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

Dette arbeidet ble støttet av Pratt School of Engineering ved Duke University, Divisjon for nefrologi ved Duke Medical School, A Chair's Research Award fra Institutt for medisin ved Duke University, og en Burroughs Wellcome Fund PDEP Career Transition Ad Hoc Award til S.M.. M.B ble støttet av National Science Foundations Graduate Research Fellowship Program. Vi takker Bursac Lab for sjenerøst å gi oss DU11 stamcellelinjen, og Varghese Lab ved Duke University for midlertidig å dele deres vevskulturanlegg med gruppen vår. Denne publikasjonen er dedikert til prof. Laura L. Kiessling, Novartis professor i kjemi ved Massachusetts Institute of Technology, i feiringen av hennes 60-årsdag.

1. Utarbeidelse av reagenser
<ol><li>Fortynn tint 5x hiPS cellekulturmedier (CCM) supplement i hiPS cellekultur basal medium for å oppnå en 1x løsning av hiPS CCM. MERK: Frossen 5x hiPS CCM supplement krever en langsom opptiningsprosess, ideelt i 4 °C for natten. Aliquots av 1x hiPS CCM kan lagres i opptil 6 måneder ved -20 °C.</li>
	<li>Tilberedning av kjellermembran (BM) matrise 1-belagte plater for hiPS cellekultur: Tine BM matrise 1 over natten på is ved 4 °C. Når de er tint, lag aliquots med passende f...

<ol>
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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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I denne rapporten beskriver vi en protokoll for generering av nyreglomerulære podocytter fra hiPS-celler. HiPS celleavledede podocytter viser morfologiske og molekylære egenskaper forbundet med den modne nyre podocyttfenotypen13. I tidligere publikasjoner viste vi at hiPS-celleavledede podocytter kan etterligne strukturen og selektiv filtreringsfunksjonen til nyreglomerulus når de er kokulturert med glomerulære mikrovaskulære endotelceller i en parfymerbar mikrofluidisk organ-på-en-chip-enhe...

Fremskritt innen menneskelig pluripotent stamcellekultur er klar til å revolusjonere regenerativ medisin, sykdomsmodellering og legemiddelscreening ved å gi forskere en fornybar, skalerbar kilde til biologisk materiale som kan konstrueres for å oppnå nesten hvilken som helst celletype i menneskekroppen1. Denne strategien er spesielt nyttig for å utlede spesialiserte og funksjonelle celletyper som ellers ville være vanskelig å få tak i. Menneskeskapte pluripotente stamceller (hiPS)2,3,4,5 er spesielt attraktive på grunn av deres somatiske celleopprinnelse og potensialet de representerer for personlig medisin. Imidlertid er utviklingsmetoder for å utlede andre celleavstamninger fra hiPS-celler fortsatt utfordrende på grunn av hyppig bruk av dårlig definerte kulturforhold som fører til lav effektivitet og ikke-spesifikk generasjon av heterogene cellepopulasjoner6,7.
Presentert her er en metode for avledning av modne nyre podocytter fra hiPS celler med spesifisitet og høy effektivitet under kjemisk definerte forhold. Ved å vurdere rollene til flere faktorer i det cellulære mikromiljøet, ble det utviklet en stamcelledifferensieringsstrategi som involverte optimalisering av løselige faktorer presentert i cellekulturmediet, samt uoppløselige faktorer, for eksempel ekstracellulære matrisekomponenter eller limsubstrater. Gitt betydningen av integrinsk signalering i podocyttutvikling og funksjon, ble uttrykket av integrinske reseptorer på celleoverflaten først undersøkt. β1 integrins ble sterkt uttrykt ikke bare i hiPS-celler, men også i deres derivater, inkludert mesoderm og mellomliggende mesodermceller8,9,10. Påfølgende eksperimenter bekreftet at ligander som binder seg til β1 integrins (inkludert laminin 511 eller laminin 511-E8 fragment) støtter adhesjon og differensiering av hiPS-celler i podocytter når de brukes sammen med det løselige induktive mediet beskrevet nedenfor.
Induksjon av cellelinjeforpliktelse ble initiert ved først å bekrefte at hiPS-celler dyrket på lamininbelagte overflater i to dager i nærvær av et medium som inneholder Activin A, CHIR99021 og Y27632 Rock-hemmer kan skille seg ut i celler som uttrykker de tidlige mesodermmarkørene HAND1, goosecoid og brachy braury8,11. Behandling av mesodermcellene i 14 dager med et medium supplert med benmorfogenetisk protein 7 (BMP-7) og CHIR99021 gjorde det mulig å avlede mellomliggende mesodermceller som uttrykte nefro cellemarkører wilm's Tumor 1 (WT1), oddetallsrelatert protein 1 (OSR1)8,11og parret boksgen 2 protein (PAX2)12. For å utlede de modne nyreglomerulære podocyttene ble de mellomliggende mesodermcellene behandlet i 4-5 dager med et nytt medium bestående av BMP-7, Activin A, vaskulær endotel vekstfaktor (VEGF),all-trans retinoinsyre og CHIR99021. Flowcytometri og immunostaining ble brukt til å bekrefte at >90% av de resulterende cellene viste de molekylære, morfologiske og funksjonelle egenskapene til den modne nyrekapocytten8,11,13. Disse egenskapene inkluderer utvikling av primære og sekundære fotprosesser; uttrykk for podocyttlinjespesifikke gener, inkludert SYNPO, PODXL, MAF, EFNB28 og uttrykk for proteiner, inkludert podocin, nephrin og WT114,15,16. I tillegg ble det funnet at hiPS celle-avledede podocytter kan opprettholdes i kultur i opptil fire uker in vitro ved hjelp av et kommersielt tilgjengelig medium8,11 som gir en ekstra fleksibilitet i tidspunktet for nedstrøms eksperimenter. Hvis du vil ha mer informasjon om strømningscytometripanelene som brukes til å bestemme renheten til hiPS-podocyttene, kan du se vår forrige publikasjon11.

