on the protocol developed by Sui L. et al.10 (2018) for the generation of hPSCs into pancreatic &#946;-cells. The protocol was adjusted to recent updates from Sui L. et al.11 (2021), as the latest research emphasizes the significance of using aphidicolin (APH) treatment to enhance the differentiation of &#946;-cells. The current protocol includes the addition of APH to the medium during the later stages of the process. Furthermore, modifications have been made to the composition of the medium during the early stages of differentiation compared to the initial protocol. A notable change is the addition of Keratinocyte Growth Factor (KGF) on Day 6 and continuing until Day 8. The keratinocyte growth factor (KGF) is introduced from day 6 to day 8, which slightly differs from the initial protocol10, where KGF was not included in the stage 4 medium.
The first and essential step in the generation of &#946;-cell-like cells is the directed differentiation of hPSCs into definitive endoderm (DE), a primitive germ layer that gives rise to the epithelial lining of various organs, including the pancreas. After the formation of DE, the cells undergo differentiation into the primitive gut tube, which is followed by the specification of the posterior foregut fate. The posterior foregut then develops into pancreatic progenitor cells, which have the potential to differentiate into all cell types of the pancreas, including the endocrine and exocrine cells. The subsequent stage in the process involves pancreatic endocrine progenitors giving rise to the hormone-secreting cells found in the islets of Langerhans. In the end, the differentiation process reaches its final stage by producing fully functional pancreatic &#946;-cell like cells9,10. It is important to note that this process is complex and often requires optimization of the culture conditions, such as specific growth factors and extracellular matrix components, to improve the efficiency and specificity of differentiation9,10. Furthermore, generating functional &#946;-cell like cells from hPSCs in vitro is still a major challenge. Ongoing research focuses on improving differentiation protocols and enhancing the maturation and function of the resulting &#946;-cells9.
In this protocol, the use of gentle cell dissociation during the culture and passage of hPSCs is essential to maintain cell viability and pluripotency, significantly improving the efficiency of differentiation into pancreatic &#946;-cells. Additionally, each stage-specific medium has been meticulously optimized following the protocol developed by Sui L. et al.10 to promote a high yield of insulin-secreting cells in clusters that closely resemble the human islet.

Forfatter Spotlight: Fremskridt og udfordringer i β-cellers differentiering fra pluripotente stamceller

Optimeret protokol til generering af funktionelle insulinudskillende celler i bugspytkirtlen fra humane pluripotente stamceller

Attaining a high and consistent efficiency in the differentiation process for converting pluripotent stem cells into human beta cells is a notable challenge. This challenge is influenced by the precision and the consistency with which the experimenter follows the protocol. Moreover, the amount of beta cells obtained in each experiment is quite variable, which may result in the reduced function of beta cells across multiple experiments.In the context of high leptin beta cell research in diabetes, the availability of cadaveric eyelet donors is quite limited. Consequently, the use of stem cell differentiated beta cells is offering an unlimited and abundant supply of insulin secreting cells. Beta cell differentiation is a pivotal advancement in our field, offering numerous benefits.It allows us to explore the development and function of beta cells, leading to a deeper understanding of diabetes causes and beta cell dysfunction. We aim to use this technique for our research focusing on the genetics of diabetes. Our primary objective is to focus on mutation of certain genes that can influence beta cells and eyelet function.So this protocol will facilitate the access to human eyelet derived from pre-potent stem cells with mutations of interest.

Protokollen beskrevet i dette papir tilbyder en yderst effektiv tilgang til differentiering af β-lignende celler fra hPSC'er10. Denne proces anvender et 2D-kultursystem, der er let skalerbart, hvilket gør det muligt at bruge det i forskellige eksperimentelle indstillinger, såsom læringsdifferentiering, mindre projekter og laboratorier og pilottest for at vurdere potentialet i en iPSC-linje til differentiering.
Det er vigtigt at karakterisere de funktionelle egenskab...

Watch this Scientific Journal Video about Author Spotlight: Advancements and Challenges in β-Cells Differentiation from Pluripotent Stem Cells at JoVE.com

Denne artikel præsenterer en protokol til rettet differentiering og funktionel analyse af β-cellelignende celler. Vi beskriver optimale dyrkningsbetingelser og passager for humane pluripotente stamceller, før der genereres insulinproducerende bugspytkirtelceller. Den seks-trins differentiering skrider frem fra endelig endodermdannelse til funktionelle β-cellelignende celler, der udskiller insulin som reaktion på glukose.

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic ...

Det er en bemærkelsesværdig udfordring at opnå en høj og konsekvent effektivitet i differentieringsprocessen til omdannelse af pluripotente stamceller til humane betaceller. Denne udfordring påvirkes af præcisionen og konsistensen, hvormed eksperimentatoren følger protokollen. Desuden er mængden af betaceller opnået i hvert eksperiment ret variabel, hvilket kan resultere i den reducerede funktion af betaceller på tværs af flere eksperimenter.I forbindelse med forskning i betaceller med højt leptinindhold i diabetes er tilgængeligheden af kadaveriske øjendonorer ret begrænset. Derfor tilbyder brugen af stamcelledifferentierede betaceller en ubegrænset og rigelig forsyning af insulinudskillende celler. Betacelledifferentiering er et afgørende fremskridt inden for vores felt og tilbyder mange fordele.Det giver os mulighed for at udforske udviklingen og funktionen af betaceller, hvilket fører til en dybere forståelse af diabetesårsager og betacelledysfunktion. Vi sigter mod at bruge denne teknik til vores forskning med fokus på genetik af diabetes. Vores primære mål er at fokusere på mutation af visse gener, der kan påvirke betaceller og øjenfunktion.Så denne protokol vil lette adgangen til menneskelige øjer afledt af præpotente stamceller med mutationer af interesse.

optimized-protocol-for-generating-functional-pancreatic-insulin

Research

JoVE Journal

Biology

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic &#946;-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for &#946;-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic &#946;-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic &#946;-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.

This article presents a protocol for directed differentiation and functional analysis of &#946;-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional &#946;-cell like cells secreting insulin in response to glucose.