Isolation of Contractile Cardiomyocytes from Human Pluripotent Stem-Cell-Derived Cardiomyogenic Cultures Using a Human NCX1-EGFP Reporter
The prospective isolation of defined contractile human pluripotent stem cell (hPSC)–derived cardiomyocytes is advantageous for regenerative medicine and drug screening applications. Currently, enrichment of cardiomyocyte populations from such cultures can be achieved by combinations of cell surface...
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| Veröffentlicht in: | Stem cells and development 2015-01, Vol.24 (1), p.11-20 |
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| creator | Ovchinnikov, Dmitry A. Hidalgo, Alejandro Yang, Seung-Kwon Zhang, Xinli Hudson, James Mazzone, Stuart B. Chen, Chen Cooper-White, Justin J. Wolvetang, Ernst J. |
| description | The prospective isolation of defined contractile human pluripotent stem cell (hPSC)–derived cardiomyocytes is advantageous for regenerative medicine and drug screening applications. Currently, enrichment of cardiomyocyte populations from such cultures can be achieved by combinations of cell surface markers or the labor-intensive genetic modification of cardiac developmental genes, such as
NKX2.5
or
MYH6
, with fluorescent reporters. To create a facile, portable method for the isolation of contractile cardiomyocytes from cardiomyogenic hPSC cultures, we employed a highly conserved cardiac enhancer sequence in the
SLC8A1 (NCX1)
gene to generate a lentivirally deliverable, antibiotic-selectable NCX1cp-EGFP reporter. We show that human embryonic stem cells (and induced pluripotent stem cells) transduced with the NCX1cp-EGFP reporter cassette exhibit enhanced green fluorescent protein (EGFP) expression in cardiac progenitors from 5 days into the directed cardiac hPSC differentiation protocol, with all reporter-positive cells transitioning to spontaneously contracting foci 3 days later. In subsequent stages of cardiomyocyte maturation,
NCX1cp
-EGFP expression was exclusively limited to contractile cells expressing high levels of cardiac troponin T (CTNT), MLC2a/v, and α-actinin proteins, and was not present in CD90/THY1
+
cardiac stromal cells or CD31/PECAM
+
endothelial cells. Flow-assisted cytometrically sorted EGFP
+
fractions of differentiated cultures were highly enriched in both early (
NKX2.5
and
TBX5
) and late (CTNT/
TNNI2
,
MYH6
,
MYH7
,
NPPA
, and
MYL2
) cardiomyocyte markers, with a significant proportion of cells displaying a ventricular-like action potential pattern in patch-clamp recordings. We conclude that the use of the cardiac-specific promoter of the human
SLC8A1
(
NCX1
) gene is an effective strategy to isolate contractile cardiac cells and their progenitors from hPSC-derived cardiomyogenic cultures. |
| doi_str_mv | 10.1089/scd.2014.0195 |
| format | Article |
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NKX2.5
or
MYH6
, with fluorescent reporters. To create a facile, portable method for the isolation of contractile cardiomyocytes from cardiomyogenic hPSC cultures, we employed a highly conserved cardiac enhancer sequence in the
SLC8A1 (NCX1)
gene to generate a lentivirally deliverable, antibiotic-selectable NCX1cp-EGFP reporter. We show that human embryonic stem cells (and induced pluripotent stem cells) transduced with the NCX1cp-EGFP reporter cassette exhibit enhanced green fluorescent protein (EGFP) expression in cardiac progenitors from 5 days into the directed cardiac hPSC differentiation protocol, with all reporter-positive cells transitioning to spontaneously contracting foci 3 days later. In subsequent stages of cardiomyocyte maturation,
NCX1cp
-EGFP expression was exclusively limited to contractile cells expressing high levels of cardiac troponin T (CTNT), MLC2a/v, and α-actinin proteins, and was not present in CD90/THY1
+
cardiac stromal cells or CD31/PECAM
+
endothelial cells. Flow-assisted cytometrically sorted EGFP
+
fractions of differentiated cultures were highly enriched in both early (
NKX2.5
and
TBX5
) and late (CTNT/
TNNI2
,
MYH6
,
MYH7
,
NPPA
, and
MYL2
) cardiomyocyte markers, with a significant proportion of cells displaying a ventricular-like action potential pattern in patch-clamp recordings. We conclude that the use of the cardiac-specific promoter of the human
SLC8A1
(
NCX1