<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

Nyresykdom påvirker mer enn 10% av den globale befolkningen og koster milliarder av dollar i føderale utgifter. De mest alvorlige former for nyresykdom og eventuell nyresvikt i sluttfasen skyldes ofte skaden på de glomerulære podocyttene, som er de høyt spesialiserte epitelcellene som fungerer sammen med endotelceller og den glomerulære kjellermembranen for å danne nyrenes filtreringsbarriere. Fremskritt innen nyremedisin har blitt hindret av begrenset tilgjengelighet av primærvev og mangel på robuste metoder for avledning av funksjonelle menneskelige nyreceller, som podocytter. Evnen til å utlede podocytter fra fornybare kilder, som stamceller, kan bidra til å fremme dagens forståelse av mekanismene for menneskelig nyreutvikling og sykdom, samt gi nye verktøy for terapeutisk oppdagelse. Målet med denne protokollen var å utvikle en metode for å utlede modne, post-mitotiske podocytter fra menneskeskapte pluripotente stamceller (hiPS) med høy effektivitet og spesifisitet, og under kjemisk definerte forhold. HiPS celle-avledede podocytter produsert av denne metoden uttrykker avgrensningsspesifikke markører (inkludert nephrin, podocin og Wilms Tumor 1) og viser de spesialiserte morfologiske egenskapene (inkludert primære og sekundære fotprosesser) forbundet med modne og funksjonelle podocytter. Interessant nok er disse spesialiserte funksjonene merkbart fraværende i den udødeliggjorte podocyttcellelinjen som er mye brukt i feltet, noe som antyder at protokollen som er beskrevet her, produserer menneskelige nyrekapocytter som har en utviklingsmessig mer moden fenotype enn de eksisterende podocytcellelinjene som vanligvis brukes til å studere menneskelig nyrebiologi.

Målet med denne protokollen var å demonstrere at modne menneskelige podocytter kan avledes fra hiPS-celler under kjemisk definerte forhold. Dataene som presenteres i dette manuskriptet ble generert ved hjelp av DU11 hiPS cellelinje17, som først ble testet for, og funnet å være fri for mykoplasma. Kromosomal analyse ble også utført, og cellene ble funnet å være karyotypisk normale. Starter med de uavklarte DU11 hiPS-cellene, differensieringsstrategien (figur 1...

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

Presentert her er en kjemisk definert protokoll for avledning av humane nyre podocytter fra induserte pluripotente stamceller med høy effektivitet (>90%) og uavhengig av genetiske manipulasjoner eller subpopulation utvalg. Denne protokollen produserer ønsket celletype innen 26 dager og kan være nyttig for nefrotoksisitetstesting og sykdomsmodellering.

Veiledet differensiering av modne nyrekapocytter fra menneskeskapte pluripotente stamceller under kjemisk definerte forhold

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

Utvikling, utvidelse, og<em&gt; In vivo</em&gt; Overvåking of Human NK-celler fra humane embryonale stamceller (hESCs) og og Induced pluripotent stamceller (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

Transplantasjon av indusert pluripotent stamcelle-avledet Mesoangioblast-lignende myogene forfedre i musemodeller av muskelregenerering

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

Isolering av Mesenchymal stamceller fra menneskelige Alveolar Periosteum og effekter av Vitamin D på Osteogenic aktivitet i Periosteum-avledet celler

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

Menneskelige Pluripotent stamceller kultur på polyvinylacetat alkohol-Co-Itaconic syre Hydrogels med varierende stivhet Under Xeno-gratis forhold

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

Isolering av voksen Human dermal fibroblaster fra abdominal hud og generering av indusert Pluripotent stamceller ved hjelp av en ikke-integrering metode

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

Skalerbar generasjon av modne cerebellar organoider fra humane pluripotente stamceller og karakterisering ved immunstaining

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

Enkeltcellet optisk virkningspotensial måling i humane induserte pluripotente stamcelleavledede kardiomyocytter

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 avledet menneskekonstruert bindevev for screeningapplikasjoner

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

Isogen nyre glomerulus chip konstruert fra humant induserte pluripotente stamceller

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

Generering av humane blodkarorganoider fra pluripotente stamceller

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 ...