Method Article
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 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 CD3ζ 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.
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-γ and TNF-α. 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, "off-the-shelf therapy"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-CD3ζ) 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.
1. Feeder-free culture method of human iPSCs
NOTE: Thaw the frozen undifferentiated human iPSCs and culture them with the use of mTeSR 1-plus on Matrigel (henceforth referred to as basement membrane matrix [BMM]) pre-coated plates, as previously described23,34. It is very important to make sure the iPSCs are not differentiated before and after engineering. Freshly thawed iPSCs should be cultured for about 2-3 passages to exhibit pluripotency morphology consistent with the human pluripotent stem cells. The following instructions are used for passaging cells from one well of a 6-well plate.
2. Engineering of human iPSCs to express anti-GPC3 CAR using piggyBac vector
3. Clonal selection and pluripotency confirmation of GPC3-CAR iPSCs
4. Generation of hematopoietic progenitor cells from engineered CAR iPSCs by spin embryoid body (EB) Formation
NOTE: A spin EB or hematopoietic organoid protocol is used to produce hematopoietic progenitors23,34. The cells within the EBs differentiate into stromal cells to support lymphocyte development38, thereby eliminating the need for xeno-derived stromal cells like OP923,34,48. Below are the instructions for collecting cells from a single well of a 6-well plate for EB formation.
5. Differentiation of GPC3 CAR iPSC derived NK cells from Spin EBs
NOTE: The GPC3 CAR-expressing Spin EBs can be transferred into either 24-well plates or 6-well plates coated with 2% gelatin or without coating. For medium changes, 6-well plates are more suitable, while 2% gelatin coating enhances EBs attachment.
6. Expansion of anti-GPC3 CAR iPSC derived NK cells
NOTE: Typically, a yield of 2-20 × 106 NK cells can be obtained from a single 6-well plate, even before expansion. To facilitate further expansion of NK cells for downstream applications, artificial antigen-presenting cells (aAPCs) are employed to generate >1 × 109 NK cells.
7. Phenotypic and functional characterization of GPC3 CAR iPSC derived NK cells
NOTE: The characterization of GPC3 CAR iPSC-derived NK cells involves a comprehensive assessment of their phenotypic profile and functional activity.
8. Troubleshooting items and solutions
NOTE: Troubleshooting the differentiation, expansion, and functional testing of iPSC-derived GPC3 CAR NK cells using the spin embryoid body (EB) method can involve several steps. Below are some common issues one may encounter, along with suggested solutions:
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 iPSCs are clonally expanded and then differentiated into functional CAR iPSC-derived NK cells. These engineered NK cells are subsequently subjected to in vitro and in vivo functional assays to evaluate their antitumor activity against various tumor cell lines (Figure 1).
Microscopic analysis of the NK differentiation process of WT and anti-GPC3-CAR engineered iPSCs at various stages
The GPC3 CAR-engineered iPSCs are differentiated into anti-GPC3-CAR-engineered iPSC NK cells. The formation of spin embryoid bodies (EBs) from GPC3 CAR engineered iPSCs, to the differentiation of GPC3-CAR iPSC NK cells were presented as a series of microscopic images documenting the differentiation stages of anti-GPC3-CAR engineered iPSCs into functional GPC3 CAR iPSC NK cells. The undifferentiated wild-type (WT) and GPC3 CAR iPSCs were cultured in mTeSR Plus medium (Figure 2A). These WT and anti-GPC3-CAR iPSCs are then dissociated into single cells and cultured in STEMdiff APEL medium containing SCF (40 ng/mL), BMP4 (20 ng/mL), VEGF (20 ng/mL) and 10 µM ROCK inhibitor for 6 days in an ultra-low attachment U-bottom 96 well plate. The formation of spin EBs was captured on day 6 from both WT and GPC3 CAR iPSCs (Figure 2B). These spin EBs are further transferred to 6 well plate containing NK differentiation medium with the following cytokines IL-3 (5 ng/mL), SCF (20 ng/mL), IL-7 (20 ng/mL), and IL-15 (10 ng/mL). The differentiation process of WT and GPC3 CAR iPSC-derived NK cells at various time points from day 3 to day 28, were captured using a microscope at 100x magnification (Figure 2C).
Phenotype of WT and anti-GPC3 CAR iPSC derived EBs (day 6) and WT and GPC3 CAR iPSC derived NK cells (day 35)
The WT and GPC3 CAR iPSC-derived cells are phenotypically characterized at different stages of NK differentiation process. Panel A shows the expression of typical hematopoietic antigens CD34, CD31, CD43, and CD45 from the spin EBs of WT and GPC3 CAR iPSCs collected on day 6 were measured by flow cytometry (Figure 3A). Panel b presents the phenotype of WT and GPC3 CAR iPSC-derived NK cells harvested from the NK differentiation medium on day 35, highlighting the NK-specific markers (CD45, CD56, CD16, and NKG2D) associated with mature NK cells were measured by flow cytometry (Figure 3B).
