Name: guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions
Uploaded: 2020-07-02T13:00:00
Description: Watch this Scientific Journal Video about Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions at JoVE.com

Dit werk werd ondersteund door de Pratt School of Engineering aan de Duke University, de afdeling Nefrologie aan de Duke Medical School, A Chair's Research Award van de afdeling Geneeskunde aan de Duke University en een Burroughs Wellcome Fund PDEP Career Transition Ad Hoc Award aan S.M.. M.B werd ondersteund door het Graduate Research Fellowship Program van de National Science Foundation. We bedanken het Bursac Lab voor het genereus verstrekken van de DU11-stamcellijn en het Varghese Lab aan de Duke University voor het tijdelijk delen van hun weefselkweekfaciliteit met onze groep. Deze publicatie is opgedragen aan Prof. Laura L. Kiessling, Novartis Professor of Chemistry aan het Massachusetts Institute of Technology, ter ere van haar 60e verjaardag.

1. Bereiding van reagentia
<ol><li>Verdun ontdooide 5x hiPS celkweek media (CCM) supplement in hiPS cel cultuur basaal medium om een 1x oplossing van hiPS CCM te verkrijgen. OPMERKING: Bevroren 5x hiPS CCM supplement vereist een langzaam ontdooiproces, idealiter in 4 °C voor een nacht. Aliquots van de 1x hiPS CCM kunnen tot 6 maanden bewaard worden bij -20 °C.</li>
	<li>Bereiding van keldermembraan (BM) matrix 1-gecoate platen voor hiPS-celkweek: Ontdooi BM matrix 1 's nachts op ijs bij 4 °C. Na ontdooiing bereid...

<ol>
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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=Tissue+Expression+of+Nephrin+in+Human+and+Pig.">Tissue Expression of Nephrin in Human and Pig.</a> Pediatric Research. 55, 774-781 (2004).</li><li>Guo, J. K., 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=WT1+is+a+key+regulator+of+podocyte+function:+reduced+expression+levels+cause+crescentic+glomerulonephritis+and+mesangial+sclerosis.">WT1 is a key regulator of podocyte function: reduced expression levels cause crescentic glomerulonephritis and mesangial sclerosis.</a> Human Molecular Genetics. 11, 651-659 (2002).</li><li>Roselli, S., 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=Podocin+localizes+in+the+kidney+to+the+slit+diaphragm+area.">Podocin localizes in the kidney to the slit diaphragm area.</a> American Journal of Pathology. 160, 131-139 (2002).</li><li>Shadrin, I. 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=Cardiopatch+platform+enables+maturation+and+scale-up+of+human+pluripotent+stem+cell-derived+engineered+heart+tissues.">Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues.</a> Nature Communications. 8, 1825 (2017).</li><li>Satoh, D., 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=aPKC&#955;+maintains+the+integrity+of+the+glomerular+slit+diaphragm+through+trafficking+of+nephrin+to+the+cell+surface.">aPKC&#955; maintains the integrity of the glomerular slit diaphragm through trafficking of nephrin to the cell surface.</a> The Journal of Biochemistry. 156, 115-128 (2014).</li><li>Mae, S. I., 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=Monitoring+and+robust+induction+of+nephrogenic+intermediate+mesoderm+from+human+pluripotent+stem+cells.">Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells.</a> Nature Communications. 4, 1367 (2013).</li><li>Reya, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+in+stem+cells+and+cancer.">Wnt signalling in stem cells and cancer.</a> Nature. 434, 843-850 (2005).</li><li>Clevers, H., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt&#946;-Catenin+Signaling+and+Disease.">Wnt&#946;-Catenin Signaling and Disease.</a> Cell. 149, 1192-1205 (2012).</li><li>Ciampi, O., 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+podocytes+from+human+induced+pluripotent+stem+cells.">Generation of functional podocytes from human induced pluripotent stem cells.</a> Stem Cell Research. 17, 130-139 (2016).</li><li>Fuente Mora, 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=Differentiation+of+Podocyte+and+Proximal+Tubule-Like+Cells+from+a+Mouse+Kidney-Derived+Stem+Cell+Line.">Differentiation of Podocyte and Proximal Tubule-Like Cells from a Mouse Kidney-Derived Stem Cell Line.</a> Stem Cells and Development. 21, 296-307 (2011).</li><li>Chuah, J. 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In dit rapport beschrijven we een protocol voor het genereren van nierglomerulaire podocyten uit hiPS-cellen. De van hiPS-cellen afgeleide podocyten vertonen morfologische en moleculaire kenmerken geassocieerd met het volwassen nierpodocytenfenotype13. In eerdere publicaties toonden we aan dat de van hiPS-cellen afgeleide podocyten de structuur en selectieve filtratiefunctie van de nierglomerulus kunnen nabootsen wanneer ze worden gekweekt met glomerulaire microvasculaire endotheelcellen in een pe...

