Los autores desean agradecer a Melinda Hexum para la iniciación del protocolo EB vuelta a nuestro laboratorio. Nos gustaría dar las gracias a otros miembros del laboratorio, incluyendo Laura E. Bendzick, Michael Lepley, y Zhenya Ni por su asistencia técnica con este trabajo. Los autores también desean agradecer a Brad Taylor en Caliper Life Sciences por su asesoramiento técnico especializado.

<ol>
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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. 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(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>

Este trabajo fue apoyado por el NIH / NHLBI-R01 HL77923 (DSK) y por el NIH subvención T32 GM008244 MSTP (DAK), Células Madre Biología Formación subvención T32 (DAK) (T32HD060536) Pregrado Programa de Oportunidades de Investigación (UROP) Grant, de la Universidad de Minnesota (AMB), el Fondo de Investigación de la Leucemia de la Universidad de Minnesota Cancer Center y la Fundación William L y Blanche Hughes.

hESCs son una plataforma ideal para estudiar diversos tipos de células y mantener un notable potencial para la traducción clínica. Utilizamos un enfoque EB giro definido para diferenciar células madre / iPSCs de células progenitoras hematopoyéticas. El enfoque EB giro ha dado derivación consistente de células progenitoras hematopoyéticas y la diferenciación a células NK, sin embargo, todavía existe variación en la eficiencia de la diferenciación a través de líneas de células y puede necesitar ser modifi...

Las células madre embrionarias humanas (hESCs) y células madre pluripotentes inducidas (iPSCs), son células indiferenciadas pluripotentes capaces de auto-renovación y multi-linaje diferenciación ilimitada. hESCs se han diferenciado con éxito en subconjuntos maduras y funcionales de cada capa germinal, incluyendo las células del sistema hematopoyético 1-3. Las células asesinas naturales (NK) son los linfocitos del sistema inmune innato que se puede derivar de hESCs por la formación de cuerpos embrioides (EB) de 4,5 o co-cultivo con líneas de células del estroma 1,2,6-8. Las células NK poseen capacidades anti-virales y anti-tumoral y tienen el potencial para ser eficaz contra una amplia gama de tumores malignos, ya que no requieren la estimulación de antígenos antes de llevar a cabo sus funciones efectoras. Por lo tanto, las células NK son células madre derivada de una atractiva fuente de células para la inmunoterapia. Además, la derivación de las células NK de hESCs proporciona un sistema genéticamente susceptibles a estudiar el desarrollo normal &lt;em&gt; in vitro. Debido a que proporcionan un sistema manejable de forma genética, las células NK células madre derivado experimentalmente pueden ser modificados para expresar reporteros fluorescentes y bioluminiscentes proporcionar un modelo óptimo para estudiar las funciones efectoras de células NK in vitro e in vivo. células NK hCME-derivados poseen actividad contra una amplia gama de objetivos, incluyendo el VIH 9, leucemia (K562) y otros tipos de cáncer 7,8. Sin embargo, la capacidad de derivar eficazmente suficientes células NK capaces de tratar a los pacientes sigue siendo un obstáculo importante para la traducción clínica y es, en menor grado, una limitación para una amplia pre-clínica en los estudios in vivo de desarrollo de las células NK y las funciones anti-cáncer. Aquí, utilizamos un enfoque EB giro para obtener progenitores hematopoyéticos de hESCs 4,5,10. Después de 11 días el giro EBs se transfieren al cultivo de células NK con o sin alimentadores para 28 días. Después de 4 semanas en medio de cultivo de las células NK, las células NK son transferred de co-cultivo con células K562 modificadas para expresar unido a la membrana interleucina 21 (IL-21), que sirven como células presentadoras de antígeno artificiales (AAPCS). Adaptación de un protocolo para la expansión de las células NK de sangre periférica utilizando estos vehículos blindados artificial 11,12, somos capaces de expandir las células NK 2-logs, manteniendo un fenotipo maduro y capacidades citotóxicas. Este proceso de desarrollo y expansión proporciona suficientes células NK células madre derivadas de una amplia caracterización in vivo. Para los estudios in vivo, que son capaces de controlar de forma no invasiva el injerto a largo plazo y la cinética de la inyección de la luciferasa de luciérnaga expresar (Fluc +), células NK células madre derivadas utilizando imágenes de bioluminiscencia. Además, somos capaces de seguir las interacciones de células NK con células tumorales utilizando un esquema de formación de imágenes de doble, bioluminiscente o fluorescente. Un estudio anterior de nuestro grupo utilizó imágenes de bioluminiscencia en un modelo anti-tumor para seguir la progresión del tumor y cleArance de células Fluc + K562 in vivo 7. Ahora, mediante la ingeniería nuestros hESCs para expresar luciferasa de luciérnaga 13,14 podemos seguir la biodistribución y el tráfico de las células NK a K562 células tumorales que expresan la proteína fluorescente recientemente caracterizado, turboFP650 15. Hemos elegido este sistema dual reportero con el fin de seguir simultáneamente las dos poblaciones de células in vivo (Figura 1). La mayoría de los modelos de doble imagen han sido los sistemas de doble luciferasa, pero estos sistemas pueden ser técnicamente difícil debido a los requisitos de entrega de coelenterazina, el sustrato necesario para la expresión de la mayoría de Renilla y Gaussia luciferase reporteros 16-18. Reporteros fluorescentes han permitido un fácil monitoreo de muchas líneas celulares y los constructos in vitro, pero ha tenido un éxito limitado para la formación de imágenes in vivo debido a la superposición entre el tejido y la autofluorescencia de piel y los espectros de emisión de muchos commonly utilizado reporteros fluorescentes como GFP, DsRed y tdTomato 15,19. Esta preocupación ha impulsado el desarrollo de las proteínas fluorescentes de rojo lejano, que permiten una mejor penetración del tejido y una mayor señal específica en comparación con el 15,19 de fondo. TurboFP650, la proteína fluorescente se muestra en este sistema, es desplazado rojo lejano y supera muchos de los problemas involucrados con las proteínas fluorescentes de imágenes de los animales que viven. Este método para el desarrollo y expansión de células NK derivadas de hESCs nos ha permitido caracterizar además células NK células madre derivado de in vitro e in vivo, lo que es necesario para comprender mejor la función de células NK y clínicamente importante para mejorar las terapias actuales adoptivos de células NK. También es susceptible a la derivación y la expansión de las células NK derivadas de IPSC. El esquema imágenes fluorescentes y bioluminiscentes dual es ampliamente aplicable a sistemas distintos del modelo antitumoral hemos demostrado aquí.