Phenotype of expanded WT and anti-GPC3-CAR iPSC derived NK cells
After the Differentiation of WT and anti-GPC3 CAR iPSC-derived NK cells, the differentiated NK cells are harvested and expanded using irradiated aAPCs in a Gibco NK Xpander medium containing IL-2 (100 U/mL) and IL-15 (10 ng/mL). The expanded WT and anti-GPC3 CAR iPSC NK cells activation receptors such as CD94, CD16, NKp30, NKp44, NKp46, NKG2D, CD226, FasL, and TRAIL were measured by using flow cytometry (Figure 4A). After confirming the NK activation phenotype on the expanded WT and anti-GPC3 CAR iPSC NK cells, we evaluated the surface expression of GPC3 antigen on various tumor cell lines, including HepG2, SNU-449, SKOV3, and CAL27, via flow cytometry (Figure 4B). We have also confirmed the CAR expression on expanded anti- GPC3 CAR iPSC-derived NK cells by flow cytometric analysis.
Functional activity of WT and anti-GPC3 CAR iPSC derived NK cells against HCC and other tumor cell lines
After confirming the phenotype of WT and anti-GPC3 CAR iPSC-derived NK cells, we evaluated the functional antitumor activity against various tumor cell lines. Caspase3/7-based killing assays were conducted to assess the cytotoxic activity of WT and anti-GPC3 CAR iPSC-derived NK cells against HepG2, SNU-449, CAL27, and SKOV3 cell lines at various effector-to-target ratios. The results indicate the enhanced antitumor efficacy of anti-GPC3 CAR iPSC-derived NK cells compared to WT iPSC NK cells, showcasing their potential in targeting GPC3-expressing tumor cells via CAR specificity and other NK activation mechanisms (Figure 5A-D).
Figure 1: Schematic diagram of anti-GPC3-CAR iPSC derived NK differentiation, expansion and clinical application. (A) Overview of the process for generating anti-GPC3 CAR iPSC-derived NK cells for preclinical and clinical use. Unmodified iPSCs are transfected with a piggyBac vector carrying anti-GPC3-CAR gene. After successful transfection, anti-GPC3-CAR-expressing iPSCs are clonally expanded and differentiated into functional NK cells. These anti-GPC3 CAR iPSC-derived NK cells are subjected to both in vitro and in vivo functional assays to assess their cytotoxic activity against various GPC3-expressing tumor cell lines39. Upon validation of their efficacy and safety, these CAR iPSC-derived NK cells are further developed as a potential "off-the-shelf" therapy for clinical applications in cancer treatment. (B) Schematic representation of the piggyBac vector containing the anti-GPC3 CAR construct used for transfecting iPSCs. (C) Typical timeline of iPSC-derived NK cell differentiation, expansion, harvesting, and performing functional assays. Please click here to view a larger version of this figure.
Figure 2: Microscopic images of CAR iPSC NK differentiation from anti-GPC3-CAR engineered iPSCs at different stages. (A) Undifferentiated WT and GPC3 CAR iPSC cultured in mTeSR plus medium. (B) WT and GPC3 CAR iPSC spin embryoid body on day 6. (C) WT and GPC3 CAR iPSC derived NK cell differentiation at different days from D3 to D28. Magnification of 100x for a to c. Please click here to view a larger version of this figure.
Figure 3: Phenotype of WT and anti-GPC3 CAR iPSC derived day 6 EB and differentiated WT and GPC3 CAR iPSC derived NK cells on Day 35. (A) Typical hematopoietic antigens expressed on WT and GPC3 CAR iPSCs on day 6 of Differentiation of hematopoietic Differentiation. (B) Phenotype of WT and GPC3 CAR iPSC derived NK cells are harvested from NK differentiation medium on day 35. Please click here to view a larger version of this figure.
Figure 4: Phenotype of WT and anti-GPC3-CAR iPSC derived NK cells after expansion. (A) WT and GPC3 CAR iPSC derived NK cells are expanded in Gibco NK Xpander medium, and NK maturation markers were assessed by using flow cytometry. (B) Surface expression of GPC3 antigen on various tumor cell lines including HepG2, SNU-449, SKOV3 and CAL27 were measured by using flow cytometry. (C) Representative histogram showing the CAR expression on the GPC3 CAR iPSC derived NK cells after expansion. The CAR construct incorporated with FLAG tag and GFP to measure the CAR by flow cytometry. Please click here to view a larger version of this figure.
