Name: Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells (Video) | JoVE Journal
Uploaded: 2025-02-14T14:10:00.000+00:00
Duration: 11 min 8 s
Description: Learn to generate CAR-expressing natural killer cells from human iPSCs using non-viral transposon-based gene integration. This protocol enhances antitumor activity and scalability for cancer immunotherapy applications.

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5 minutes

We thank all Kaufman lab members for their support, scientific insights and discussions. These studies were supported by the NIH/NCI grants U01CA217885, P30CA023100 (administrative supplement) and the Sanford Stem Cell Institute at UCSD. JT: writing and revision of manuscript. DSK: reviewed and edited the manuscript.

DSK is a co-founder and advisor to Shoreline Biosciences and has an equity interest in the company. DSK also consults for Therabest and RedC Bio for which he receives income and/or equity. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. The remaining authors declare no competing interests.

This protocol outlines a standardized and reproducible approach for generating CAR-expressing, iPSC-derived NK cells from a consistent cell source aimed at facilitating targeted &#34;off-the-shelf&#34; cancer immunotherapy. Multiple preclinical and clinical studies have shown the efficacy of adoptive NK cell-based immunotherapy in treating cancers while minimizing toxicities, like graft versus host diseases (GvHD) or cytokine-release syndrome (CRS)23,42,</su...

NK cells, a type of lymphocyte within the innate immune system, are pivotal in the early defense against tumors and virally infected cells1,2,3. Unlike T cells, NK cells do not require antigen presentation via major histocompatibility complex (MHC) molecules. Instead, NK cells have a repertoire of activating and inhibitory receptors that regulate their activity3. NK cell-mediated cytotoxic activity utilizes various mechanisms, including the release of perforin and granzymes, engagement of death receptors, and the production of pro-inflammatory cytokines such as IFN-&#947; and TNF-&#945;. This unique mode of action positions NK cells as an attractive candidate for cancer immunotherapy, particularly in the treatment of solid cancers where immune evasion is a significant hurdle1,2,3,4.
Hepatocellular carcinoma (HCC) is one of the most common and deadly forms of liver cancer worldwide5. Traditional therapeutic approaches, including surgery, chemotherapy, and radiotherapy, typically provide limited clinical benefit and result in high recurrence rates6,7. Recent advances in immunotherapy and targeted therapy have significantly impacted the treatment of HCC8,9. Immune checkpoint inhibitors, like nivolumab and pembrolizumab, have shown promising results by enhancing the immune cell response against cancer cells10,11. These therapies have led to improved survival rates and better quality of life for some patients. However, they can also cause immune-related adverse effects, which may limit their use in certain patients12. Targeted therapies, such as sorafenib and lenvatinib, specifically inhibit pathways that promote cancer cell growth and angiogenesis13. These treatments have known efficacy to control disease progression and prolong survival8,14. Nonetheless, resistance to targeted therapies typically develops, and side effects of treatment are common15,16. Among the promising avenues in cancer immunotherapeutic strategies, the advent of chimeric antigen receptor (CAR) CAR T cells has revolutionized cancer treatment, especially in the treatment of hematologic malignancies such as lymphoma and multiple myeloma17,18,19.
CAR-expressing NK cells, combining the innate cytotoxic activity of NK cells with the precision targeting of CAR technology, represent an innovative and potentially transformative approach for solid tumors such as HCC20,21,22,23,24. CAR-engineered cells can specifically recognize and kill tumor cells expressing the target antigen while sparing normal tissues, thereby reducing the risk of off-target effects associated with conventional therapies25,26,27. CAR-NK cells produced from NK cells isolated from peripheral blood or cord blood are typically produced by transducing NK cells with CAR constructs that consist of an extracellular antigen-recognition domain, a transmembrane domain, and intracellular signaling domains necessary for activation and proliferation28,29,30.
The challenge of engineering a stable and homogenous population of functionally engineered NK cells for clinical treatment can be addressed by employing induced pluripotent stem cells (iPSCs)31,32. Engineering of human iPSCs with NK cell-optimized CARs provides an improved NK cell activation and proliferation signal and provides an inexhaustible and homogenous population of CAR-expressing NK cells as a standardized, &#34;off-the-shelf therapy&#34;20,33. In this protocol, genetic modification of iPSCs to generate CAR-expressing iPSC-derived NK cells involves integrating an NK cell optimized CAR construct that includes NK cell-derived transmembrane and signaling domains (NKG2D-2B4-CD3&#950;) and an anti-glypican-3 (GPC3) scFv as described in our previous studies23. The genetically engineered iPSCs are then differentiated and expanded using an NK cell differentiation protocol developed previously34. These engineered CAR-expressing iPSC-derived NK cells have the ability to recognize and eliminate HCC and other tumor cells expressing specific tumor-associated antigens, such as Glypican 3 (GPC3), which is overexpressed in HCC and other malignancies35,36,37,38.
The application of iPSC-derived CAR-NK cells for the treatment of diverse cancers holds significant promise30,39,40; many of these iPSC-derived CAR-NK cells are currently in clinical trials41,42,43. To facilitate advancement in this area, this protocol enables efficient production of engineered iPSC-derived CAR-NK cells, from cell engineering to differentiation into mature NK cells and in vitro expansion.