<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)

Se presenta un método para derivar las células asesinas naturales (NK) de hESCs indiferenciadas y iPSCs utilizando un enfoque de enlace de forma gratuita. Este método da lugar a altos niveles de células NK después de 4 semanas la cultura y puede someterse a una mayor expansión 2-registro con las células presentadoras de antígeno artificiales. células NK y células madre IPSC derivado desarrollados en este sistema tienen un fenotipo y función maduro. La producción de un gran número de células NK modificables genéticamente es aplicable tanto para mecanicista básica, así como los estudios antitumorales. La expresión de la luciferasa de luciérnaga en células madre derivadas de células NK permite un enfoque no invasivo para seguir el injerto de células NK, la distribución, y la función. También se describe un esquema de doble imagen que permite el monitoreo independiente de dos poblaciones celulares diferentes para caracterizar con más claridad sus interacciones in vivo. Este método de derivación, la expansión, y de doble en vivo de imágenes proporciona un enfoque fiable para la producción de células NK y su evaluaciónación que es necesaria para mejorar las terapias actuales adoptivos de células NK.

La generación de células progenitoras hematopoyéticas utilizando el enfoque de EB giro permite el desarrollo de las células NK óptima de hESCs y iPSCs. Como se demuestra en la Figura 2, día 11 espín EBs contienen altos porcentajes de células progenitoras que expresan CD34, CD45, CD43, y CD31. Los altos niveles de CD34 y CD45 permite la transferencia directa a las condiciones de NK sin necesidad de clasificación o el apoyo a las células del estroma. Si no es subóptima diferenciación EB giro, ...

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

Este protocolo describe el desarrollo, la expansión, y en la formación de imágenes in vivo de células NK derivadas de hESCs y iPSCs.

Desarrollo, la expansión y<em&gt; En vivo</em&gt; Monitoreo de las células NK humanas a partir de células madre embrionarias humanas (hESCs) y y células madre pluripotentes inducidas (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 ...

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 vistas.  University of Minnesota, Minneapolis. Este protocolo describe el desarrollo, la expansión, y en la formación de imágenes in vivo de células NK derivadas de hESCs y iPSCs.

Video: 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.

Trasplante De InducidaS Pluripotentes Derivadas De Células Madre Mesoangioblastos Como Progenitores Miógenos En Modelos De Ratón De La Regeneración Muscular

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

Investigación

Bioingeniería

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.

Ordenando las células individuales y solo los embriones en 3D Confinamiento: un nuevo dispositivo para la selección de alto contenido

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.

Eficiente generación y edición de IPSCs alimentador-libre de las células pancreáticas humanas utilizando el sistema CRISPR Cas9

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.

Aislamiento de fibroblastos dérmicos humanos adultos de la piel abdominal y generación de células madre pluripotentes inducidas utilizando un método no integrador

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.

Generación escalable de organoides cerebelosos maduros a partir de células madre pluripotentes humanas y caracterización por inmunosuización

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.

Diferenciación guiada de podocitos renales maduros de células madre pluripotentes inducidas por humanos en condiciones químicamente definidas

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.

Medición del potencial de acción óptica unicelular en cardiomiocitos derivados de células madre pluripotentes inducidas por humanos

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 Inducción de células madre de pulpa dental humana hacia linajes pancreáticos

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.

Tejido conectivo de ingeniería humana derivado de fibroblastos para aplicaciones de detección

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.