Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression
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 transdu...
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| Veröffentlicht in: | PloS one 2011-08, Vol.6 (8), p.e23657-e23657 |
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| creator | Uosaki, Hideki Fukushima, Hiroyuki Takeuchi, Ayako Matsuoka, Satoshi Nakatsuji, Norio Yamanaka, Shinya Yamashita, Jun K |
| description | 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.
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.
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. |
| doi_str_mv | 10.1371/journal.pone.0023657 |
| format | Article |
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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.
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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0023657</identifier><identifier>PMID: 21876760</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Antibodies ; Biology ; Biomarkers - metabolism ; Bone marrow ; Calcium-binding protein ; Cardiomyocytes ; Cell adhesion ; Cell adhesion & migration ; Cell adhesion molecules ; Cell culture ; Cell growth ; Cell Line ; Cell Membrane - metabolism ; Cell Separation - methods ; Cell surface ; Differentiation ; Drug resistance ; Embryo cells ; Embryonic Stem Cells - cytology ; Embryonic Stem Cells - metabolism ; Embryos ; Endothelium ; Fibroblasts ; Fluorescence ; Genetic modification ; Genetically modified organisms ; Growth factors ; Heart cells ; Heart diseases ; Humans ; Induced Pluripotent Stem Cells - cytology ; Induced Pluripotent Stem Cells - metabolism ; Laboratories ; Medical research ; Methods ; Monomolecular films ; Myocytes, Cardiac - cytology ; Myocytes, Cardiac - metabolism ; Pericytes ; Pluripotency ; Purification ; R&D ; Regenerative medicine ; Research & development ; Stem cells ; Surface markers ; Therapy ; Transplantation ; Troponin ; Vascular cell adhesion molecule 1 ; Vascular Cell Adhesion Molecule-1 - metabolism</subject><ispartof>PloS one, 2011-08, Vol.6 (8), p.e23657-e23657</ispartof><rights>COPYRIGHT 2011 Public Library of Science</rights><rights>2011 Uosaki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Uosaki et al. 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c757t-95c3c77618cef6887010814edc08408a4f835fb4e7af82371c466541bb6322b83</citedby><cites>FETCH-LOGICAL-c757t-95c3c77618cef6887010814edc08408a4f835fb4e7af82371c466541bb6322b83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158088/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158088/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21876760$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Uosaki, Hideki</creatorcontrib><creatorcontrib>Fukushima, Hiroyuki</creatorcontrib><creatorcontrib>Takeuchi, Ayako</creatorcontrib><creatorcontrib>Matsuoka, Satoshi</creatorcontrib><creatorcontrib>Nakatsuji, Norio</creatorcontrib><creatorcontrib>Yamanaka, Shinya</creatorcontrib><creatorcontrib>Yamashita, Jun K</creatorcontrib><title>Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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.
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.
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.</description><subject>Antibodies</subject><subject>Biology</subject><subject>Biomarkers - metabolism</subject><subject>Bone marrow</subject><subject>Calcium-binding protein</subject><subject>Cardiomyocytes</subject><subject>Cell adhesion</subject><subject>Cell adhesion & migration</subject><subject>Cell adhesion molecules</subject><subject>Cell culture</subject><subject>Cell growth</subject><subject>Cell Line</subject><subject>Cell Membrane - metabolism</subject><subject>Cell Separation - methods</subject><subject>Cell surface</subject><subject>Differentiation</subject><subject>Drug resistance</subject><subject>Embryo cells</subject><subject>Embryonic Stem Cells - cytology</subject><subject>Embryonic Stem Cells - metabolism</subject><subject>Embryos</subject><subject>Endothelium</subject><subject>Fibroblasts</subject><subject>Fluorescence</subject><subject>Genetic modification</subject><subject>Genetically modified organisms</subject><subject>Growth factors</subject><subject>Heart cells</subject><subject>Heart diseases</subject><subject>Humans</subject><subject>Induced Pluripotent Stem Cells - cytology</subject><subject>Induced Pluripotent Stem Cells - metabolism</subject><subject>Laboratories</subject><subject>Medical research</subject><subject>Methods</subject><subject>Monomolecular films</subject><subject>Myocytes, Cardiac - cytology</subject><subject>Myocytes, Cardiac - metabolism</subject><subject>Pericytes</subject><subject>Pluripotency</subject><subject>Purification</subject><subject>R&D</subject><subject>Regenerative medicine</subject><subject>Research & development</subject><subject>Stem cells</subject><subject>Surface markers</subject><subject>Therapy</subject><subject>Transplantation</subject><subject>Troponin</subject><subject>Vascular cell adhesion molecule 1</subject><subject>Vascular Cell Adhesion Molecule-1 - metabolism</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk11v0zAUhiMEYmPwDxBYQgJx0WLHn71BqqoBlYYm8bFby3Hs1pMTBztB60_gX-Os3dSgXaBcOPJ5znuOX_sUxUsE5whz9OE6DLFVft6F1swhLDGj_FFxiha4nLES4sdH_yfFs5SuIaRYMPa0OCmR4IwzeFr8ObfWaWfaHqi2BkkrrypvQDdElwOqd6EFwQKtYu1Cswt615sEbAwN2A6NaoFpqrgLrdO3Aq6tB21q0Pks0IV-FE69aYA23idQ7cDVavkVgTREq7QB5qaLJqVc5XnxxCqfzIvDelb8_HT-Y_VldnH5eb1aXsw0p7yfLajGmnOGhDaWCcEhggIRU2soCBSKWIGprYjhyooyG6UJY5SgqmK4LCuBz4rXe93OhyQPLiaJMBQlJITSTKz3RB3Uteyia1TcyaCcvN0IcSNV7J32RjJa0dKyheUlI5DZynINcy88G8x1hbPWx0O1oWpyIPsRlZ-ITiOt28pN-C0xogKKsd13B4EYfg0m9bJxaTRTtSYMSQrB6EIQxDP55h_y4cMdqI3K_bvWhlxWj5pySXg2tMymZWr-AJW_2jRO5xdnXd6fJLyfJGSmNzf9Rg0pyfX3b__PXl5N2bdH7NYo329T8MP4LtMUJHtQx5BSNPbeYwTlODB3bshxYORhYHLaq-P7uU-6mxD8FyoqEek</recordid><startdate>20110818</startdate><enddate>20110818</enddate><creator>Uosaki, Hideki</creator><creator>Fukushima, Hiroyuki</creator><creator>Takeuchi, Ayako</creator><creator>Matsuoka, Satoshi</creator><creator>Nakatsuji, Norio</creator><creator>Yamanaka, Shinya</creator><creator>Yamashita, Jun K</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20110818</creationdate><title>Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression</title><author>Uosaki, Hideki ; Fukushima, Hiroyuki ; Takeuchi, Ayako ; Matsuoka, Satoshi ; Nakatsuji, Norio ; Yamanaka, Shinya ; Yamashita, Jun K</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c757t-95c3c77618cef6887010814edc08408a4f835fb4e7af82371c466541bb6322b83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Antibodies</topic><topic>Biology</topic><topic>Biomarkers - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Uosaki, Hideki</au><au>Fukushima, Hiroyuki</au><au>Takeuchi, Ayako</au><au>Matsuoka, Satoshi</au><au>Nakatsuji, Norio</au><au>Yamanaka, Shinya</au><au>Yamashita, Jun K</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2011-08-18</date><risdate>2011</risdate><volume>6</volume><issue>8</issue><spage>e23657</spage><epage>e23657</epage><pages>e23657-e23657</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>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.
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.
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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>21876760</pmid><doi>10.1371/journal.pone.0023657</doi><tpages>e23657</tpages><oa>free_for_read</oa></addata></record> |
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| language | eng |
| recordid | cdi_plos_journals_1308204455 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Antibodies Biology Biomarkers - metabolism Bone marrow Calcium-binding protein Cardiomyocytes Cell adhesion Cell adhesion & migration Cell adhesion molecules Cell culture Cell growth Cell Line Cell Membrane - metabolism Cell Separation - methods Cell surface Differentiation Drug resistance Embryo cells Embryonic Stem Cells - cytology Embryonic Stem Cells - metabolism Embryos Endothelium Fibroblasts Fluorescence Genetic modification Genetically modified organisms Growth factors Heart cells Heart diseases Humans Induced Pluripotent Stem Cells - cytology Induced Pluripotent Stem Cells - metabolism Laboratories Medical research Methods Monomolecular films Myocytes, Cardiac - cytology Myocytes, Cardiac - metabolism Pericytes Pluripotency Purification R&D Regenerative medicine Research & development Stem cells Surface markers Therapy Transplantation Troponin Vascular cell adhesion molecule 1 Vascular Cell Adhesion Molecule-1 - metabolism |
| title | Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-08T10%3A09%3A00IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Efficient%20and%20scalable%20purification%20of%20cardiomyocytes%20from%20human%20embryonic%20and%20induced%20pluripotent%20stem%20cells%20by%20VCAM1%20surface%20expression&rft.jtitle=PloS%20one&rft.au=Uosaki,%20Hideki&rft.date=2011-08-18&rft.volume=6&rft.issue=8&rft.spage=e23657&rft.epage=e23657&rft.pages=e23657-e23657&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0023657&rft_dat=%3Cgale_plos_%3EA476882237%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1308204455&rft_id=info:pmid/21876760&rft_galeid=A476882237&rft_doaj_id=oai_doaj_org_article_65b52f69f726406fbf7c0edc71877cb3&rfr_iscdi=true |