Die Autoren bedanken sich bei Melinda Hexum zur Initiierung der Spin EB Protokoll danken in unserem Labor. Wir möchten an die anderen Mitglieder des Labors, darunter Laura E. Bendzick, Michael Lepley und Zhenya Ni für ihre technische Unterstützung bei dieser Arbeit danken. Die Autoren möchte auch Brad Taylor bei Caliper Life Sciences danken für seine kompetente technische Beratung.

<ol>
	<li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+embryonic+stem+cells.">In vitro differentiation of embryonic stem cells.</a> Current Opinion in Cell Biology. 7, 862-869 (1995).</li><li>Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+colony-forming+cells+derived+from+human+embryonic+stem+cells.">Hematopoietic colony-forming cells derived from human embryonic stem cells.</a> Proceedings of the National Academy of Sciences. 98, 10716-10721 (2001).</li><li>Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+clinical+therapies+using+hematopoietic+cells+derived+from+human+pluripotent+stem+cells.">Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells.</a> Blood. 114, 3513-3523 (2009).</li><li>Ng, E. S., Davis, R., Stanley, E. G., Elefanty, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+describing+the+use+of+a+recombinant+protein-based,+animal+product-free+medium+(APEL)+for+human+embryonic+stem+cell+differentiation+as+spin+embryoid+bodies.">A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies.</a> Nature Protocols. 3, 768-776 (2008).</li><li>Ng, E. S., Davis, R. P., Azzola, L., Stanley, E. G., Elefanty, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forced+aggregation+of+defined+numbers+of+human+embryonic+stem+cells+into+embryoid+bodies+fosters+robust,+reproducible+hematopoietic+differentiation.">Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation.</a> Blood. 106, 1601-1603 (2005).</li><li>Vodyanik, M. A., Bork, J. A., Thomson, J. A., Slukvin, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+CD34++cells:+efficient+production+in+the+coculture+with+OP9+stromal+cells+and+analysis+of+lymphohematopoietic+potential.">Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential.</a> Blood. 105, 617-626 (2005).</li><li>Woll, P. 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=Human+embryonic+stem+cells+differentiate+into+a+homogeneous+population+of+natural+killer+cells+with+potent+in+vivo+antitumor+activity.">Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity.</a> Blood. 113, 6094-6101 (2009).</li><li>Woll, P. S., Martin, C. H., Miller, J. S., Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+NK+cells+acquire+functional+receptors+and+cytolytic+activity.">Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.</a> Journal of Immunology. 175, 5095-5103 (2005).</li><li>Ni, Z., 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=Human+Pluripotent+Stem+Cells+Produce+Natural+Killer+Cells+That+Mediate+Anti-HIV-1+Activity+by+Utilizing+Diverse+Cellular+Mechanisms.">Human Pluripotent Stem Cells Produce Natural Killer Cells That Mediate Anti-HIV-1 Activity by Utilizing Diverse Cellular Mechanisms.</a> Journal of Virology. 85, 43-50 (2011).</li><li>Ng, E. S., Davis, R. P., Hatzistavrou, T., Stanley, E. G., Elefanty, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+as+spin+embryoid+bodies+and+a+description+of+the+hematopoietic+blast+colony+forming+assay.">Directed differentiation of human embryonic stem cells as spin embryoid bodies and a description of the hematopoietic blast colony forming assay.</a> Current Protocols in Stem Cell Biology. Chapter 1, Unit 1D.3 (2008).</li><li>Denman, C. 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=Membrane-Bound+IL-21+Promotes+Sustained+Ex+Vivo+Proliferation+of+Human+Natural+Killer+Cells.">Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells.</a> PLoS ONE. 7, e30264 (2012).</li><li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion,+Purification,+and+Functional+Assessment+of+Human+Peripheral+Blood+NK+Cells.">Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells.</a> J. Vis. Exp. (48), e2540 (2011).</li><li>Tian, X., 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=Bioluminescent+imaging+demonstrates+that+transplanted+human+embryonic+stem+cell-derived+CD34++cells+preferentially+develop+into+endothelial+cells.">Bioluminescent imaging demonstrates that transplanted human embryonic stem cell-derived CD34+ cells preferentially develop into endothelial cells.</a> Stem Cells. 27, 2675-2685 (2009).</li><li>Wilber, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+stable+transgene+expression+in+human+embryonic+stem+cells+using+transposon-mediated+gene+transfer.">Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated gene transfer.</a> Stem Cells. 25, 2919-2927 (2007).</li><li>Shcherbo, 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=Near-infrared+fluorescent+proteins.">Near-infrared fluorescent proteins.</a> Nature Methods. 7, 827-829 (2010).</li><li>Nguyen, V. T., Morange, M., Bensaude, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firefly+luciferase+luminescence+assays+using+scintillation+counters+for+quantitation+in+transfected+mammalian+cells.">Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells.</a> Analytical Biochemistry. 171, 404-408 (1988).</li><li>Sadikot, R. T., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+Imaging.">Bioluminescence Imaging.</a> Proceedings of the American Thoracic Society. 2, 537-540 (2005).</li><li>Santos, E. B., 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=Sensitive+in+vivo+imaging+of+T+cells+using+a+membrane-bound+Gaussia+princeps+luciferase.">Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase.</a> Nature Medicine. 15, 338-344 (2009).</li><li>Chudakov, D. M., Matz, M. V., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Proteins+and+Their+Applications+in+Imaging+Living+Cells+and+Tissues.">Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues.</a> Physiological Reviews. 90, 1103-1163 (2010).</li><li>Knorr, D. A., Bock, A., Brentjens, R. J., Kaufman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+Human+Embryonic+Stem+Cell-Derived+Lymphocytes+to+Study+In+Vivo+Trafficking+and+Immunotherapy.">Engineered Human Embryonic Stem Cell-Derived Lymphocytes to Study In Vivo Trafficking and Immunotherapy.</a> Stem Cells Dev. 28, 28 (2013).</li><li>Germain, R. N., Robey, E. A., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Decade+of+Imaging+Cellular+Motility+and+Interaction+Dynamics+in+the+Immune+System.">A Decade of Imaging Cellular Motility and Interaction Dynamics in the Immune System.</a> Science. 336, 1676-1681 (2012).</li></ol>

