Name: immunological elimination of undifferentiated human pluripotent stem cells using pluripotent state-specific antigen, glypican-3
Uploaded: 2019-12-10T10:36:16
Description: Much attention has been focused on human pluripotent stem cells (hPSCs) due to their potential as cell sources in regenerative therapies. Especially in ...

This work was mainly supported by the Research Project for Practical Application of Regenerative Medicine from the Japan Agency for Medical Research and Development (AMED) and partly supported by the National Cancer Center Research and Development Fund (25-A-7) and (28-A-8), as well as Health and Labor Science Research Grants for Clinical Research on Applying Health Technology and Research for Promotion of Cancer Control Programmes, Japan.

1. Immunofluorescence staining of hiPSCs with GPC3 and OCT4
<ol>
	<li>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 &#176;C in a 5% CO2 incubator overnight.</li>
	<li>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.</li>
	<li>Fix cells with 500 &#181;L of 4% paraform...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypicans:+proteoglycans+with+a+surprise.">Glypicans: proteoglycans with a surprise.</a> Journal of Clinical Investigation. 108 (4), 497-501 (2001).</li><li>Baumhoer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypican+3+expression+in+human+nonneoplastic,+preneoplastic,+and+neoplastic+tissues:+a+tissue+microarray+analysis+of+4,387+tissue+samples.">Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples.</a> American Journal of Clinical Pathology. 129 (6), 899-906 (2008).</li><li>Kandil, D., Leiman, G., Allegretta, M., Evans, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypican-3+protein+expression+in+primary+and+metastatic+melanoma:+a+combined+immunohistochemistry+and+immunocytochemistry+study.">Glypican-3 protein expression in primary and metastatic melanoma: a combined immunohistochemistry and immunocytochemistry study.</a> Cancer. 117 (4), 271-278 (2009).</li><li>Kinoshita, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypican+3+expression+in+pediatric+malignant+solid+tumors.">Glypican 3 expression in pediatric malignant solid tumors.</a> European Journal of Pediatric Surgery. 25 (1), 138-144 (2015).</li><li>Maeda, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypican-3+expression+in+clear+cell+adenocarcinoma+of+the+ovary.">Glypican-3 expression in clear cell adenocarcinoma of the ovary.</a> Modern Pathology. 22 (6), 824-832 (2009).</li><li>Saikali, Z., Sinnett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+glypican+3+(GPC3)+in+embryonal+tumors.">Expression of glypican 3 (GPC3) in embryonal tumors.</a> International Journal of Cancer. 89 (5), 418-422 (2000).</li><li>Stadlmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypican-3+expression+in+primary+and+recurrent+ovarian+carcinomas.">Glypican-3 expression in primary and recurrent ovarian carcinomas.</a> International Journal of Gynecological Pathology. 26 (3), 341-344 (2007).</li><li>Toretsky, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypican-3+expression+in+Wilms+tumor+and+hepatoblastoma.">Glypican-3 expression in Wilms tumor and hepatoblastoma.</a> Journal of Pediatric Hematology/Oncology. 23 (8), 496-499 (2001).</li><li>Tohyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Large-Scale+2D+Culture+System+for+Human+Induced+Pluripotent+Stem+Cells+and+Differentiated+Cardiomyocytes.">Efficient Large-Scale 2D Culture System for Human Induced Pluripotent Stem Cells and Differentiated Cardiomyocytes.</a> Stem Cell Reports. 9 (5), 1406-1414 (2017).</li><li>Tohyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+Oxidation+Is+Indispensable+for+Survival+of+Human+Pluripotent+Stem+Cells.">Glutamine Oxidation Is Indispensable for Survival of Human Pluripotent Stem Cells.</a> Cell Metabolism. 23 (4), 663-674 (2016).</li><li>Sawada, Y., Sakai, M., Yoshikawa, T., Ofuji, K., Nakatsura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glypican-3-derived+peptide+vaccine+against+hepatocellular+carcinoma.">A glypican-3-derived peptide vaccine against hepatocellular carcinoma.</a> Oncoimmunology. 1 (8), 1448-1450 (2012).</li><li>Hattori, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nongenetic+method+for+purifying+stem+cell-derived+cardiomyocytes.">Nongenetic method for purifying stem cell-derived cardiomyocytes.</a> Nature Methods. 7 (1), 61-66 (2010).</li><li>Tohyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+metabolic+flow+enables+large-scale+purification+of+mouse+and+human+pluripotent+stem+cell-derived+cardiomyocytes.">Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.</a> Cell Stem Cell. 12 (1), 127-137 (2013).</li></ol>

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 antigens3,4,5,<sup class=...

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 shortage1,2.
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 derivatives3,4,5,6,7. 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 embryos9,10,11. 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 carcinomas11,13,14,15,16,17,18,19. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100-mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15ml Centrifuge Tube</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Centrifuge Tube</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353078</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-21200</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-rat IgG</td>
			<td>Invitrogen</td>
			<td>A-21470</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11003</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 anti-rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-11010</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td>Thaw completely at 4 &#176;C overnight and dilute it 50 times with Dulbecco&#39;s Modified Eagle&#39;s Medium before coating culture dishes</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein-AM</td>
			<td>Dojindo</td>
			<td>C396</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Tracker Green CMFDA</td>
			<td>Thermo Fisher Scientific</td>
			<td>C7025</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cTroponin I antibody</td>
			<td>abcam</td>
			<td>ab52862</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS(&#8211;)</td>
			<td>Wako</td>
			<td>045-29795</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GPC3 antibody</td>
			<td>biomosaics</td>
			<td>B0025R</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human/mouse OCT-3/4 antibody</td>
			<td>R&#38;D</td>
			<td>MAB1759</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>STEM CELL</td>
			<td>5850</td>
			<td>Warm at room temperature before use</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum, Fetal Bovine</td>
			<td>biowest</td>
			<td>S1780</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>StemProAccutase</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1110501</td>
			<td>dissociation buffer</td>
			<td></td>
		</tr>
		<tr>
			<td>troponin T antibody</td>
			<td>thermo</td>
			<td>MA5-12960</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Wako Pure Chemical Industries</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>12571048</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunological elimination of undifferentiated human pluripotent stem cells using pluripotent state-specific antigen, glypican-3

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.

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...

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.

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

Much attention has been focused on human pluripotent stem cells (hPSCs) due to their potential as cell sources in regenerative therapies. Especially in ...