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 t....

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% paraform.......

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