Diese Arbeit wurde vom NIH / NHLBI R01-HL77923 (DSK) und NIH MSTP Zuschuss T32 GM008244 (DAK), Stem Cell Biology Ausbildungsförderung T32 (DAK) (T32HD060536), Undergraduate Research Opportunities Program (UROP) Grant, University of Minnesota unterstützt (AMB), der Leukemia Research Fund der University of Minnesota Cancer Center, und der William L und Blanche Hughes Foundation.

hESCs sind eine ideale Plattform, um verschiedene Zelltypen zu studieren und halten bemerkenswerte Potenzial für die klinische Übersetzung. Wir verwenden eine definierte, Spin EB Ansatz hESC / iPSCs auf hämatopoetischen Vorläuferzellen zu differenzieren. Der Spin EB Ansatz konsistente Ableitung von hämatopoetischen Vorläuferzellen und Differenzierung auf NK-Zellen ergab, doch, Variation existiert noch in Differenzierung Effizienz in Zelllinien und müssen für die Erzeugung von anderen hämatopoetischen Zelllinien...

Menschliche embryonale Stammzellen (hES) und induzierten pluripotenten Stammzellen (iPS-Zellen) sind undifferenzierte, pluripotente Zellen, die sich unbegrenzt Selbsterneuerung und Differenzierung multi-Linie. hESCs wurden erfolgreich in reifen und funktionale Untergruppen von jedem Keimblatt, einschließlich Zellen des blutbildenden Systems 1-3 unterschieden. Natürliche Killerzellen (NK)-Zellen sind Lymphozyten des angeborenen Immunsystems, von HES durch Bildung von embryoid bodies (EBs) 4,5 oder Co-Kultur mit Zelllinien 1,2,6-8 Stromazellen abgeleitet werden. NK-Zellen besitzen anti-virale und anti-Tumor-Fähigkeiten und das Potential haben, sich gegen ein breites Spektrum von Tumoren wirksam, da sie keiner vorherigen Antigen-Stimulation zu ihren Effektorfunktionen durchzuführen. Somit sind hESC abgeleiteten NK-Zellen eine attraktive Quelle von Zellen für die Immuntherapie. Zusätzlich stellt die Ableitung von NK-Zellen aus HES ein genetisch zugänglich, um den normalen Entwicklung zu studieren &lt;em&gt; in vitro. Weil sie eine genetisch manipulierbaren System bereitzustellen, kann hESC abgeleiteten NK Zellen experimentell modifiziert werden, um Fluoreszenz-und Biolumineszenz Reporter exprimieren einen optimalen Modell NK-Zell-Effektorfunktionen in vitro und in vivo zu studieren. hESC abgeleiteten NK-Zellen besitzen Wirkung gegen eine Reihe von Zielen, einschließlich HIV 9, Leukämie (K562) und andere Krebsarten 7,8. Jedoch ist die Fähigkeit, effizient ableiten genug NK Zellen, zur Behandlung von Patienten vor ein erhebliches Hindernis für die klinische Übersetzung und zu einem geringeren Grad, eine Beschränkung für umfangreiche präklinischen in vivo-Untersuchungen der NK-Zell-Entwicklung und Anti-Krebs-Funktionen. Hier verwenden wir ein Spin EB Ansatz zur Ableitung von hämatopoetischen Vorläuferzellen aus hESCs 4,5,10. Nach 11 Tage der Spin EBs sind NK-Zell-Kultur mit oder ohne Feeder für 28 Tage übertragen. Nach 4 Wochen in NK Zellkulturmedium, NK-Zellen transferred zu Co-Kultur mit K562-Zellen modifiziert werden, um membrangebundene Interleukin 21 (IL-21), die als künstliche Antigen präsentierende Zellen (aAPCs) dienen auszudrücken. Anpassung ein Protokoll für den Ausbau der peripheren Blut NK-Zellen mit diesen künstlichen APCs 11,12, sind wir in der Lage zu expandieren NK-Zellen 2-Protokolle unter Beibehaltung einer reifen Phänotyp und zytotoxische Fähigkeiten. Dieser Prozess der Entwicklung und Expansion bietet ausreichend hESC abgeleiteten NK-Zellen für umfangreiche in vivo Charakterisierung. Für in-vivo-Studien, können wir nicht-invasiv überwacht langfristig Transplantation und die Kinetik der injizierten Glühwürmchenluciferase sind Ausdruck (Fluc +), hESC abgeleiteten NK-Zellen mit Biolumineszenz-Bildgebung. Darüber hinaus sind wir in der Lage, NK-Zell-Interaktionen mit Tumorzellen mit einem Dual, Biolumineszenz oder Fluoreszenz-Imaging-Schema folgen. Eine frühere Studie von unserer Gruppe verwendet Biolumineszenz-Bildgebung in einem