Immunological Elimination of Undifferentiated Human Pluripotent Stem Cells Using Pluripotent State-Specific Antigen, Glypican-3

Summary

Regenerative therapies using human induced pluripotent stem cells (hiPSCs) have recently attracted much attention. In this study, we use anticancer immunotherapy with peptide vaccination to prevent hiPSC-derived tumorigenesis. Our results show that glypican-3 (GPC3) works as a pluripotent state-specific immunogenic antigen in hiPSCs.

Abstract

Much attention has been focused on human pluripotent stem cells (hPSCs) due to their potential as cell sources in regenerative therapies. Especially in the area of cardiac regenerative medicine, which is challenged by organ shortage, transplantation of human induced pluripotent stem cells (hiPSC)-derived cardiomyocytes has potential to treat many patients with severe heart failure. However, to achieve transplantation of hiPSC-derived cardiomyocytes, removal of contaminated immature cells with high accuracy is essential to eliminate the risk of teratoma formation caused by residual undifferentiated hiPSCs. Peptide vaccination is well-known as an effective anticancer immunotherapy because of selective cellular cytotoxicity. To establish immunological elimination of contaminated immature hiPSCs, we identified glypican-3 (GPC3) as a pluripotent state-specific carcinoembryonic antigen. Immunostaining showed that hiPSCs expressed GPC3, especially in pluripotent states. Undifferentiated hiPSCs were rejected by cytotoxic T cell (CTL) clones sensitized with HLA-class I-restricted GPC3 peptides. These results indicate that GPC3-specific CTLs can prevent hiPSC-derived tumorigenesis, which may occur by contamination by undifferentiated cells. Our results indicate that GPC3 works as a pluripotent state-specific immunogenic antigen in hiPSCs. These results show the applicability of GPC3-mediated immunotherapy to ensure safety in regenerative medical procedures using hiPSCs.

Introduction

Recently, regenerative therapies using human induced pluripotent stem cells (hiPSCs) have attracted much attention as new cell sources for regenerative therapies. Especially in cardiac regenerative medicine, transplantation of hiPSC-derived cardiomyocytes is expected to resolve the challenge of organ shortage^1,2.

Retinal regeneration requires a small number of cells; hence, the possibilities of tumor formation due to residual undifferentiated stem cells are negligible. In contrast, regenerative procedures of the heart and liver, which require a large number of cells, are difficult to perform safely.

Until now, many different methods of eliminating undifferentiated human pluripotent stem cells (hPSCs) from hiPSC derivatives have been reported because of teratoma formation induced by undifferentiated hPSC contamination in hiPSC derivatives^3,4,5,6,7. However, to achieve transplantation of hiPSC-derived cardiomyocytes, complete removal of residual undifferentiated cells in vivo is important because a massive number of cells is required for transplantation.

Peptide vaccination has been used for cancer patients as an anticancer immunotherapy with selective cellular cytotoxicity⁸. In this study, we aim to prevent hiPSC-derived tumorigenesis with CTLs using peptide vaccination methods.

GPC3 is one of the carcinoembryonic antigens and is widely expressed in human embryos^9,10,11. It is also overexpressed in 72-81% of patients with hepatocellular carcinoma (HCC)¹², and expression of GPC3 has been reported in melanoma, Wilms tumor, hepatoblastoma, ovarian clear cell adenocarcinoma, yolk sac tumor, and other carcinomas^{11,13,14,15,16,17,18,19}. In this study, we report that hiPSCs uniquely express the oncofetal antigen GPC3, and GPC3-specific CTLs can be used in immunotherapy for removal of undifferentiated hiPSCs from hiPSC derivatives for future regenerative medical procedures.

