Name: Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs) (Video) | JoVE Journal
Uploaded: 2013-04-23T13:00:00.000+00:00
Duration: 9 min 2 s
Description: Learn to generate and expand human NK cells from hESCs and iPSCs using a feeder-free approach with defined cytokines. This protocol enables functional assays and dual in vivo imaging for tumor targeting studies.

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5 minutes

The authors would like to thank Melinda Hexum for initiation of the spin EB protocol within our lab. We would like to thank other members of the lab, including Laura E. Bendzick, Michael Lepley, and Zhenya Ni for their technical assistance with this work. The authors would also like to thank Brad Taylor at Caliper Life Sciences for his expert technical advice.

<ol><li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+differentiation+of+embryonic+stem+cells%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Hematopoietic+colony-forming+cells+derived+from+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">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/pubmed?term=((Toward+clinical+therapies+using+hematopoietic+cells+derived+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((A+protocol+describing+the+use+of+a+recombinant+protein-based%2C+animal+product-free+medium+%28APEL%29+for+human+embryonic+stem+cell+differentiation+as+spin+embryoid+bodies%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Forced+aggregation+of+defined+numbers+of+human+embryonic+stem+cells+into+embryoid+bodies+fosters+robust%2C+reproducible+hematopoietic+differentiation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Human+embryonic+stem+cell-derived+CD34%2B+cells%3A+efficient+production+in+the+coculture+with+OP9+stromal+cells+and+analysis+of+lymphohematopoietic+potential%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Human+embryonic+stem+cells+differentiate+into+a+homogeneous+population+of+natural+killer+cells+with+potent+in+vivo+antitumor+activity%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Human+embryonic+stem+cell-derived+NK+cells+acquire+functional+receptors+and+cytolytic+activity%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">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/pubmed?term=((Human+Pluripotent+Stem+Cells+Produce+Natural+Killer+Cells+That+Mediate+Anti-HIV-1+Activity+by+Utilizing+Diverse+Cellular+Mechanisms%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">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/pubmed?term=((Directed+differentiation+of+human+embryonic+stem+cells+as+spin+embryoid+bodies+and+a+description+of+the+hematopoietic+blast+colony+forming+assay%5BTitle%5D)+AND+%22Current+Protocols+in+Stem+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Membrane-Bound+IL-21+Promotes+Sustained+Ex+Vivo+Proliferation+of+Human+Natural+Killer+Cells%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Expansion%2C+Purification%2C+and+Functional+Assessment+of+Human+Peripheral+Blood+NK+Cells%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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/pubmed?term=((Bioluminescent+imaging+demonstrates+that+transplanted+human+embryonic+stem+cell-derived+CD34%2B+cells+preferentially+develop+into+endothelial+cells%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">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/pubmed?term=((Efficient+and+stable+transgene+expression+in+human+embryonic+stem+cells+using+transposon-mediated+gene+transfer%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">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/pubmed?term=((Near-infrared+fluorescent+proteins%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">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/pubmed?term=((Firefly+luciferase+luminescence+assays+using+scintillation+counters+for+quantitation+in+transfected+mammalian+cells%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Bioluminescence+Imaging%5BTitle%5D)+AND+%22Proceedings+of+the+American+Thoracic+Society%22%5BJournal%5D)">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/pubmed?term=((Sensitive+in+vivo+imaging+of+T+cells+using+a+membrane-bound+Gaussia+princeps+luciferase%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">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/pubmed?term=((Fluorescent+Proteins+and+Their+Applications+in+Imaging+Living+Cells+and+Tissues%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">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/pubmed?term=((Engineered+Human+Embryonic+Stem+Cell-Derived+Lymphocytes+to+Study+In+Vivo+Trafficking+and+Immunotherapy%5BTitle%5D)+AND+%22Stem+Cells+Dev%22%5BJournal%5D)">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/pubmed?term=((A+Decade+of+Imaging+Cellular+Motility+and+Interaction+Dynamics+in+the+Immune+System%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A Decade of Imaging Cellular Motility and Interaction Dynamics in the Immune System.</a> Science. 336, 1676-1681 (2012).</li>
</ol>

This work was supported by NIH/NHLBI R01-HL77923 (D.S.K.) and by NIH MSTP grant T32 GM008244 (D.A.K.), Stem Cell Biology Training Grant T32 (D.A.K.)(T32HD060536), Undergraduate Research Opportunities Program (UROP) Grant, University of Minnesota (A.M.B), the Leukemia Research Fund of the University of Minnesota Cancer Center, and the William L and Blanche Hughes Foundation.

hESCs are an ideal platform to study diverse cell types and hold remarkable potential for clinical translation. We use a defined, spin EB approach to differentiate hESC/iPSCs to hematopoietic progenitor cells. The spin EB approach has yielded consistent derivation of hematopoietic progenitor cells and differentiation to NK cells; yet, variation still exists in differentiation efficiency across cell lines and may need to be modified for generation of other hematopoietic cell lineages. While comparable results can be obtai...