Anti-Tumor-Modell bis zur Tumorprogression und cle folgenarance von Fluc + K562-Zellen in vivo 7. Nun, von Engineering bis hESCs unsere ausdrückliche Glühwürmchenluciferase 13,14 können wir die biologische Verteilung und Handel von NK Zellen K562 Tumorzellen, die das kürzlich charakterisierten fluoreszierendes Protein exprimieren turboFP650 15 folgen. Wir haben dieses duale System Reporter ausgewählt, um gleichzeitig folgen die beiden Zellpopulationen in vivo (Abbildung 1). Die meisten Dual Imaging Modelle haben Dual-Luciferase-Systeme, aber diese Systeme technisch eine Herausforderung aufgrund der Lieferbedingungen von Coelenterazin, das Substrat für die Expression der meisten Renilla Luciferase Reporter und Gaussia 16-18 erforderlich. Fluorescent Reporter haben eine einfache Überwachung von vielen Zelllinien und Konstrukte in vitro erlaubt, hat aber nur begrenzten Erfolg hatten in-vivo-Bildgebung aufgrund der Überlappung zwischen Gewebe und Fell Autofluoreszenz und Emissionsspektren von vielen commonly verwendet fluoreszierenden Reporter einschließlich GFP, DsRed und tdTomato 15,19. Diese Sorge hat die Entwicklung der weit rot fluoreszierende Proteine, die für eine bessere Gewebe Penetranz und höhere spezifische Signal im Vergleich zu 15,19 Hintergrund erlauben gefördert. TurboFP650, das fluoreszierende Protein in diesem System gezeigt, ist weit-Rot verschoben und überwindet viele der Probleme, mit bildgebenden fluoreszierenden Proteinen in lebenden Tieren. Dieses Verfahren für die Entwicklung und den Ausbau von NK-Zellen hESCs abgeleitet hat uns erlaubt, weiter zu charakterisieren hESC abgeleiteten NK-Zellen in vitro und in vivo, die erforderlich sind, um besser zu verstehen, NK-Zell-Funktion und klinisch wichtig, aktuelle NK-Zell-Therapien Adoptiveltern zu verbessern. Es ist auch offen für die Ableitung und Erweiterung iPSC abgeleiteten NK-Zellen. Das duale Fluoreszenz und Biolumineszenz-Imaging-System ist breit anwendbar auf andere Systeme als die Anti-Tumor-Modell, das wir hier gezeigt haben.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>BPEL media for Spin EB generation</td>
		</tr>
		<tr>
			<td>Iscove's Modified Dulbecco's Medium (IMDM)</td>
			<td>Fisher Scientific</td>
			<td>SH3022801</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F-12 Nutrient Mixture w/ Glutamax I</td>
			<td>Invitrogen</td>
			<td>31765035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A3311</td>
			<td>Commercially available BSA can be cytotoxic to ES cells. Deionizing the solution can reduce the potential for cytotoxicity.</td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol)</td>
			<td>Sigma-Aldrich</td>
			<td>P8136</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Linoleic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L1012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Linolenic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>L2376</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Synthechol 500X solution</td>
			<td>Sigma-Aldrich</td>
			<td>S5442</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>a-monothioglycerol (a-MTG)</td>
			<td>Sigma-Aldrich</td>
			<td>S5442</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protein-free hybridoma mix II</td>
			<td>Invitrogen</td>
			<td>12040077</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ascorbic Acid 2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutamax I</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Insulin, Transferrin, Selenium 100X solution (ITS)</td>
			<td>Invitrogen</td>
			<td>41400-045</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>NK Differentiation Media </td>
		</tr>
		<tr>
			<td>Dubecco's Modified Eagle Medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>11965-118</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F-12 Media</td>
			<td>Invitrogen</td>
