Name: a novel feeder-free system for mass production of murine natural killer cells in vitro
Uploaded: 2018-01-09T14:00:00
Description: Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro Video at JoVE.com

This study was supported by the Research Grants Council of Hong Kong (GRF 468513, CUHK3/CRF/12R) and the Innovation and Technology Fund of Hong Kong (ITS/227/15, ITS InP/164/16, ITS-InP/242/16), Direct Grant for Research-CUHK (2016.035), and Hong Kong Scholar Program.
H.-Y.L. designed and supervised all experiments and contributed to manuscript preparation. P.M.-K.T. performed experiments, analyzed data and contributed to manuscript preparation. P.C.-T. T., J.Y.-F.C., J.S.-C.,H., Q.-M.W., and G.-Y.L. collected animal samples and participated in animal experiments. J.S., X.-R.H., and K.-F.T. contributed to manuscript preparation.

The protocol for obtaining and differentiating bone marrow-derived NK cells (BM-NK) is based on previously published methods20,21,22. All procedures with mice have been approved by the Animal Ethics Experimental Committee (AEEC) at the Chinese University of Hong Kong.
1. Preparation of OP9 Conditional Medium
<ol>
	<li>Culture the murine stroma cell line OP9 in alpha-MEM containing 20% FBS, 100 U/mL ...