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are undifferentiated, pluripotent cells capable of unlimited self-renewal and multi-lineage differentiation. hESCs have been successfully differentiated into mature and functional subsets of each germ layer, including cells of the hematopoietic system1-3. Natural killer (NK) cells are lymphocytes of the innate immune system that can be derived from hESCs by formation of embryoid bodies (EBs)4,5 or co-culture with stromal cell lines1,2,6-8. NK cells possess anti-viral and anti-tumor capabilities and have the potential to be effective against a broad range of malignancies, as they do not require prior antigen stimulation to perform their effector functions. Thus, hESC-derived NK cells are an attractive source of cells for immunotherapy. Additionally, the derivation of NK cells from hESCs provides a genetically amenable system to study normal development in vitro.
Because they provide a genetically tractable system, hESC-derived NK cells can be experimentally modified to express fluorescent and bioluminescent reporters providing an optimal model to study NK cell effector functions in vitro and in vivo. hESC-derived NK cells possess activity against a range of targets including HIV 9, leukemia (K562) and other cancer types 7,8. However, the ability to efficiently derive enough NK cells capable of treating patients remains an important barrier for clinical translation and is, to a lesser degree, a limitation for extensive pre-clinical in vivo studies of NK cell development and anti-cancer functions. Here, we use a spin EB approach to derive hematopoietic progenitors from hESCs 4,5,10. Following 11 days the spin EBs are transferred to NK cell culture with or without feeders for 28 days. After 4 weeks in NK cell culture medium, NK cells are transferred to co-culture with K562 cells modified to express membrane-bound interleukin 21 (IL-21), which serve as artificial antigen presenting cells (aAPCs). Adapting a protocol for the expansion of peripheral blood NK cells using these artificial APCs 11,12, we are able to expand NK cells 2-logs while retaining a mature phenotype and cytotoxic capabilities.
This process of development and expansion provides sufficient hESC-derived NK cells for extensive in vivo characterization. For in vivo studies, we are able to non-invasively monitor long term engraftment and kinetics of injected firefly luciferase expressing (Fluc+), hESC-derived NK cells using bioluminescence imaging. Furthermore, we are able to follow NK cell interactions with tumor cells using a dual, bioluminescent or fluorescent imaging scheme. An earlier study by our group used bioluminescence imaging in an anti-tumor model to follow tumor progression and clearance of Fluc+ K562 cells in vivo 7. Now, by engineering our hESCs to express firefly luciferase 13,14 we can follow the biodistribution and trafficking of NK cells to K562 tumor cells that express the recently characterized fluorescent protein, turboFP650 15. We have chosen this dual reporter system in order to simultaneously follow the two cell populations in vivo (Figure 1). Most dual imaging models have been dual-luciferase systems, but these systems can be technically challenging due to the delivery requirements of coelenterazine, the substrate required for expression of most Renilla and Gaussia luciferase reporters 16-18. Fluorescent reporters have allowed easy monitoring of many cell lines and constructs in vitro, but has had limited success for in vivo imaging due to the overlap between tissue and fur autofluorescence and the emission spectra of many commonly used fluorescent reporters including GFP, DsRed, and TdTomato 15,19. This concern has encouraged the development of far-red fluorescent proteins, which allow for better tissue penetrance and higher specific signal compared to background 15,19. TurboFP650, the fluorescent protein shown in this system, is far-red shifted and overcomes many of the issues involved with imaging fluorescent proteins in living animals.