Protocol

1. Immunofluorescence staining of hiPSCs with GPC3 and OCT4

1. Grow cultured hiPSCs in stabilized feeder-free maintenance medium (mTESR1; Table of Materials) at a density of 3 x 10⁴ cells/cm² using 12 well plates. Incubate the dish at 37 °C in a 5% CO₂ incubator overnight.
2. Remove the medium of each well and wash with 1 mL of phosphate-buffered saline (PBS) for each well 1x and then remove the PBS.
3. Fix cells with 500 µL of 4% paraformaldehyde for 30 min at 4 °C.
4. Wash 3x with 1 mL of PBS for each well and then remove PBS.
5. Permeabilize with 500 µL of 0.2% triton X-100 in PBS for 15 min at room temperature (RT).
6. Remove triton X-100 in PBS, wash cells with 1 mL of PBS 3x for 5 min, and then remove PBS.
7. Incubate cells in the anti-GPC3 and anti-OCT4 antibodies diluted 1:200 in 2% fetal bovine serum (FBS) in PBS overnight at 4 °C.
8. Decant the solution and wash the cells 3x for 5 min in PBS.
9. Incubate the cells with the secondary antibodies (Alexa Fluor 488 anti-mouse IgG, Alexa Fluor 546 anti-rat IgG) diluted 1:200 in 2% FBS in PBS for 1 h at RT in the dark.
10. Decant the secondary antibody solution and wash with PBS 1x for 5 min in the dark.
11. Stain cells with 4',6-diamidino-2-phenylindole (DAPI; 1 µg/mL final concentration) for 5 min at RT.
12. Decant the solution containing DAPI and then wash the cells 2x with PBS for 5 min in the dark. After the wash, observe by fluorescence microscopy.

2. Cell viability assays

1. Culture hiPSCs in 96 well plates at a density of 1 x 10⁴ cells/cm² in mTESR1 with ROCK inhibitor at a final concentration of 10 µM at 37 °C.
2. Culture hiPSC-derived cardiomyocytes in 96 well plates at a density of 2 x 10⁴ cells per well with alpha minimum essential medium (αMEM) with 5% FBS at 37 °C in static culture.
   NOTE: hiPSC-derived cardiomyocytes were generated as previously described^20,21.
3. After 24 h, remove the medium in steps 2.1 and 2.2.
4. Adjust HLA-A2 restricted-GPC3 specific CTLs to 5 x 10⁵ cells in 100 µL of each medium and add to both plates in steps 2.1 and 2.2.
   NOTE: HLA-A*02:01-restricted GPC3_144-152 (FVGEFFTDV)-reactive CTL clones were previously established using peripheral blood mononuclear cells from HCC patients administered a GPC3 vaccination²².
5. After 48 h of coculture with CTLs, stain cells with 1 µM calcein-AM by adding 1 µL of calcein-AM to 100 µL of αMEM (cardiomyocytes) or mTESR1 (hiPSCs) in each well.
6. Incubate plates for 15 min at 37 °C.
7. Remove each medium with calcein-AM and wash the wells 2x with PBS.
8. Observe live cells by fluorescence microscopy.