<ol>
	<li>Schreiber, R. D., Old, L. J., Smyth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunoediting:+integrating+immunity's+roles+in+cancer+suppression+and+promotion.">Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion.</a> Science. 331 (6024), 1565-1570 (2011).</li><li>Junttila, M. R., de Sauvage, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+tumour+micro-environment+heterogeneity+on+therapeutic+response.">Influence of tumour micro-environment heterogeneity on therapeutic response.</a> Nature. 501 (7467), 346-354 (2013).</li><li>Rusakiewicz, 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=Immune+infiltrates+are+prognostic+factors+in+localized+gastrointestinal+stromal+tumors.">Immune infiltrates are prognostic factors in localized gastrointestinal stromal tumors.</a> Cancer Res. 73 (12), 3499-3510 (2013).</li><li>Mamessier, E., 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+breast+cancer+cells+enhance+self+tolerance+by+promoting+evasion+from+NK+cell+antitumor+immunity.">Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity.</a> J Clin Invest. 121 (9), 3609-3622 (2011).</li><li>Stern, M., 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=Pre-emptive+immunotherapy+with+purified+natural+killer+cells+after+haploidentical+SCT:+a+prospective+phase+II+study+in+two+centers.">Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers.</a> Bone Marrow Transplant. 48 (3), 433-438 (2013).</li><li>Miller, J. 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=Successful+adoptive+transfer+and+in+vivo+expansion+of+human+haploidentical+NK+cells+in+patients+with+cancer.">Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer.</a> Blood. 105 (8), 3051-3057 (2005).</li><li>Rubnitz, J. E., 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=NKAML:+a+pilot+study+to+determine+the+safety+and+feasibility+of+haploidentical+natural+killer+cell+transplantation+in+childhood+acute+myeloid+leukemia.">NKAML: a pilot study to determine the safety and feasibility of haploidentical natural killer cell transplantation in childhood acute myeloid leukemia.</a> J Clin Oncol. 28 (6), 955-959 (2010).</li><li>Curti, 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=Successful+transfer+of+alloreactive+haploidentical+KIR+ligand-mismatched+natural+killer+cells+after+infusion+in+elderly+high+risk+acute+myeloid+leukemia+patients.">Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients.</a> Blood. 118 (12), 3273-3279 (2011).</li><li>Bachanova, V., 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=Clearance+of+acute+myeloid+leukemia+by+haploidentical+natural+killer+cells+is+improved+using+IL-2+diphtheria+toxin+fusion+protein.">Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein.</a> Blood. 123 (25), 3855-3863 (2014).</li><li>Stringaris, K., 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=Leukemia-induced+phenotypic+and+functional+defects+in+natural+killer+cells+predict+failure+to+achieve+remission+in+acute+myeloid+leukemia.">Leukemia-induced phenotypic and functional defects in natural killer cells predict failure to achieve remission in acute myeloid leukemia.</a> Haematologica. 99 (5), 836-847 (2014).</li><li>Rouce, R. H., 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=The+TGF-&#946;/SMAD+pathway+is+an+important+mechanism+for+NK+cell+immune+evasion+in+childhood+B-acute+lymphoblastic+leukemia.">The TGF-&#946;/SMAD pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia.</a> Leukemia. 30 (4), 800-811 (2016).</li><li>Derynck, R., Akhurst, R. J., Balmain, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling+in+tumor+suppression+and+cancer+progression.">TGF-&#946; signaling in tumor suppression and cancer progression.</a> Nature Genet. 29 (2), 117-129 (2001).</li><li>Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+in+cancer.">TGFbeta in cancer.</a> Cell. 134 (2), 215-230 (2008).</li><li>Ikushima, H., Miyazono, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+signalling:+a+complex+web+in+cancer+progression.">TGFbeta signalling: a complex web in cancer progression.</a> Nat Rev Cancer. 10 (6), 415-424 (2010).</li><li>Pickup, M., Novitskiy, S., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+TGF&#946;+in+the+tumour+microenvironment.">The roles of TGF&#946; in the tumour microenvironment.</a> Nat Rev Cancer. 13 (11), 788-799 (2013).</li><li>Rook, A. H., 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=Effects+of+transforming+growth+factor+beta+on+the+functions+of+natural+killer+cells:+depressed+cytolytic+activity+and+blunting+of+interferon+responsiveness.">Effects of transforming growth factor beta on the functions of natural killer cells: depressed cytolytic activity and blunting of interferon responsiveness.</a> J Immunol. 136 (10), 3916-3920 (1986).</li><li>Bellone, G., Aste-Amezaga, M., Trinchieri, G., Rodeck, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+NK+cell+functions+by+TGF-beta+1.">Regulation of NK cell functions by TGF-beta 1.</a> J Immunol. 155 (3), 1066-1073 (1995).</li><li>Trotta, R., 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=TGF-&#946;+utilizes+SMAD3+to+inhibit+CD16-mediated+IFN-&#947;+production+and+antibody-dependent+cellular+cytotoxicity+in+human+NK+cells.">TGF-&#946; utilizes SMAD3 to inhibit CD16-mediated IFN-&#947; production and antibody-dependent cellular cytotoxicity in human NK cells.</a> J Immunol. 181 (6), 3784-3792 (2008).</li><li>Shull, M. M., 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=Targeted+disruption+of+the+mouse+transforming+growth+factor-&#946;1+gene+results+in+multifocal+inflammatory+disease.">Targeted disruption of the mouse transforming growth factor-&#946;1 gene results in multifocal inflammatory disease.</a> Nature. 359 (6397), 693-699 (1992).</li><li>Chen, T. J., Kotecha, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytobank:+providing+an+analytics+platform+for+community+cytometry+data+analysis+and+collaboration.">Cytobank: providing an analytics platform for community cytometry data analysis and collaboration.</a> Curr Top Microbiol Immunol. 377, 127-157 (2014).</li><li>Williams, N. 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=Differentiation+of+NK1.1+,+Ly49++NK+cells+from+flt3++multipotent+marrow+progenitor+cells.">Differentiation of NK1.1+, Ly49+ NK cells from flt3+ multipotent marrow progenitor cells.</a> J Immunol. 163 (5), 2648-2656 (1999).</li><li>Fathman, J. W., 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=Identification+of+the+earliest+natural+killer+cell-committed+progenitor+in+murine+bone+marrow.">Identification of the earliest natural killer cell-committed progenitor in murine bone marrow.</a> Blood. 118 (20), 5439-5447 (2011).</li><li>Tang, P. M., 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=Smad3+promotes+cancer+progression+by+inhibiting+E4BP4-mediated+NK+cell+development.">Smad3 promotes cancer progression by inhibiting E4BP4-mediated NK cell development.</a> Nat Commun. 6 (8), 14677 (2017).</li></ol>

In the present work, we have described a novel method for producing bone marrow-derived murine NK cells in vitro. The cell feeder in the original system21,22 is successfully replaced by the conditional medium of OP9 cells, which largely increased the stability of the differentiation system. In addition, the system can produce high quantity and purity of mature NK cells for in vitro as well as in vivo assays, which can facilitate the mec...