This method for developing and expanding NK cells derived from hESCs has allowed us to further characterize hESC-derived NK cells in vitro and in vivo, which is necessary to better understand NK cell function and clinically important to improve current NK cell adoptive therapies. It is also amenable to the derivation and expansion of iPSC-derived NK cells. The dual fluorescent and bioluminescent imaging scheme is broadly applicable to systems other than the anti-tumor model we have shown here.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Media&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;BPEL media for Spin EB generation&lt;/strong&gt;</td></tr><tr><td>Iscove's Modified Dulbecco's Medium (IMDM)</td><td>Fisher Scientific</td><td>SH3022801</td><td></td></tr><tr><td>F-12 Nutrient Mixture w/ Glutamax I</td><td>Invitrogen</td><td>31765035</td><td></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></td></tr><tr><td>Linoleic Acid</td><td>Sigma-Aldrich</td><td>L1012</td><td></td></tr><tr><td>Linolenic Acid</td><td>Sigma-Aldrich</td><td>L2376</td><td></td></tr><tr><td>Synthechol 500X solution</td><td>Sigma-Aldrich</td><td>S5442</td><td></td></tr><tr><td>a-monothioglycerol (a-MTG)</td><td>Sigma-Aldrich</td><td>S5442</td><td></td></tr><tr><td>Protein-free hybridoma mix II</td><td>Invitrogen</td><td>12040077</td><td></td></tr><tr><td>Ascorbic Acid 2-phosphate</td><td>Sigma-Aldrich</td><td>A8960</td><td></td></tr><tr><td>Glutamax I</td><td>Invitrogen</td><td>35050061</td><td></td></tr><tr><td>Insulin, Transferrin, Selenium 100X solution (ITS)</td><td>Invitrogen</td><td>41400-045</td><td></td></tr><tr><td>Pen-Strep</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;NK Differentiation Media &lt;/strong&gt;</td></tr><tr><td>Dubecco's Modified Eagle Medium (DMEM)</td><td>Invitrogen</td><td>11965-118</td><td></td></tr><tr><td>F-12 Media</td><td>Invitrogen</td><td>CX30315</td><td></td></tr><tr><td>15% Human AB serum</td><td>Valley Biomed</td><td>HP1022HI</td><td></td></tr><tr><td>5 ng/ml Sodium Selenite</td><td>Sigma-Aldrich</td><td>S5261</td><td></td></tr><tr><td>50 uM ethanolamine</td><td>Sigma-Aldrich</td><td>E9508</td><td></td></tr><tr><td>20 ng/ml ascorbic acid</td><td>Sigma-Aldrich</td><td>A8960</td><td></td></tr><tr><td>25 uM 2-mercapt&amp;#8211;thanol (BME)</td><td>Gibco</td><td>21985</td><td></td></tr><tr><td>2 mM L-glutamine</td><td>Gibco</td><td>CX30310</td><td></td></tr><tr><td>1% Pen-Strep</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Cytokines&lt;/strong&gt;</td></tr><tr><td colspan="4">(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;amp; D Systems</td><td>314-BP</td><td>20 ng/ml in BPEL media</td></tr><tr><td>rhVEGF</td><td>R&amp;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 10&lt;sup&gt;5&lt;/sup&gt; 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 colspan="4">&lt;strong&gt;&lt;em&gt;In vivo&lt;/em&gt; Imaging&lt;/strong&gt;</td></tr><tr><td>D-Luciferin Sodium Salt</td><td>Gold BioTechnology</td><td>Lucna-500</td><td></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 colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>IVIS Spectrum Imaging System</td><td>Caliper Life Sciences</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Cells&lt;/strong&gt;</td></tr><tr><td>Membrane bound IL-21 expressing artificial antigen presenting cells</td><td><a href="http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood" target="_blank"><img src="/img/new_window.png"> MD Anderson, Houston, TX</a></td><td></td><td>contact: Dean A. Lee. &lt;a href="http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood" target="_blank"&gt;http://www.jove.com/video/2540/expansion-purification-functional-assessment-human-peripheral-blood&lt;/a&gt;</td></tr><tr><td>Firefly luciferase expressing hESCs</td><td>University of Minnesota, Minneapolis, MN</td><td></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></td><td>contact: Dan S. Kaufman. Description under plasmid comments section</td></tr><tr><td colspan="4">&lt;p class="jove_content"&gt;&lt;strong&gt;Materials Table.&lt;/strong&gt;&lt;/p&gt;</td></tr></tbody></table>

