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

The scope of our research mainly focuses on the development of human iPSC CAR NK cells with improved anti-tumor activity and in-vivo persistence to better treat diverse maligns. With this novel CAR iPSC NK generation protocol, we are aiming to answer how these cells can overcome tumor micro environmental resistance and improve efficacy and ensure safe and scalable production. The main experimental challenges in developing iPSC derived CAR NK cells include achieving consistent and efficient differentiation of iPSC into functional in cases ensuring stable CAR expression without any adverse effects and improving in-vivo persistence.

Additionally, researchers face challenges in scalability, immune rejections and regulatory holders for clinical applications. We have significantly advanced the iPSC derived CAR NK cell research by establishing efficient non-viral based CAR gene integration by using transposon-based gene modifications. Additionally, we have optimized CRISPR-Cas9 at the iPSC level to delete immune checkpoints, TMA factors to improve and cases cells and tumor efficacy and in-vivo persistence in solid tumor models.

With our protocol using non-viral piggyback vector mediated CAR gene expression, we are aiming to address the gap for the development of safe, efficient and scalable gene modification for iPSC CAR NK cells, this method reduces the risk of insertional mutagenesis enhances the reproducibility and cost effectiveness of generating CAR NK cells for cancer immunotherapy. In future our laboratory will focuses on the development of novel engineered iPSC derived NK cells targeting immune checkpoints. We will also investigate targeting various tumor micro environmental factors by using bispecific or trispecific antibodies to enhance specificity in vivo persistence and anti-tumor activity.

To begin spin embryo body or EB formation passage, approximately 200, 000 engineered CAR iPSCs per well in a six well basement membrane matrix pre-coded plate containing MT surplus, incubate the cells at 37 degrees Celsius with 5%carbon dioxide, after two days, remove the culture medium and detach the cells by incubating with one milliliter of prewarm TrypLE Select at 37 degrees Celsius for five minutes. Then carefully dissociate the cell aggregates into a single cell suspension and transfer the suspension to a 15 milliliter conical tube. Add three milliliters of PBS to stop the dissociation reaction.

Next, re-suspend the cells in MK SR-plus medium and filter them through a 70 micrometer cell strainer to remove aggregates. Centrifuge the cells. Wash the pellet with five milliliters of PBS and then re-suspend them in one milliliter of EB formation medium.

After counting, dilute the cells to the appropriate density using an EB formation medium containing cytokines and 10 micromolar ROCK inhibitor. Using a multi-channel pipette seed 3000 to 10, 000 cells per well in 100 microliters of EB media in a 96 well plate. Centrifuge the cells at 300 G for five minutes and incubate at 37 degrees Celsius with 5%carbon dioxide for six days.

To check embryo body quality first prepare four milliliters of pre-warmed 0.25%trypsin with 0.4%chicken serum for dissociating the EBs. after six days transfer 10 to 30 EBs to a 15 milliliter conical tube. Add trypsin chicken serum solution to the EBs and incubate them in a water bath.

Vortexing the EBs during disassociation every three to five minutes for 10 minutes. Pipette the trypsinized EBs mixing up and down several times to ensure thorough dissociation. Then add five milliliters of PBS to stop the trypsinization process.

Filter the cell solution through a 70 micrometer cell strainer into a new conical tube. Next centrifuge the cells at 300 G for five minutes and re-suspend the pellet in three to five milliliters of fresh EB media. After counting, stain the cells with typical EB markers.

Add the recommended volume of antibodies to each tube containing dissociated single cells of EB in 100 microliters of flow buffer and incubate on ice for 30 minutes. After incubation, wash the cells twice with flow buffer. Add SYTOX blue live dead stain and analyze using flow cytometry analysis.

Begin by coding each well of a six well plate with five to 10 microliters of a 2%sterilized gelatin solution. Incubate the plate at 37 degrees Celsius for two to four hours. After incubation, aspirate any remaining gelatin solution and add three milliliters of the natural killer or NK differentiation medium containing cytokines to each well of the gelatin coated six well plate.

Next using a one milliliter pipette, carefully transfer spin EBs derived from engineered CAR iPSCs to a 10 centimeter dish. Add three milliliters of NK differentiation medium containing cytokines to each well of the six well plate. Using a one milliliter micro pipette distributes 16 to 20 EBs into each well of the six well plate and incubate at 37 degrees Celsius with 5%carbon dioxide for three to four weeks.

After incubation evaluate the expression of NK cell markers CD45 positive and CD56 positive by flow cytometry on suspension cells. When more than 80%of suspension cells express CD45 positive and CD56 positive. Harvest the cells by passing them through a 70 micrometer filter to remove clumps.

Prior to co-culturing artificial antigen presenting cells or artificial APCs within NK cells irradiate the artificial APCs with 100 gray and preserve them as frozen stalks. After harvesting NK cells, culture them at a density of five times 10 to the power of five cells per milliliter in NK expansion medium supplemented with 50 units per milliliter of Interleukin-2 and Interleukin-15. Add thaw irradiated artificial APCs to the NK cells at a one to one ratio and incubate at 37 degrees Celsius with 5%carbon dioxide.

To begin turn on the flow cytometer. Evaluate the NK activation receptor's expression on the anti GPC3 CAR iPSC derived NK cells to determine the car case cell activity and maturation status. For the Caspase-3/7 assays.

Count the target cells in GPC3 CAR iPSC derived NK cells and pre-stain them with CellTrace Violet dye at a final concentration of five micromolar in PBS under light protection for 15 minutes at 37 degrees Celsius. Wash the stain target cells in a complete culture medium Before co-culture with CAR iPSC derived NK cells at various effector to target ratios at 37 degrees Celsius. After three hours and 30 minutes of co-culture.

Add Caspase-3/7 green detection reagent and incubate for another 30 minutes. Add SYTOX dead cell staining solution during the final five minutes of staining and mix gently. Analyze the stained cells using flow cytometry.

The formation of spin EBs from GPC3 CAR engineered IPCs was observed on day six. Confirming early differentiation. Differentiated natural killer or NK cells from GPC3 CAR IPCs showed distinct morphology at various stages from day three to day 28.

Flow cytometric analysis demonstrates the expression of hematopoietic antigens CD34 and CD31 as well as a small amount of CD43 but not yet expressing CD45 in both wild type and GPC3 CAR iPSCs derived spin EBs on day six. In case-specific markers were detected in mature wild type in GPC3 CAR iPSC derived NK cells on day 35. Confirming successful differentiation into NK cells.

Expanded wild type and anti GPC3 CAR iPSC derived NK cells exhibited various activation markers, demonstrating NK cell maturation. GPC3 antigen expression was confirmed on tumor cell lines using flow cytometry, validating the target specificity for anti GPC3 CAR iPSC derived NK cells. Anti GPC3 CAR iPSC derived NK cells demonstrated higher cytotoxic activity against GPC3 expressing HepG2, SNU-449, CAL 27 and SKOV3 cell lines compared to wild type iPSC NK cells.