Cancer progression is largely dependent on the tumor microenvironment1,2, including host-derived immunocytes, e.g., NK cells. Several studies demonstrated that intratumoral NK cells are negatively correlated with the tumor progression3,4. In addition, clinical studies showed that NK cell adoptive therapy is a possible strategy for cancer5,6,7,8,9. NK cell-based cancer immunotherapy was recently suggested as a therapeutic option for solid tumors, but challenges exist due to the secretion of immunosuppressive cytokines and downregulation of activating ligands in the microenvironment of solid tumors10,11. Transforming growth factor-&#946; (TGF-&#946;) has been suggested to play a suppressive role in carcinogenesis, but paradoxically cancer cells also produce TGF-&#946;1 to support the tumor development12,13,14,15. TGF-&#946; signaling can suppress the cytolytic activity of NK cells via down-regulating interferon responsiveness and CD16-mediated interferon-gamma (IFN-&#947;) production in vitro16,17,18.
Although disruption of TGF-&#946; signaling in the tumor microenvironment may be a possible way for eliminating cancers, completely blocking TGF-&#946; signaling will cause autoimmune diseases due to its anti-inflammatory function, as evidenced by the development of adverse side effects including systemic inflammation, cardiovascular defects, and autoimmunity in mouse models19. Thus, understanding the working mechanism of TGF-&#946;-mediated immunosuppression will lead to the identification of an accessible therapeutic target for treating cancer.
To elucidate the molecular events necessary for NK cell development, Williams et al. established an in vitro system for differentiating murine bone marrow hematopoietic stem cells into NK cells20. This system largely facilitates the mechanistic study of NK cell development, including the identification of novel progenitors of NK cells21. However, the bone marrow progenitors should be cultured in the system with supporting OP9 stromal cells as a feeder layer20,21, and this heterogeneous cell population largely limits the further application of gene-disrupting tools (e.g., siRNA-mediated gene silencing) specifically applied to the differentiating NK cells.
Here, we describe a feeder-free system that has been developed by further modifying the in vitro system of Williams et al20. In our system, the OP9 stromal feeder cells are not required, and instead OP9 conditional medium is used without affecting the differentiation of NK cells in vitro, and this recently lead us to uncover that TGF-&#946; is able to promote cancer progression via suppressing E4bp4-dependent NK cell development in the tumor microenvironment22. This novel system successfully provides a background-free method for elucidating the molecular mechanism of NK cell development under specific conditions (e.g., high TGF- &#946;1, siRNA-mediated gene silencing, etc.) in vitro.

<table border="0" cellpadding="0" cellspacing="0" style="width:1113px;" width="1113">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">OP9 cell line</td>
			<td style="width:129px;">ATCC</td>
			<td style="width:344px;">ATCC&#174; CRL-2749&#8482;</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MEM &#945;, no nucleosides</td>
			<td>Gibco</td>
			<td>22561021</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine IL-7</td>
			<td>PEPROTECH</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine SCF</td>
			<td>PEPROTECH</td>
			<td>250-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine Flt3-Ligand</td>
			<td>PEPROTECH</td>
			<td>250-31L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine IL-2</td>
			<td>PEPROTECH</td>
			<td>212-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamin RNAiMAX Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>1377815</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IC Fixation Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-8222-49</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow Cytometry Staining Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-4222-26</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-conjugated anti-mouse CD244</td>
			<td>eBioscience</td>
			<td>12-2441-83</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cy3-conjugated anti-mouse NKp46</td>
			<td>Bioss</td>
			<td>bs-2417R-cy3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nonsense control (NC)</td>
			<td>Ribobio</td>
			<td>siN05815122147</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">siRNA against mouse E4BP4 mRNA</td>
			<td>Ribobio</td>
			<td>N/A</td>
			<td>5&#8242;-GAUGAGGGUGUA 
			GUGGGCAAGUCUU-3&#8242;</td>
		</tr>
	</tbody>
</table>

a novel feeder-free system for mass production of murine natural killer cells in vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

Representative results are obtained following the described protocol. Total bone marrow suspension cells were cultivated under the feeder-free differentiation system for 11 days; significant increase in proliferation rate was observed by day 7 compared with the number of total cells on day 0 (Figure 1A). Mature NK cells with high nuclear to cytoplasmic ratio and granule-rich cytoplasm morphology were found by day 6 in the system (<strong clas...

Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro Video at JoVE.com

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro Video (Video) | JoVE

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Research

JoVE Journal

Immunology and Infection

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays (Video) | JoVE 

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Artificial Antigen Presenting Cell AAPC Mediated Activation and Expansion of Natural Killer T Cells Video (Video) | JoVE

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells Video (Video) | JoVE

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells Video (Video) | JoVE

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer Video (Video) | JoVE

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System Video (Video) | JoVE

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen Video (Video) | JoVE

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood Video (Video) | JoVE

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4 Video (Video) | JoVE

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration Video (Video) | JoVE

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...