			<td>CX30315</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15% Human AB serum</td>
			<td>Valley Biomed</td>
			<td>HP1022HI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 ng/ml Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 uM ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>E9508</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20 ng/ml ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25 uM 2-mercaptoethanol (BME)</td>
			<td>Gibco</td>
			<td>21985</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2 mM L-glutamine</td>
			<td>Gibco</td>
			<td>CX30310</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1% Pen-Strep</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Cytokines</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>(all cytokines used fresh from frozen aliquots)</td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>PeproTech, Inc.</td>
			<td>300-07</td>
			<td>40 ng/ml in BPEL media; 20 ng/ml in NK medium). As noted by the Elefanty protocol, and as we discovered with a bad lot of BMP4, there can be lot differences with cytokines in regards to hematopoietic differentiation.Especially if buying cytokines in bulk, obtain a sample of the lot to test prior to purchase. Compare head-to-head with old lot.</td>
		</tr>
		<tr>
			<td>rhBMP-4</td>
			<td>R &amp;D Systems</td>
			<td>314-BP</td>
			<td>20 ng/ml in BPEL media</td>
		</tr>
		<tr>
			<td>rhVEGF</td>
			<td>R&amp;D Systems</td>
			<td>293-VE</td>
			<td>20 ng/ml in BPEL media</td>
		</tr>
		<tr>
			<td>IL-2</td>
			<td>PeproTech, Inc.</td>
			<td>200-02</td>
			<td>1 x 105 U/ml given to mice after NK cell injection; 50 U/ml used in NK cell expansion protocol</td>
		</tr>
		<tr>
			<td>IL-15</td>
			<td>PeproTech, Inc.</td>
			<td>200-15</td>
			<td>10 ng/ml given to mice after NK cell injection (first 7 days only)</td>
		</tr>
		<tr>
			<td>IL-3</td>
			<td>PeproTech, Inc.</td>
			<td>200-03</td>
			<td>5 ng/ml in NK media</td>
		</tr>
		<tr>
			<td>IL-7</td>
			<td>PeproTech, Inc.</td>
			<td>200-07</td>
			<td>20 ng/ml in NK media</td>
		</tr>
		<tr>
			<td>Flt-3-Ligand</td>
			<td>PeproTech, Inc.</td>
			<td>300-19</td>
			<td>10 ng/ml in NK media</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>In vivo Imaging</td>
		</tr>
		<tr>
			<td>D-Luciferin Sodium Salt</td>
			<td>Gold BioTechnology</td>
			<td>Lucna-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TurboFP650 plasmid</td>
			<td>Evrogen, Moscow, Russia</td>
			<td>FP731</td>
			<td>Subcloned into a Sleeping Beauty transposon based plasmid driven by the mCAGs promoter. Cells were then sorted on their expression of turboFP650 by FACS. Cells and plasmids can be obtained from our lab.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>IVIS Spectrum Imaging System</td>
			<td>Caliper Life Sciences</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Cells</td>
		</tr>
		<tr>
			<td>Membrane bound IL-21 expressing artificial antigen presenting cells</td>
			<td>MD Anderson, Houston, TX</td>
			<td>&nbsp;</td>
			<td>contact: Dean A. Lee. <a href="http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood" target="_blank">http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood</a></td>
		</tr>
		<tr>
			<td>Firefly luciferase expressing hESCs</td>
			<td>University of Minnesota, Minneapolis, MN</td>
			<td>&nbsp;</td>
			<td>contact: Dan S. Kaufman . H9 cells modified with a Sleeping Beauty transposon based method (references 13 and 14). Expression of firefly luciferase is driven by the mCAGGS promoter. Following the firefly luciferase gene is an IRES element at the 5' end of a GFP:zeocin fusion construct.</td>
		</tr>
		<tr>
			<td>TurboFP650 expressing K562 cells</td>
			<td>University of Minnesota, Minneapolis, MN</td>
			<td>&nbsp;</td>
			<td>contact: Dan S. Kaufman. Description under plasmid comments section</td>
		</tr>
	</tbody>
</table>
Materials Table.

