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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)

Published: April 23rd, 2013



1Department of Medicine (Hematology, Oncology, and Transplant), University of Minnesota, Minneapolis, 2Stem Cell Institute, University of Minnesota, Minneapolis
* These authors contributed equally

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 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.

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 m....

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1. Adapting hESCs or iPSCs in TrypLE for Spin EB Cultures

  1. The initial dissociation with TrypLE works best if the ES/iPS colonies from the collagenase-passaged cultures are relatively small. The starting ES/iPS populations should be cells passed no longer than 4-5 days previous. 4-5 days before initiation of TrypLE passage, pass ES cells at a density that will allow the cells to be ~70% confluent in 4 days time. Use regular ES media for culture of TrypLE-passaged ES cells. Here, we are using hESCs stably modif.......

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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.......

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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.......

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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.


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Name Company Catalog Number Comments
Name of Reagent Company Catalog Number Comments
      BPEL media for Spin EB generation
Iscove's Modified Dulbecco's Medium (IMDM) Fisher Scientific SH3022801  
F-12 Nutrient Mixture w/ Glutamax I Invitrogen 31765035  
Bovine Serum Albumin (BSA) Sigma-Aldrich A3311 Commercially available BSA can be cytotoxic to ES cells. Deionizing the solution can reduce the potential for cytotoxicity.
Poly(vinyl alcohol) Sigma-Aldrich P8136  
Linoleic Acid Sigma-Aldrich L1012  
Linolenic Acid Sigma-Aldrich L2376  
Synthechol 500X solution Sigma-Aldrich S5442  
a-monothioglycerol (a-MTG) Sigma-Aldrich S5442  
Protein-free hybridoma mix II Invitrogen 12040077  
Ascorbic Acid 2-phosphate Sigma-Aldrich A8960  
Glutamax I Invitrogen 35050061  
Insulin, Transferrin, Selenium 100X solution (ITS) Invitrogen 41400-045  
Pen-Strep Invitrogen 15140122  
      NK Differentiation Media
Dubecco's Modified Eagle Medium (DMEM) Invitrogen 11965-118  
F-12 Media Invitrogen CX30315  
15% Human AB serum Valley Biomed HP1022HI  
5 ng/ml Sodium Selenite Sigma-Aldrich S5261  
50 uM ethanolamine Sigma-Aldrich E9508  
20 ng/ml ascorbic acid Sigma-Aldrich A8960  
25 uM 2-mercaptoethanol (BME) Gibco 21985  
2 mM L-glutamine Gibco CX30310  
1% Pen-Strep Invitrogen 15140122  
      (all cytokines used fresh from frozen aliquots)
SCF PeproTech, Inc. 300-07 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.
rhBMP-4 R &D Systems 314-BP 20 ng/ml in BPEL media
rhVEGF R&D Systems 293-VE 20 ng/ml in BPEL media
IL-2 PeproTech, Inc. 200-02 1 x 105 U/ml given to mice after NK cell injection; 50 U/ml used in NK cell expansion protocol
IL-15 PeproTech, Inc. 200-15 10 ng/ml given to mice after NK cell injection (first 7 days only)
IL-3 PeproTech, Inc. 200-03 5 ng/ml in NK media
IL-7 PeproTech, Inc. 200-07 20 ng/ml in NK media
Flt-3-Ligand PeproTech, Inc. 300-19 10 ng/ml in NK media
      In vivo Imaging
D-Luciferin Sodium Salt Gold BioTechnology Lucna-500  
TurboFP650 plasmid Evrogen, Moscow, Russia FP731 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.
IVIS Spectrum Imaging System Caliper Life Sciences    
Membrane bound IL-21 expressing artificial antigen presenting cells MD Anderson, Houston, TX   contact: Dean A. Lee.
Firefly luciferase expressing hESCs University of Minnesota, Minneapolis, MN   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.
TurboFP650 expressing K562 cells University of Minnesota, Minneapolis, MN   contact: Dan S. Kaufman. Description under plasmid comments section

Materials Table.

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

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