Figure 5: Functional activity of WT and anti-GPC3 CAR iPSC derived NK cells against HCC and other tumor cell lines. (A-D) Antitumor activity of WT and GPC3 CAR iPSC-derived NK cells were tested against various tumor cell lines, including HepG2, SNU-449, CAL27, and SKOV3. The X-axis represents the E:T ratios of the WT and anti-GPC3 CAR iPSC-derived NK cells tested against HepG2, SNU-449, CAL27, and SKOV3. The specific E: T ratios indicated on the x-axis are 10:1, 5:1, 2.5:1, 1:1, 0.5:1, and 0.25:1. Please click here to view a larger version of this figure.
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 "off-the-shelf" 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, 49,50,51,52,53,54,55,56,57. This approach utilizes an efficient and well-defined system to produce homogeneous and well-characterized CAR iPSC-derived NK cells can be scaled for clinical application. Additionally, iPSC-derived NK cells engineered to express NK-optimized CAR retain typical NK cell phenotypes and cytolytic functions 23,39,42,50,51,52,58.
Using human iPSCs for CAR iPSC-derived NK cell production offers a more efficient method for genetic modification, including CAR expression, compared to primary NK cells isolated from peripheral blood31,59,60. In addition to CAR expression, further enhancements to the antitumor activity of these cells can be achieved through modifications such as the deletion of inhibitory receptors or the introduction of cytokine expression41,39,61. This can be achieved through a single genetic modification event, eliminating the need for patient-specific modifications as seen in current CAR-T cell therapies31,62,63. Additionally, NK cell-based therapies with novel CAR-expressing cells can potentially be used in treating chronic infectious diseases30,64,65,66,67,68.
This protocol collectively demonstrates the potential of using human iPSCs to create homogeneous populations of CAR-expressing NK cells to enhance in vitro and in vivo antitumor activity. NK cell-optimized CARs enable antigen-specific activation of signaling pathways, improving the function of these cells. Modifications and troubleshooting are essential for optimizing the yield and functionality of CAR NK cells. Adjusting the cytokine cocktail and timing can improve differentiation efficiency. Ensuring the stability and expression of the CAR construct in NK cells may require optimizing transfection methods or vector design. Additional troubleshooting could address issues such as low transduction efficiency, cell viability, and functional activity of CAR iPSC derived NK cells, and may involve iterative testing of different culture conditions or genetic modifications to enhance persistence, cytotoxicity, and safety.
The significance of our method lies in its ability to generate a potentially unlimited supply of CAR NK cells from a standardized iPSC source. iPSC-derived CAR NK cells offer a stable platform for multiple gene edits, reducing variability and improving the consistency of the engineered cells. This method also enables the creation of "off-the-shelf" NK cell therapies that are ready for immediate use, bypassing the need for individualized cell sourcing and processing.
In addition, this protocol has certain limitations in maintaining the stability of CAR expression when using piggyBac vectors, as the transient nature of transgene integration may result in variable expression levels over time. Furthermore, the reliance on aAPCs and specific cytokines for expansion can complicate the scalability of these processes for clinical applications. Lastly, potential immune responses against the piggyBac elements may pose risks in therapeutic settings.
In conclusion, the differentiation and expansion of CAR-NK cells from human iPSCs hold significant promise for treating hepatocellular carcinoma (HCC) and other malignancies. This innovative approach leverages the regenerative potential of iPSCs and the innate immune properties of NK cells to create a potent and targeted cancer immunotherapy. Advances in this field could lead to more effective and widely available NK cell-based therapies, offering new hope for patients with HCC and other hard-to-treat diseases.
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.
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.