<table><tbody><tr><td>aAPC&amp;nbsp;</td><td>Dean A. 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Kaufman lab</td><td>N/A</td><td></td></tr><tr><td>Human: iPS cells&amp;nbsp;</td><td>Dan S. Kaufman lab&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>Human: SNU-449 cells&amp;nbsp;</td><td>ATCC</td><td>Cat#CRL-2234</td><td></td></tr><tr><td>Human: SNU-449-td-tomato-luc cells&amp;nbsp;</td><td>Dan S. Kaufman lab</td><td>N/A</td><td></td></tr><tr><td>IncuCyte Caspase-3/7 Green Apoptosis Assay&amp;nbsp;</td><td>Essenbioscience&amp;nbsp;</td><td>Cat#4440</td><td></td></tr><tr><td>L-Ascorbic acid</td><td>Sigma Aldrich</td><td>A5960</td><td></td></tr><tr><td>MP Biomedicals&amp;nbsp;Human Serum, Type AB</td><td>MP Biomedicals</td><td>ICN2938249</td><td></td></tr><tr><td>mTeSR plus</td><td>StemCell Technologies, Inc.&amp;nbsp;</td><td>100-0276</td><td></td></tr><tr><td>NovoExpress software&amp;nbsp;</td><td>ACEA Biosciences&amp;nbsp;</td><td>https://www.agilent.com/en/product/research-flow-cytometry/flow-cytometry-software/novocyte-novoexpress-software-1320805</td><td></td></tr><tr><td>PE/Cy7 anti-human SSEA-4 Antibody&amp;nbsp;</td><td>Biolegend</td><td>330420</td><td></td></tr><tr><td>PE-CD16&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#560995&amp;nbsp;</td><td></td></tr><tr><td>PE-CD226</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#559789</td><td></td></tr><tr><td>PE-CD34&amp;nbsp;</td><td>BD Biosciences</td><td>Cat# 555822&amp;nbsp;</td><td></td></tr><tr><td>PE-CD45&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat# 555483&amp;nbsp;</td><td></td></tr><tr><td>PE-CD94&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#555888&amp;nbsp;</td><td></td></tr><tr><td>PE-cy7-CD56&amp;nbsp;</td><td>BioLegend</td><td>Cat# 318318&amp;nbsp;</td><td></td></tr><tr><td>PE-FAS Ligand&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#564261&amp;nbsp;</td><td></td></tr><tr><td>PE-NKp30</td><td>BD Biosciences</td><td>Cat# 558407</td><td></td></tr><tr><td>PE-NKp44&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#558563&amp;nbsp;</td><td></td></tr><tr><td>PE-NKp46</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#331908</td><td></td></tr><tr><td>PE-NKp46&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#557991&amp;nbsp;</td><td></td></tr><tr><td>Peripheral blood buffy coat&amp;nbsp;</td><td>San Diego Blood Bank (https://www. sandiegobloodbank.org/)&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>PE-TRAIL&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>Cat#565499&amp;nbsp;</td><td></td></tr><tr><td>pKT2-mCAG-IRES-GFP-ZEO&amp;nbsp;</td><td>Branden Moriarity lab&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>pMAX-GFP plasmid</td><td>Lonza</td><td>N/A</td><td>GFP positive control&amp;nbsp;</td></tr><tr><td>Prism 9</td><td>Graphpad&amp;nbsp;</td><td>Version 9</td><td></td></tr><tr><td>pSpCas9&amp;nbsp;</td><td>GenScript</td><td>PX165</td><td></td></tr><tr><td>RBC Lysis Buffer (10x)</td><td>Biolegend</td><td>Cat#420301</td><td></td></tr><tr><td>Recombinant human bFGF basic&amp;nbsp;</td><td>R&amp;amp;D Systems&amp;nbsp;</td><td>Cat#4114-TC</td><td></td></tr><tr><td>Recombinant human BMP-4&amp;nbsp;</td><td>PeproTech</td><td>Cat#120-05</td><td></td></tr><tr><td>Recombinant human FLT-3 Ligand&amp;nbsp;</td><td>PeproTech</td><td>Cat# 300-19</td><td></td></tr><tr><td>Recombinant human IL-15&amp;nbsp;</td><td>PeproTech</td><td>Cat# 200-15</td><td></td></tr><tr><td>Recombinant human IL-2&amp;nbsp;</td><td>PeproTech</td><td>Cat# 200-02</td><td></td></tr><tr><td>Recombinant human IL-3&amp;nbsp;</td><td>PeproTech</td><td>Cat#200-03</td><td></td></tr><tr><td>Recombinant human IL-7&amp;nbsp;</td><td>PeproTech</td><td>Cat# 200-07</td><td></td></tr><tr><td>Recombinant Human Nodal Protein</td><td>R&amp;amp;D Systems&amp;nbsp;</td><td>Cat#3218-ND-025</td><td></td></tr><tr><td>Recombinant human SCF&amp;nbsp;</td><td>PeproTech</td><td>Cat# 300-07</td><td></td></tr><tr><td>Recombinant Human TGF-&amp;beta;1</td><td>PeproTech</td><td>Cat#100-21</td><td></td></tr><tr><td>Recombinant human VEGF&amp;nbsp;</td><td>PeproTech</td><td>Cat# 100-20</td><td></td></tr><tr><td>RPMI1640</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma Aldrich</td><td>214485</td><td></td></tr><tr><td>STEMdiff APEL 2 Medium</td><td>StemCell Technologies, Inc.&amp;nbsp;</td><td>5270</td><td>EB formation medium</td></tr><tr><td>STEMdiff APEL2 Medium&amp;nbsp;</td><td>StemCell Technologies, Inc.&amp;nbsp;</td><td>Cat#05270</td><td></td></tr><tr><td>Super piggyBac Transposase expression vector</td><td>SBI</td><td>Cat#PB210PA-1</td><td></td></tr><tr><td>SYTOX AADvanced Dead Cell Stain Kit</td><td>ThermoFisher Scientific</td><td>S10274, S10349</td><td>Dead cell staining solution kit</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr></tbody></table>