Vooruitgang in menselijke pluripotente stamcelcultuur staat klaar om een revolutie teweeg te brengen in regeneratieve geneeskunde, ziektemodellering en medicijnscreening door onderzoekers een hernieuwbare, schaalbare bron van biologisch materiaal te bieden die kan worden ontworpen om bijna elk celtype in het menselijk lichaam te verkrijgen1. Deze strategie is vooral nuttig voor het afleiden van gespecialiseerde en functionele celtypen die anders moeilijk te verkrijgen zouden zijn. Door de mens geïnduceerde pluripotente stamcellen (hiPS)2,3,4,5 zijn bijzonder aantrekkelijk vanwege hun somatische celoorsprong en het potentieel dat ze vertegenwoordigen voor gepersonaliseerde geneeskunde. Het ontwikkelen van methoden om andere cellijningen af te leiden uit hiPS-cellen blijft echter een uitdaging vanwege het frequente gebruik van slecht gedefinieerde kweekomstandigheden die leiden tot een lage efficiëntie en niet-specifieke generatie van heterogene celpopulaties6,7.
Hier gepresenteerd is een methode voor de afleiding van volwassen nierpodocyten uit hiPS-cellen met specificiteit en hoge efficiëntie onder chemisch gedefinieerde omstandigheden. Door rekening te houden met de rol van meerdere factoren binnen de cellulaire micro-omgeving, werd een stamceldifferentiatiestrategie ontwikkeld die de optimalisatie van oplosbare factoren in het celkweekmedium omvatte, evenals onoplosbare factoren, zoals extracellulaire matrixcomponenten of kleefsubstraten. Gezien het belang van integrinesignalering in de ontwikkeling en functie van podocyten, werd de expressie van integrinereceptoren op het celoppervlak aanvankelijk onderzocht. β1-integrinen kwamen niet alleen sterk tot expressie in hiPS-cellen, maar ook in hun derivaten, waaronder mesoderm- en intermediaire mesodermcellen8,9,10. Latere experimenten bevestigden dat liganden die binden aan β1-integrinen (inclusief laminine 511 of laminine 511-E8-fragment) de hechting en differentiatie van hiPS-cellen in podocyten ondersteunen bij gebruik in combinatie met de oplosbare inductieve media die hieronder worden beschreven.
Inductie van cellijn commitment werd geïnitieerd door eerst te bevestigen dat hiPS-cellen gekweekt op de laminine-gecoate oppervlakken gedurende twee dagen in aanwezigheid van een medium met Activine A, CHIR99021 en Y27632 Rock-remmer kunnen differentiëren in cellen die de vroege mesodermmarkers HAND1, goosecoid en brachyury tot expressie brengen8,11. Behandeling van de mesodermcellen gedurende 14 dagen met een medium aangevuld met botmorfogenetisch eiwit 7 (BMP-7) en CHIR99021 maakte de afleiding mogelijk van intermediaire mesodermcellen die de nefron-voorlopercelmarkers Wilm's Tumor 1 (WT1), oneven overgeslagen gerelateerd eiwit 1 (OSR1)8,11en gepaard box gen 2-eiwit (PAX2)12tot expressie riepen. Om de rijpe nierglomerulaire podocyten af te leiden, werden de intermediaire mesodermcellen gedurende 4-5 dagen behandeld met een nieuw medium bestaande uit BMP-7, Activine A, vasculaire endotheelgroeifactor (VEGF), transtrans-retinoïnezuur en CHIR99021. Flowcytometrie en immunostaining werden gebruikt om te bevestigen dat >90% van de resulterende cellen de moleculaire, morfologische en functionele kenmerken van de volwassen nierpodocyt8,11,13vertoonden. Deze kenmerken omvatten de ontwikkeling van primaire en secundaire voetprocessen; de expressie van podocytenlijnspecifieke genen, waaronder SYNPO, PODXL, MAF, EFNB28 en de expressie van eiwitten waaronder podocine, nefrine en WT114,15,16. Bovendien bleek dat de van hiPS-cellen afgeleide podocyten tot vier weken in vitro in cultuur kunnen worden gehouden door een commercieel beschikbaar medium8,11 te gebruiken dat een extra flexibiliteit biedt in de timing van downstream-experimenten. Voor meer informatie over de flowcytometriepanelen die worden gebruikt voor het bepalen van de zuiverheid van de hiPS-podocyten, verwijzen wij u naar onze vorige publicatie11.