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

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

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 high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

The generation of hematopoietic progenitor cells using the spin EB approach allows for optimal NK cell development from hESCs and iPSCs. As demonstrated in Figure 2, day 11 spin EBs contain high percentages of progenitor cells expressing CD34, CD45, CD43, and CD31. High levels of CD34 and CD45 allows direct transfer to NK conditions without need for sorting or supporting stromal cells. If there is suboptimal spin EB differentiation, it is recommended that stromal cells such as EL08-1D2 are used in the se...

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and 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 ...

The overall goal of this procedure is to generate human natural killer or NK cells from blurry, potent stem cells. This is accomplished by first generating hematopoietic progenitor cells. In the second step, the hematopoietic progenitor cells are differentiated into NK cells through the use of defined cytokines.Next, NK cells can be tested in a variety of functional assays. In the final step, dual imaging is used to monitor the ability of NK cells to kill tumor targets in vivo. The main advantage of this technique over traditional existing methods such as stromal cell mediated differentiation or regular EB formation, is that the spiny B system provides a completely defined feeder free approach for the large scale expansion of progenitor cells that can be differentiated into natural killer cells.The implications of this technique expand towards the treatment of chronic viral infections, as well as cancer, as NK cells have been shown to be important effector cells in both of these diseases. To prepare for embryo body or EB plating, first pipette 150 microliters of sterile water into the 36 outer wells of a round bottom low attachment 96 well plate to minimize evaporation. Next, aspirate the medium off of the embryonic stem and induced pluripotent stem cell cultures and add one milliliter of Prewarm trip.Select place the trip alley containing plates into a 37 degrees Celsius incubator, and then after five minutes, gently pipette the cells off of the plate, transfer the dissociated cells to a conical tube and pipette the cell solution up and down to break apart any clumps. Now dilute the trip Ali with one equal volume of BPEL media and at least one equal volume of DPBS and spin down the cells for five minutes at 480 times G and eight degrees Celsius. Then remove the supernatant and resuspend the pellet in five milliliters, BPEL media plus five milliliters DPBS.After spinning down the cells again, resuspend the pellet in five to 10 milliliters of BPEL media and then pass the cells through a 70 micron filter into a fresh 50 milliliter conical tube to remove any clumps. After counting and spinning down the filtered cells Resus, suspend the palate at three times 10 to the fourth cells per milliliter plate, 100 microliters of cells into each of the inner 60 wells of the previously prepared 96 well plates, and then spin the 96 well plates for four minutes at 480 times G and eight degrees Celsius. Then carefully place the plates in a 37 degrees Celsius incubator using a multi-channel pipette sent to 100 microliters.Begin this next step by transferring six spin EBS to each well of a 24 well plate. Then add 400 microliters of NK differentiation media containing cytokines to each well to bring the total volume in each well to approximately one milliliter. Incubate the cells for 28 days, feeding the cultures every five to seven days with NK differentiation, media containing cytokines.At day 28, the NK cells will express a mature phenotype following a transfer to NK cell differentiation culture. To test NK cell cytotoxicity, perform a four hour chromium release assay to generate large numbers of NK cells for the in vivo experiments. Co-culture the cells with artificial antigen presenting cells using the protocol previously demonstrated by Sochi et al.Begin this step by Resus suspending the desired number of tumor cells in 200 microliters of is cov modified delco medium, or IM DM supplemented with 20%FBS. Then using a 27 or 28 gauge needle, inject the tumor cells subcutaneously into the upper left thorax of each six to eight week old, non-obese diabetic. Severe combined immunodeficiency with gamma chain knockout mouse.Allow the tumor cells to engraft for four days. The cells should form a bubble under the skin of the mouse. Then resuspend the desired number of HESC derived NK cells in 300 microliters of IMDM, supplemented with 20%FBS without antibiotics.Next, using a new 27 or 28 gauge needle intraperitoneal, inject the HESC derived NK cells into each mouse with a tumor. Then to monitor the engraftment of the cells by bioluminescent imaging, inject each mouse peritoneal with 120 microliters of D luciferian. After anesthetizing the mice, place the animals in an IVUS spectrum within 10 minutes after injection of the D luciferian, secure each mouse on its back using Velcro, and then set the IVUS machine to the correct imaging platform.Now set the benning to medium and the F stop to one, and then acquire the image for one minute to perform fluorescent imaging for turbo FP six 50 positive cells. First, use the imaging wizard to set up an emission scan to measure the probe of interest, as well as background signal from the list of probes. Choose input EM ex and enter 605 for the excitation and 660 to 720 for the emission for the tumor signal.Then select add probe for the background signal. Set the excitation to 570 and the emission to 640 to 720. Filters can also be set through the filter config button.Finally, allow the mice to fully recover from the anesthesia before transporting them back to the animal facility. The images can be analyzed using the living image software package version 4.2. Day 11, spin EBS contain high percentages of progenitor cells expressing CD 34, CD 43, CD 45, and CD 31.High levels of CD 34 and CD 45 allow direct transfer to NK cell conditions without the need for sorting or for supporting stromal cells. NK cells can be detected by flow cytometry. After four weeks of culture.Cells within the lymphocyte gait are analyzed for the expression of CD 56. CD 56 positive cells can then be further analyzed for co-expression of markers such as CD one 17, CD 94, or NK P 46. NK cells maintain the expression of reporter genes that were modified into the parent E-S-I-P-S line and therefore can be followed in vivo.Here, the IVUS spectrum has been used to demonstrate the simultaneous imaging of both NK cells and tumor cells. In the same mouse bioluminescent imaging of Firefly Luciferase, HESC derived nnk cells on day zero seven and 14 after NK cell injection is shown fluorescent imaging of turbo FP six 50 positive K 5 6 2 cells on day zero seven and 14. After NK cell injection is shown here, a sequence showing the tissue autofluorescence and fluorescence signal from the turbo FP six 50 positive cells generated.Using the spectral unmixing feature in living image software is shown. These mice are non injection controls and show only the background signal. While attempting this technique, it is important to maintain high quality ES and IPSL culture, as well as performing DYS spinny b differentiation as described.

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JoVE Journal

Bioengineering

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Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates.  iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

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Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

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

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

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