development, expansion, and in vivo monitoring of human nk cells from human embryonic stem cells (hescs) and induced pluripotent stem cells (ipscs)

Wir präsentieren eine Methode zur Ableitung natürlichen Killerzellen (NK-Zellen) aus undifferenzierten hES und iPS mit einem Feeder-freien Ansatz. Diese Methode ergibt sich ein hohes Maß an NK-Zellen nach 4 Wochen Kultur und weitere 2-log Expansion mit künstlichen Antigen-präsentierenden Zellen zu unterziehen. hESC-und iPS-abgeleiteten NK-Zellen in diesem System entwickelt haben eine reife Phänotyp und Funktion. Die Produktion einer großen Anzahl von NK-Zellen genetisch veränderbar gilt sowohl für grundlegende mechanistische sowie Anti-Tumor-Studien. Expression der Firefly-Luciferase in hES-Zellen abgeleiteten NK ermöglicht eine nicht-invasive Ansatz zur NK-Zell-Transplantation, Verteilung und Funktion folgen. Wir beschreiben auch ein Dual-Bildgebungsverfahren, das eine getrennte Überwachung von zwei verschiedenen Zellpopulationen zu deutlicher charakterisieren ihre Wechselwirkungen in vivo ermöglicht. Diese Art der Ableitung, Expansion und zwei in-vivo-Bildgebung eine zuverlässige Methode zur Herstellung von NK-Zellen und deren ausgewertetation, die zur aktuellen NK-Zell-Therapien Adoptiveltern zu verbessern.