<table>
	<tbody>
		<tr>
			<td>Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DU11 human iPS cells</td>
			<td></td>
			<td></td>
			<td>The DU11(Duke University clone #11) iPS cell line was generated at the Duke University iPSC Core Facility and provided to us by the Bursac Lab at Duke University. This line has been tested for mycoplasma and was last karyotyped in July 2019 by our lab, and found to be karyotypically normal.</td>
		</tr>
		<tr>
			<td>Growth Factors and Media Supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans retinoic acid (500 mg)</td>
			<td>72262</td>
			<td>Stem Cell Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 serum-free supplement</td>
			<td>17504044</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>04-0004</td>
			<td>Stemgent</td>
			<td>May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Complete Medium Kit with CultureBoost-R</td>
			<td>4Z0-500-R</td>
			<td>Cell Systems</td>
			<td>Podocyte maintenance media</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>12634028</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with GlutaMAX supplement</td>
			<td>10565042</td>
			<td>Thermo/Life Technologies</td>
			<td>DMEM/F12 with glutamine supplement</td>
		</tr>
		<tr>
			<td>Heat-inactivated FBS</td>
			<td>10082147</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human activin A</td>
			<td>PHC9564</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BMP7</td>
			<td>PHC9544</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF</td>
			<td>PHC9394</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 medium</td>
			<td>05850</td>
			<td>Stem Cell Technologies</td>
			<td>hiPS cell culture media (CCM)</td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin, liquid (100&#215;)</td>
			<td>15140-163</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632 ROCK inhibitor</td>
			<td>1254</td>
			<td>Tocris</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488&#8211; and Alexa Fluor 594&#8211;conjugated secondary antibodies</td>
			<td>A32744; A32754; A-11076; A32790</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Brachyury(T&#65289;</td>
			<td>ab20680</td>
			<td>Abcam</td>
			<td></td>
		</tr>
		<tr>
			<td>Nephrin</td>
			<td>GP-N2</td>
			<td>Progen</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT4</td>
			<td>AF1759</td>
			<td>R&#38;D Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>PAX2</td>
			<td>71-6000</td>
			<td>Invitrogen</td>
			<td></td>
		</tr>
		<tr>
			<td>WT1</td>
			<td>MAB4234</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM Molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iMatrix-511 Laminin-E8 (LM-E8) fragment</td>
			<td>N-892012</td>
			<td>Iwai North America</td>
			<td>Basement membrane (BM) matrix 2</td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified matrix, 5-mL vial</td>
			<td>354277</td>
			<td>BD Biosciences</td>
			<td>Basement membrane (BM) matrix 1. May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Enzymes and Other Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>A1110501</td>
			<td>Thermo/Life Technologies</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>A9418</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>D2438</td>
			<td>Sigma-Aldrich</td>
			<td>DMSO is toxic. Should be handled in chemical safety hood</td>
		</tr>
		<tr>
			<td>Enzyme-free cell dissociation buffer, Hank&#8217;s balanced salt</td>
			<td>13150016</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol solution, 70% (vol/vol), biotechnology grade</td>
			<td>97065-058</td>
			<td>VWR</td>
			<td>Ethanol is flammable and toxic</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>431097</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>28906</td>
			<td>Thermo/Life Technologies</td>
			<td>PFA should be handled in a chemical fume hood with proper personal protection equipment, including gloves, lab coat, and safety eye glasses. Avoid inhalation and contact with skin.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>14190-250</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Distilled Water</td>
			<td>15230162</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>97062-208</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05%</td>
			<td>25300-120</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirating pipettes, individually wrapped</td>
			<td>29442-462</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-15R Centrifuge</td>
			<td>B99516</td>
			<td>Beckman Coulter</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 15 mL</td>
			<td>352097</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 50 mL</td>
			<td>352098</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoboxes</td>
			<td>3395465</td>
			<td>Thermo/Life Technologies</td>
			<td>For storing frozen aliquots</td>
		</tr>
		<tr>
			<td>EVOS M7000</td>
			<td>AMF7000</td>
			<td>Thermo/Life Technologies</td>
			<td>Flourescent microscope used to acquire images of fixed and stained iPS cells and their derivatives</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>100503-092</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell VIOS 160i CO2 incubator</td>
			<td>51030403</td>
			<td>Thermo/Life Technologies</td>
			<td>For the routine culture and maintenace of iPS cells and their derivatives</td>
		</tr>
		<tr>
			<td>Inverted Zeiss Axio Observer equipped with AxioCam 503 camera</td>
			<td>491916-0001-000(microscope) ; 426558-0000-000(camera)</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Used to acquire phase contrast images of live iPS cells and their derivatives at each stage of podocyte differentiation</td>
		</tr>
		<tr>
			<td>Kimberly-Clark nitrile gloves</td>
			<td>40101-346</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, large</td>
			<td>21905-049</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, small</td>
			<td>21905-026</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 precision barrier pipette tips</td>
			<td>P1096-FR</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P100 barrier pipette tips</td>
			<td>P1125</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 barrier pipette tips</td>
			<td>P1126</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 barrier pipette tips</td>
			<td>P1121</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 barrier pipette tips</td>
			<td>P1122</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 10 mL, individually wrapped</td>
			<td>356551</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 25 mL, individually wrapped</td>
			<td>356525</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 5 mL, individually wrapped</td>
			<td>356543</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip, 0.22 &#956;m, PES</td>
			<td>SCGP00525</td>
			<td>EMD Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Microcentrifuge Tubes</td>
			<td>1138W14</td>
			<td>Thomas Scientific</td>
			<td>For aliquoting growth factors</td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated 12-well plates</td>
			<td>353043</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated six-well plates</td>
			<td>353046</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR white techuni lab coat</td>
			<td>10141-342</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-beveled cell lifter</td>
			<td>3008</td>
			<td>Corning</td>
			<td></td>
		</tr>
	</tbody>
</table>

guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions

Nierziekte treft meer dan 10% van de wereldbevolking en kost miljarden dollars aan federale uitgaven. De meest ernstige vormen van nierziekte en uiteindelijk eindstadium nierfalen worden vaak veroorzaakt door de schade aan de glomerulaire podocyten, de zeer gespecialiseerde epitheelcellen die samen met endotheelcellen en het glomerulaire keldermembraan functioneren om de filtratiebarrière van de nier te vormen. Vooruitgang in de niergeneeskunde is gehinderd door de beperkte beschikbaarheid van primaire weefsels en het gebrek aan robuuste methoden voor de afleiding van functionele menselijke niercellen, zoals podocyten. Het vermogen om podocyten af te leiden uit hernieuwbare bronnen, zoals stamcellen, kan helpen het huidige begrip van de mechanismen van menselijke nierontwikkeling en -ziekte te bevorderen, en nieuwe hulpmiddelen bieden voor therapeutische ontdekking. Het doel van dit protocol was om een methode te ontwikkelen om volwassen, post-mitotische podocyten af te leiden uit door de mens geïnduceerde pluripotente stam (hiPS) cellen met een hoge efficiëntie en specificiteit, en onder chemisch gedefinieerde omstandigheden. De van hiPS-cellen afgeleide podocyten die door deze methode worden geproduceerd, drukken afstammingsspecifieke markers uit (waaronder nefrine, podocine en Wilm's Tumor 1) en vertonen de gespecialiseerde morfologische kenmerken (inclusief primaire en secundaire voetprocessen) geassocieerd met volwassen en functionele podocyten. Intrigerend genoeg zijn deze gespecialiseerde kenmerken met name afwezig in de onsterfelijke podocytencellijn die veel in het veld wordt gebruikt, wat suggereert dat het hierin beschreven protocol menselijke nierpodocyten produceert die een ontwikkelingsrijp fenotype hebben dan de bestaande podocytcellijnen die meestal worden gebruikt om de menselijke nierbiologie te bestuderen.