Name | Company | Catalog Number | Comments |
aAPC | Dean A. Lee lab | N/A | |
a-MEM culture medium | Fisher Scientific | Cat#12634 | |
APC anti-DYKDDDDK (FLAG Tag) | BD Biosciences | Cat#637308 | |
APC-anti-human TRA-1-81 | ThermoFisher | 17-8883-42 | |
APC-CD16 | BD Biosciences | Cat#302015 | |
APC-CD43 | BD Biosciences | Cat# 560198 | |
APC-CD45 | BD Biosciences | Cat# 555485 | |
APC-GPC3 | BD Biosciences | Cat#DB100B | |
APC-NKG2D | BD Biosciences | Cat# 558071 | |
CellEvent Caspase-3/7 Green Detection Reagent | Thermo fisher | Cat#C10423 | |
CellTrace Violet Cell Proliferation Kit | Thermo fisher | Cat#C34571 | Cell tracing fluorescent dye |
CryoStor solution | Stem Cell Technologies | https://www.stemcell.com/products/cryostor-cs10.html | Cryopreservation medium |
CTS NK Xpander | Gibco | A5019001 | |
CTS NK-Xpander Medium | Life Technologies | Cat#A5019001 | |
DMEM | Gibco | 11965084 | |
DMEM, high glucose, GlutaMAX Supplement, pyruvate | Gibco | 10569010 | |
EasySep Human NK Cell Enrichment Kit | StemCell Technologies, Inc. | Cat#19055 | |
Ethanolamine | Sigma Aldrich | E9508 | |
Fetal bovine serum | Fisher Scientific | Cat# 10437010 | |
FITC-CD94 | BD Biosciences | Cat#555888 | |
GlutaMAX Supplement | Gibco | 35050061 | |
GolgiPlug | BD Biosciences | Cat#555029 | |
GolgiStop | BD Biosciences | Cat#554724 | |
Ham's F-12 Nutrient Mix, GlutaMAX Supplement | Gibco | 31765035 | |
Horse serum | Fisher Scientific | Cat#16050130 | |
Human Serum | AB Sigma-Aldrich | Cat#BP2525100 | |
Human Stem Cell NucleofectorTM Kit | Lonza | Cat# VPH-5012 | |
Human: HePG2 cells | ATCC | Cat#HB-8065 | |
Human: HePG2-td-tomato-luc cells | Dan S. Kaufman lab | N/A | |
Human: iPS cells | Dan S. Kaufman lab | N/A | |
Human: SNU-449 cells | ATCC | Cat#CRL-2234 | |
Human: SNU-449-td-tomato-luc cells | Dan S. Kaufman lab | N/A | |
IncuCyte Caspase-3/7 Green Apoptosis Assay | Essenbioscience | Cat#4440 | |
L-Ascorbic acid | Sigma Aldrich | A5960 | |
MP Biomedicals Human Serum, Type AB | MP Biomedicals | ICN2938249 | |
mTeSR plus | StemCell Technologies, Inc. | 100-0276 | |
NovoExpress software | ACEA Biosciences | https://www.agilent.com/en/product/research-flow-cytometry/flow-cytometry-software/novocyte-novoexpress-software-1320805 | |
PE/Cy7 anti-human SSEA-4 Antibody | Biolegend | 330420 | |
PE-CD16 | BD Biosciences | Cat#560995 | |
PE-CD226 | BD Biosciences | Cat#559789 | |
PE-CD34 | BD Biosciences | Cat# 555822 | |
PE-CD45 | BD Biosciences | Cat# 555483 | |
PE-CD94 | BD Biosciences | Cat#555888 | |
PE-cy7-CD56 | BioLegend | Cat# 318318 | |
PE-FAS Ligand | BD Biosciences | Cat#564261 | |
PE-NKp30 | BD Biosciences | Cat# 558407 | |
PE-NKp44 | BD Biosciences | Cat#558563 | |
PE-NKp46 | BD Biosciences | Cat#331908 | |
PE-NKp46 | BD Biosciences | Cat#557991 | |
Peripheral blood buffy coat | San Diego Blood Bank (https://www. sandiegobloodbank.org/) | N/A | |
PE-TRAIL | BD Biosciences | Cat#565499 | |
pKT2-mCAG-IRES-GFP-ZEO | Branden Moriarity lab | N/A | |
pMAX-GFP plasmid | Lonza | N/A | GFP positive control |
Prism 9 | Graphpad | Version 9 | |
pSpCas9 | GenScript | PX165 | |
RBC Lysis Buffer (10x) | Biolegend | Cat#420301 | |
Recombinant human bFGF basic | R&D Systems | Cat#4114-TC | |
Recombinant human BMP-4 | PeproTech | Cat#120-05 | |
Recombinant human FLT-3 Ligand | PeproTech | Cat# 300-19 | |
Recombinant human IL-15 | PeproTech | Cat# 200-15 | |
Recombinant human IL-2 | PeproTech | Cat# 200-02 | |
Recombinant human IL-3 | PeproTech | Cat#200-03 | |
Recombinant human IL-7 | PeproTech | Cat# 200-07 | |
Recombinant Human Nodal Protein | R&D Systems | Cat#3218-ND-025 | |
Recombinant human SCF | PeproTech | Cat# 300-07 | |
Recombinant Human TGF-β1 | PeproTech | Cat#100-21 | |
Recombinant human VEGF | PeproTech | Cat# 100-20 | |
RPMI1640 | Gibco | 11875093 | |
Sodium selenite | Sigma Aldrich | 214485 | |
STEMdiff APEL 2 Medium | StemCell Technologies, Inc. | 5270 | EB formation medium |
STEMdiff APEL2 Medium | StemCell Technologies, Inc. | Cat#05270 | |
Super piggyBac Transposase expression vector | SBI | Cat#PB210PA-1 | |
SYTOX AADvanced Dead Cell Stain Kit | ThermoFisher Scientific | S10274, S10349 | Dead cell staining solution kit |
β-mercaptoethanol | Gibco | 21985023 |
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