advances in human induced pluripotent stem cell-derived chimeric antigen receptor-expressing natural killer cells

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Schematic diagram of anti-GPC3-CAR iPSC derived NK cells differentiation and expansion 
The schematic diagram illustrates the in vitro differentiation of anti-GPC3-CAR engineered NK cells derived from human induced pluripotent stem cells (iPSCs) and a schematic representation of piggyBac vector carrying GPC3 CAR construct. Initially, unmodified iPSCs are transfected with a piggyBac vector encoding the GPC3 CAR (chimeric antigen receptor). Following transfection, these GPC3 CAR-expressing iPSC...

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...

advances-human-induced-pluripotent-stem-cell-derived-chimeric-antigen

Research

JoVE Journal

Immunology and Infection

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

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

This video demonstrates a technique for the ex vivo generation of tumor antigen-specific chimeric antigen receptor (CAR) T cells. Retroviral vectors carrying transgenic RNA are mixed with spleen cells in a medium supplemented with interleukin-2 to promote T-cell survival and proliferation. The mixture is then transferred to a plate coated with recombinant human fibronectin fragments and centrifuged to facilitate the fusion of the virus with the cell, allowing the RNA to integrate into the host...

An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Production of Human CRISPR-Engineered CAR-T Cells

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

The present protocol shows how to differentiate CD3−/CD45+CD56+ cells with mild cytotoxicity from human expanded potential stem cells (hEPSCs) under both 3D and 2D culture conditions. This allows for routine phenotypical validation without the destruction of the complex microenvironment.

Generation of Natural Killer Cells from Human Expanded Potential Stem Cells

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

We outline a transfection protocol for producing chimeric antigen receptor-natural killer (CAR-NK) cells targeting fungal pathogens using the non-viral Sleeping Beauty transposon system. To assess antigen-specific activation, we co-cultured the engineered cells with Aspergillus fumigatus germ tubes and measured IFN-γ secretion.

Engineering Chimeric Antigen Receptor-Natural Killer Cells Targeting Fungal Infections Using the Non-viral Sleeping Beauty Transposon System

This protocol provides a standardized in vitro. workflow for consistent measurement of natural, chimeric antigen receptor–mediated, and antibody-dependent cytotoxicity of human natural killer cells using live-cell imaging and flow cytometry.

Quantifying NK-Cell Cytotoxicity Potentiated by Chimeric Antigen Receptors and Therapeutic Antibodies Using Live-Cell Imaging and Flow Cytometry

Learn to generate CAR-expressing natural killer cells from human iPSCs using non-viral transposon-based gene integration. This protocol enhances antitumor activity and scalability for cancer immunotherapy applications.

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

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