Het doel van dit protocol was om aan te tonen dat volwassen menselijke podocyten kunnen worden afgeleid van hiPS-cellen onder chemisch gedefinieerde omstandigheden. De gegevens die in dit manuscript worden gepresenteerd, werden gegenereerd met behulp van de DU11 hiPS-cellijn17, die eerst werden getest op en vrij bleken te zijn van mycoplasma. Chromosomale analyse werd ook uitgevoerd en de cellen bleken karyotypisch normaal te zijn. Beginnend met de ongedifferentieerde DU11 hiPS-cellen, werd de dif...

Watch this Scientific Journal Video about Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions at JoVE.com

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Hier wordt een chemisch gedefinieerd protocol gepresenteerd voor de afleiding van menselijke nierpodocyten uit geïnduceerde pluripotente stamcellen met een hoge efficiëntie (>90%) en onafhankelijk van genetische manipulaties of subpopulatieselectie. Dit protocol produceert het gewenste celtype binnen 26 dagen en kan nuttig zijn voor nefrotoxiciteitstests en ziektemodellering.

Geleide differentiatie van rijpe nierpodocyten uit door de mens geïnduceerde pluripotente stamcellen onder chemisch gedefinieerde omstandigheden

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Protocols for the differentiation of stem cells into functional kidney cells remain elusive. This method produces mature podocytes with specificity and efficiency, providing new tools for studying kidney disease mechanisms. This method employs cell culture medium and extracellular matrix proteins with a chemically-defined composition to produce human iPS cell derived podocytes with a high purity without using subpopulation selection or genetic manipulation.There are limited therapeutic options for patients with kidney disease. This method has implications for modeling and understanding disease mechanisms, which could facilitate the development of novel biomarkers and therapeutics. To set up a feeder-free human iPSC culture, aspirate the residual solution of basement membrane matrix 1 from pre-coated plates and wash the wells three times with one to two milliliters per well of warmed basal medium.Aspirate the spent cell culture medium from a human iPSC culture and rinse the cells three times with warmed medium. Add one milliliter of warm cell detachment solution to each well for a one-minute incubation at 37 degrees Celsius. After confirming rounded cell colony edges under a tissue culture microscope, quickly aspirate the cell detachment solution and gently rinse the cells with cell culture medium.Add three milliliters of human iPSC culture medium to the human-induced pluripotent stem cells and use a cell lifter to scrape the colonies. Gently pipette the cell suspension to dislodge any loosely adhered cells and wash the plate thoroughly to ensure harvesting of all of the cells. Transfer 500 microliters of cells into each well of a new basement membrane matrix 1 coated six-well plate containing two milliliters of human-induced pluripotent stem cells per well and move the plate in a figure eight fashion to evenly distribute the cell colonies within the wells.Place the plate in the cell culture incubator, refreshing the medium daily until the cells reach approximately 70%confluency. If necessary, aseptically scrape off areas of differentiation before aspirating the supernatant from the wells. After washing the wells three times, dissociate the cells as demonstrated and pull the resulting cell suspensions in a 15 milliliter conical tube.After mixing by pipetting, bring the final volume in the tube to 15 milliliters with warm medium and collect the cells by centrifugation. Resuspend the pellet in fresh medium for a second centrifugation and resuspend the cells in one milliliter of mesoderm induction medium for counting. After counting, resuspend the cells to a one times 10 to the fifth cells per milliliter of mesoderm induction medium concentration and aspirate the extracellular matrix solution from the basement membrane matrix 2 coated plates.Rinse the plates two times with warm medium and mix the human-induced pluripotent stem cell suspension with gentle pipetting. Add one milliliter of cells to each well of the basement membrane matrix 2 coated 12-well plates and gently shake the plates to distribute the cells more evenly. Then place the plate into the cell culture incubator.On days 2-15 of the differentiation, replace the mesoderm induction medium with one milliliter of intermediate mesoderm induction medium per well. If substantial