Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.

<h4>Rationale</h4>Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are requir...

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Autores principales:	Hideki Uosaki, Hiroyuki Fukushima, Ayako Takeuchi, Satoshi Matsuoka, Norio Nakatsuji, Shinya Yamanaka, Jun K Yamashita
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/65b52f69f726406fbf7c0edc71877cb3
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spelling	oai:doaj.org-article:65b52f69f726406fbf7c0edc71877cb32021-11-18T06:47:39ZEfficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.1932-620310.1371/journal.pone.0023657https://doaj.org/article/65b52f69f726406fbf7c0edc71877cb32011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21876760/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Rationale</h4>Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs.<h4>Method and result</h4>We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2)-positive cardiomyocytes appeared robustly with 30-70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1) antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7-8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95-98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium), CD140b (pericytes) and TRA-1-60 (undifferentiated hESCs/hiPSCs). 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5-10×10(5) VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines.<h4>Conclusion</h4>We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from hESC/hiPSCs. These findings would offer a valuable technological basis for hESC/hiPSC-based cell therapy.Hideki UosakiHiroyuki FukushimaAyako TakeuchiSatoshi MatsuokaNorio NakatsujiShinya YamanakaJun K YamashitaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 8, p e23657 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Hideki Uosaki Hiroyuki Fukushima Ayako Takeuchi Satoshi Matsuoka Norio Nakatsuji Shinya Yamanaka Jun K Yamashita Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.
description	<h4>Rationale</h4>Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs.<h4>Method and result</h4>We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2)-positive cardiomyocytes appeared robustly with 30-70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1) antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7-8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95-98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium), CD140b (pericytes) and TRA-1-60 (undifferentiated hESCs/hiPSCs). 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5-10×10(5) VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines.<h4>Conclusion</h4>We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from hESC/hiPSCs. These findings would offer a valuable technological basis for hESC/hiPSC-based cell therapy.
format	article
author	Hideki Uosaki Hiroyuki Fukushima Ayako Takeuchi Satoshi Matsuoka Norio Nakatsuji Shinya Yamanaka Jun K Yamashita
author_facet	Hideki Uosaki Hiroyuki Fukushima Ayako Takeuchi Satoshi Matsuoka Norio Nakatsuji Shinya Yamanaka Jun K Yamashita
author_sort	Hideki Uosaki
title	Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.
title_short	Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.
title_full	Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.
title_fullStr	Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.
title_full_unstemmed	Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.
title_sort	efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by vcam1 surface expression.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/65b52f69f726406fbf7c0edc71877cb3
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Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.

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