) gene is an effective strategy to isolate contractile cardiac cells and their progenitors from hPSC-derived cardiomyogenic cultures.</description><identifier>ISSN: 1547-3287</identifier><identifier>EISSN: 1557-8534</identifier><identifier>DOI: 10.1089/scd.2014.0195</identifier><identifier>PMID: 25075536</identifier><language>eng</language><publisher>United States: Mary Ann Liebert, Inc</publisher><subject>Cell Culture Techniques ; Cell Line ; Flow Cytometry ; Genes, Reporter ; Green Fluorescent Proteins - biosynthesis ; Green Fluorescent Proteins - genetics ; Humans ; Induced Pluripotent Stem Cells - cytology ; Induced Pluripotent Stem Cells - metabolism ; Myocardial Contraction ; Myocytes, Cardiac - cytology ; Myocytes, Cardiac - metabolism ; Original Research Reports ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - genetics ; Sodium-Calcium Exchanger - biosynthesis ; Sodium-Calcium Exchanger - genetics</subject><ispartof>Stem cells and development, 2015-01, Vol.24 (1), p.11-20</ispartof><rights>2015, Mary Ann Liebert, Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c337t-792cbd168949d08ab2cd4766577ddae14291c34f2a0f7fe77f92fcfb7659e55b3</citedby><cites>FETCH-LOGICAL-c337t-792cbd168949d08ab2cd4766577ddae14291c34f2a0f7fe77f92fcfb7659e55b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25075536$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ovchinnikov, Dmitry A.</creatorcontrib><creatorcontrib>Hidalgo, Alejandro</creatorcontrib><creatorcontrib>Yang, Seung-Kwon</creatorcontrib><creatorcontrib>Zhang, Xinli</creatorcontrib><creatorcontrib>Hudson, James</creatorcontrib><creatorcontrib>Mazzone, Stuart B.</creatorcontrib><creatorcontrib>Chen, Chen</creatorcontrib><creatorcontrib>Cooper-White, Justin J.</creatorcontrib><creatorcontrib>Wolvetang, Ernst J.</creatorcontrib><title>Isolation of Contractile Cardiomyocytes from Human Pluripotent Stem-Cell-Derived Cardiomyogenic Cultures Using a Human NCX1-EGFP Reporter</title><title>Stem cells and development</title><addtitle>Stem Cells Dev</addtitle><description>The prospective isolation of defined contractile human pluripotent stem cell (hPSC)–derived cardiomyocytes is advantageous for regenerative medicine and drug screening applications. Currently, enrichment of cardiomyocyte populations from such cultures can be achieved by combinations of cell surface markers or the labor-intensive genetic modification of cardiac developmental genes, such as
NKX2.5
or
MYH6
, with fluorescent reporters. To create a facile, portable method for the isolation of contractile cardiomyocytes from cardiomyogenic hPSC cultures, we employed a highly conserved cardiac enhancer sequence in the
SLC8A1 (NCX1)
gene to generate a lentivirally deliverable, antibiotic-selectable NCX1cp-EGFP reporter. We show that human embryonic stem cells (and induced pluripotent stem cells) transduced with the NCX1cp-EGFP reporter cassette exhibit enhanced green fluorescent protein (EGFP) expression in cardiac progenitors from 5 days into the directed cardiac hPSC differentiation protocol, with all reporter-positive cells transitioning to spontaneously contracting foci 3 days later. In subsequent stages of cardiomyocyte maturation,
NCX1cp
-EGFP expression was exclusively limited to contractile cells expressing high levels of cardiac troponin T (CTNT), MLC2a/v, and α-actinin proteins, and was not present in CD90/THY1
+
cardiac stromal cells or CD31/PECAM
+
endothelial cells. Flow-assisted cytometrically sorted EGFP
+
fractions of differentiated cultures were highly enriched in both early (
NKX2.5
and
TBX5
) and late (CTNT/
TNNI2
,
MYH6
,
MYH7
,
NPPA
, and
MYL2
) cardiomyocyte markers, with a significant proportion of cells displaying a ventricular-like action potential pattern in patch-clamp recordings. We conclude that the use of the cardiac-specific promoter of the human
SLC8A1
(
NCX1
) gene is an effective strategy to isolate contractile cardiac cells and their progenitors from hPSC-derived cardiomyogenic cultures.