cell growth and a rapid depletion of nutrients is observed as indicated by yellowing of the medium, the volume of the intermediate mesoderm differentiation medium can be increased to 1.3 milliliters per well. On day 16 of culture, rinse the intermediate mesoderm cells with warm medium and incubate the cells with 500 microliters of 0.05%Trypsin EDTA per well for three minutes at 37 degrees Celsius.When the cells begin to dissociate, scrape the cells with a cell lifter and gently mix the cells by pipetting. Stop the reaction with about two milliliters of Trypsin neutralizing solution per well and transfer the cells to a 50 milliliter conical tube. Bring the volume up to 50 milliliters with medium and collect the cells by centrifugation.Resuspend the pellet in podocyte induction medium at a one times 10 to the fifth cells per milliliter of medium concentration and add the cells to basement membrane matrix 2 coated plates. Then gently shake the plate to help distribute the cells more evenly and place the cells into the incubator for up to five days. Using the culture strategy as demonstrated, the stem cells can be first differentiated into mesoderm cells that express Brachyury, followed by differentiation into PAX2 positive intermediate mesoderm cells, and eventually mature kidney glomerular podocytes.Stem cell derived podocytes stain positive for WT1, as well as for the lineage identification marker nephrin. Intriguingly, the subcellular localization of nephrin is predominantly within the podocyte foot processes and cell cytoplasm, consistent with a mature podocyte phenotype. Cells seeded at densities that significantly exceed the recommended density of one times 10 to the fifth cells per well of a 12-well plate result in large clusters of cells that lack the expected morphological phenotype of mature podocytes within the standard timeline of the protocol.Seeding at the densities recommended in this protocol, however, results in cultures of cells with the desired morphology. We recommend using the appropriate basement membrane protein for cell detachment and maintaining the correct cell seeding density for mesoderm and podocyte induction. This podocyte differentiation method can be integrated with organs-on-chips microfluidic systems or 3D bioprinting technologies to develop functional models of the human kidney for drug screening and nephrotoxicity testing.The lack of appropriate cell culture models has impeded progress in understanding kidney disease mechanisms. This protocol provides researchers with an inexhaustible source of patient-derived podocytes for disease modeling.

Research

JoVE Journal

Bioengineering

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

Ontwikkeling, uitbreiding en<em&gt; In vivo</em&gt; Controle van Human NK cellen uit menselijke embryonale stamcellen (hESC &#39;s) en en geïnduceerde pluripotente stamcellen (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Transplantatie van geïnduceerde pluripotente stamcel-afgeleide mesoangioblast-achtige myogene voorlopers in muismodellen van spierregeneratie

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Isolatie van mesenchymale stamcellen uit menselijke alveolaire beenvlies en effecten van vitamine D op Osteogenic activiteit van beenvlies-afgeleide cellen

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

Menselijke pluripotente stamcel cultuur op Polyvinyl Alcohol-Co-itacon zuur Hydrogels met verschillende stijfheid onder voorwaarden Xeno-gratis

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Isolatie van volwassen menselijke dermale fibroblasten uit de buikhuid en de aanmaak van geïnduceerde pluripotente stamcellen met behulp van een niet-integrerende methode

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

Schaalbare generatie van volwassen cerebellar organoïden uit menselijke pluripotente stamcellen en karakterisering door immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Eencellige optische actiepotentiaalmeting in door de mens geïnduceerde pluripotente van stamcellen afgeleide cardiomyocyten

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblast afgeleide human engineered bindweefsel voor screeningstoepassingen

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Isogene nierglomeruluschip ontworpen uit door mensen geïnduceerde pluripotente stamcellen

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

Generatie van menselijke bloedvatorganoïden uit pluripotente stamcellen

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...