3. Coculture of hiPSCs and hiPSC-derived cardiomyocytes with GPC3-specific CTLs

1. Culture hiPSCs at a density of 1 x 10⁵ cells/cm² in 6 well plates with mTESR1 for 5−7 days at 37 °C in a 5% CO₂ incubator.
2. Culture hiPSC-derived cardiomyocytes at a density of 5 x 10⁴ cells/cm² in 12 well plates with αMEM for 3 days at 37 °C in a 5% CO₂ incubator.
3. Remove the mTESR1 medium from hiPSC culture and collect the cells with 500 µL of dissociation buffer for 5 min at 37 °C.Centrifuge cells in a 10 mL conical tube at 200 x g for 5 min at RT.
4. Remove the supernatant and fluorescently label hiPSCs with 1 mL of mTESR1 containing 1 µM green 5-chloromethylfluorescein diacetate (CMFDA), then incubate at 37 °C for 1 h.
5. After 1 h of incubation, replace the mTESR1 medium containing CMFDA with basic mTESR1 medium.
6. Seed labeled hiPSCs from step 3.5 (at a density of 1.5 x 10³ cells/cm²) in the 12 well plates where the cardiomyocytes are cultured. Then return the plates to the incubator.
7. After 24 h from seeding, adjust HLA-A2 restricted-GPC3 specific CTLs to 3 x 10⁶ cells in 100 µL of mTESR1 and coculture cells at 37 °C in a 5% CO₂ incubator in either the presence or absence of GPC3 CTLs.
8. After 48 h of coculture with CTLs, wash cells with PBS 3x for 5 min to remove CTLs. Then analyze by flow cytometry or immunofluorescence staining.
9. Dissociate with 500 µL of dissociation buffer for 5 min and collect the cells by centrifugation in 10 mL conical tubes at 200 x g for 5 min at RT.
10. After aspirating the supernatant, suspend the cells in 2% FBS in PBS using a pipette to disaggregate cells.
11. Prior to data analysis, vortex each tube to avoid cell aggregates.
12. Use flow cytometry to calculate the percentage viability of hiPSCs in cocultured cells (fluorescently labeled cells are hiPSCs).Collect a minimum of 10,000 events. For analyses, gate the live cell population excluding the dead cells. Determine the percentage of positive cells within the gated population.
13. To analyze using immunofluorescence staining (similar to section 1), incubate the cells from step 3.8 with diluted anti-troponin T and anti-OCT4 antibodies (use each at a dilution of 1:200) at 37 °C in a 5% CO₂ incubator. Then wash the cells 2x with PBS for 5 min in the dark. After the wash, observe by fluorescence microscopy.

Results

Immunofluorescent staining showed that GPC3 and the typical pluripotent stem cell marker OCT4 were expressed in pluripotent states of hiPSCs (Figure 1A). GPC3-specific CTL clones revealed cytotoxic effects against hiPSCs after coculture but not against hiPSC-derived cardiomyocytes (Figure 1B).

After 48 h of coculture, using flow cytomet...

Discussion

In this study, we identified glypican-3 (GPC3) as a pluripotent state-specific immunogenic antigen and validated the applicability of GPC3 to remove undifferentiated cells from hPSC derivatives.

To date, many methods for eliminating undifferentiated hPSCs in hiPSC derivatives have been reported, such as use of toxins, small molecules, and pluripotent state-specific antigens^3,4,5,

Materials

Name	Company	Catalog Number	Comments
100-mm tissue culture dish	Falcon	353003
15ml Centrifuge Tube	Greiner Bio-One	188271
50ml Centrifuge Tube	Greiner Bio-One	227261
96-well tissue culture plate	Falcon	353078
Alexa Fluor 488 anti-mouse IgG	Invitrogen	A-21200
Alexa Fluor 488 anti-rat IgG	Invitrogen	A-21470
Alexa Fluor 546 anti-mouse IgG	Invitrogen	A-11003
Alexa Fluor 546 anti-rabbit IgG	Invitrogen	A-11010
BD Matrigel Matrix Growth Factor Reduced	BD Biosciences	354230	Thaw completely at 4 °C overnight and dilute it 50 times with Dulbecco's Modified Eagle's Medium before coating culture dishes
Calcein-AM	Dojindo	C396
Cell Tracker Green CMFDA	Thermo Fisher Scientific	C7025
cTroponin I antibody	abcam	ab52862
DAPI	Thermo Fisher Scientific	D1306
D-PBS(–)	Wako	045-29795
GPC3 antibody	biomosaics	B0025R
Human/mouse OCT-3/4 antibody	R&D	MAB1759
mTeSR1 medium kit	STEM CELL	5850	Warm at room temperature before use
Serum, Fetal Bovine	biowest	S1780
StemProAccutase	Thermo Fisher Scientific	A1110501	dissociation buffer
troponin T antibody	thermo	MA5-12960
Y-27632	Wako Pure Chemical Industries
αMEM	Thermo Fisher Scientific	12571048