Die Erzeugung von hämatopoetischen Vorläuferzellen mit dem Spin EB Ansatz ermöglicht eine optimale NK-Zell-Entwicklung von hES und iPS-Zellen. Wie in 2 gezeigt, enthält Tag 11 Spin EBs hohe Anteile an Vorläuferzellen CD34 exprimieren, CD45, CD43, CD31 und. Hohe Konzentrationen von CD34 und CD45 ermöglicht die direkte Übertragung auf NK Bedingungen, ohne dass für die Sortierung oder die Unterstützung Stromazellen. Wenn es nicht optimal Spin EB Differenzierung, wird empfohlen, dass Stromazellen w...

Watch this Scientific Journal Video about Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs at JoVE.com

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

Dieses Protokoll beschreibt die Entwicklung, Expansion und in-vivo-Bildgebung von NK-Zellen aus hES-und iPS-Zellen abgeleitet.

Entwicklung, Erweiterung und<em&gt; In vivo</em&gt; Monitoring von humanen NK Zellen aus humanen embryonalen Stammzellen (hES-Zellen) und und induzierten pluripotenten Stammzellen (iPS-Zellen)

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 ...

development-expansion-vivo-monitoring-human-nk-cells-from-human

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)

16.7K Aufrufe.  University of Minnesota, Minneapolis. Dieses Protokoll beschreibt die Entwicklung, Expansion und in-vivo-Bildgebung von NK-Zellen aus hES-und iPS-Zellen abgeleitet.

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

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

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplantation von induzierten pluripotenten Stammzellen-abgeleiteten mesoangioblast-ähnlichen myogenen Vorläufern in Mausmodellen der Muskelregeneration

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

Forschung

Biotechnik

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Bestellen Einzelzellen und Einzel Embryos in 3D Confinement: Ein neues Gerät für High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Effiziente Erzeugung und Bearbeitung von Feeder-freie IPSCs aus menschlichen Zellen der Bauchspeicheldrüse mit dem CRISPR-Cas9-System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

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

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Isolierung von adulten menschlichen Dermal-Fibroblasten aus der Bauchhaut und Generierung induzierter pluripotenter Stammzellen mit einer nicht-integrierenden 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

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

Skalierbare Generierung von reifen Zerebellar-Organoiden aus humanen pluripotenten Stammzellen und Charakterisierung durch Immunostainierung

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

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

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Geführte Differenzierung reifer Nierenpodozyten aus human induzierten pluripotenten Stammzellen unter chemisch definierten Bedingungen

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

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

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Einzelzellige optische Aktionspotentialmessung in human induzierten pluripotenten Stammzell-abgeleiteten Kardiomyozyten

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

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

In vitro Induktion von menschlichen Zellpulpa-Stammzellen in Pankreaslinien

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasten-abgeleitetes Human Engineered Connective Tissue für Screening-Anwendungen

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

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 functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.