</description><subject>Cell Culture Techniques</subject><subject>Cell Line</subject><subject>Flow Cytometry</subject><subject>Genes, Reporter</subject><subject>Green Fluorescent Proteins - biosynthesis</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Humans</subject><subject>Induced Pluripotent Stem Cells - cytology</subject><subject>Induced Pluripotent Stem Cells - metabolism</subject><subject>Myocardial Contraction</subject><subject>Myocytes, Cardiac - cytology</subject><subject>Myocytes, Cardiac - metabolism</subject><subject>Original Research Reports</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Sodium-Calcium Exchanger - biosynthesis</subject><subject>Sodium-Calcium Exchanger - genetics</subject><issn>1547-3287</issn><issn>1557-8534</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkE2L1TAUQIMozji6dCtZuskzn027lDpfMOigDrgraXIzRNrkmaTC-wn-a1veU5eucgnnHrgHodeM7hhtu3fFuh2nTO4o69QTdM6U0qRVQj7dZqmJ4K0-Qy9K-U4pb3grn6MzrqhWSjTn6NdtSZOpIUWcPO5TrNnYGibAvckupPmQ7KFCwT6nGd8ss4n4flpy2KcKseIvFWbSwzSRD5DDT3D_9h4hBov7ZapLXgUPJcRHbE6Oj_03Ri6vr-7xZ9inXCG_RM-8mQq8Or0X6OHq8mt_Q-4-Xd_27--IFUJXojtuR8eatpOdo60ZuXVSN43S2jkDTPKOWSE9N9RrD1r7jnvrR92oDpQaxQV6e_Tuc_qxQKnDHIpdLzAR0lIG1kgqW8EavaLkiNqcSsngh30Os8mHgdFhqz-s9Yet_rDVX_k3J_UyzuD-0n9yr4A4Atu3iXEKMEKu_9H-Bvk-kzE</recordid><startdate>20150101</startdate><enddate>20150101</enddate><creator>Ovchinnikov, Dmitry A.</creator><creator>Hidalgo, Alejandro</creator><creator>Yang, Seung-Kwon</creator><creator>Zhang, Xinli</creator><creator>Hudson, James</creator><creator>Mazzone, Stuart B.</creator><creator>Chen, Chen</creator><creator>Cooper-White, Justin J.</creator><creator>Wolvetang, Ernst J.</creator><general>Mary Ann Liebert, Inc</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>7X8</scope></search><sort><creationdate>20150101</creationdate><title>Isolation of Contractile Cardiomyocytes from Human Pluripotent Stem-Cell-Derived Cardiomyogenic Cultures Using a Human NCX1-EGFP Reporter</title><author>Ovchinnikov, Dmitry A. ; Hidalgo, Alejandro ; Yang, Seung-Kwon ; Zhang, Xinli ; Hudson, James ; Mazzone, Stuart B. ; Chen, Chen ; Cooper-White, Justin J. ; Wolvetang, Ernst J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c337t-792cbd168949d08ab2cd4766577ddae14291c34f2a0f7fe77f92fcfb7659e55b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Cell Culture Techniques</topic><topic>Cell Line</topic><topic>Flow Cytometry</topic><topic>Genes, Reporter</topic><topic>Green Fluorescent Proteins - biosynthesis</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Humans</topic><topic>Induced Pluripotent Stem Cells - cytology</topic><topic>Induced Pluripotent Stem Cells - metabolism</topic><topic>Myocardial Contraction</topic><topic>Myocytes, Cardiac - cytology</topic><topic>Myocytes, Cardiac - metabolism</topic><topic>Original Research Reports</topic><topic>Recombinant Fusion Proteins - biosynthesis</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Sodium-Calcium Exchanger - biosynthesis</topic><topic>Sodium-Calcium Exchanger - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ovchinnikov, Dmitry A.</creatorcontrib><creatorcontrib>Hidalgo, Alejandro</creatorcontrib><creatorcontrib>Yang, Seung-Kwon</creatorcontrib><creatorcontrib>Zhang, Xinli</creatorcontrib><creatorcontrib>Hudson, James</creatorcontrib><creatorcontrib>Mazzone, Stuart B.</creatorcontrib><creatorcontrib>Chen, Chen</creatorcontrib><creatorcontrib>Cooper-White, Justin J.</creatorcontrib><creatorcontrib>Wolvetang, Ernst J.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Stem cells and development</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ovchinnikov, Dmitry A.</au><au>Hidalgo, Alejandro</au><au>Yang, Seung-Kwon</au><au>Zhang, Xinli</au><au>Hudson, James</au><au>Mazzone, Stuart B.</au><au>Chen, Chen</au><au>Cooper-White, Justin J.</au><au>Wolvetang, Ernst J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Isolation of Contractile Cardiomyocytes from Human Pluripotent Stem-Cell-Derived Cardiomyogenic Cultures Using a Human NCX1-EGFP Reporter</atitle><jtitle>Stem cells and development</jtitle><addtitle>Stem Cells Dev</addtitle><date>2015-01-01</date><risdate>2015</risdate><volume>24</volume><issue>1</issue><spage>11</spage><epage>20</epage><pages>11-20</pages><issn>1547-3287</issn><eissn>1557-8534</eissn><abstract>The prospective isolation of defined contractile human pluripotent stem cell (hPSC)–derived cardiomyocytes is advantageous for regenerative medicine and drug screening applications. Currently, enrichment of cardiomyocyte populations from such cultures can be achieved by combinations of cell surface markers or the labor-intensive genetic modification of cardiac developmental genes, such as
NKX2.5
or
MYH6
, with fluorescent reporters. To create a facile, portable method for the isolation of contractile cardiomyocytes from cardiomyogenic hPSC cultures, we employed a highly conserved cardiac enhancer sequence in the
SLC8A1 (NCX1)
gene to generate a lentivirally deliverable, antibiotic-selectable NCX1cp-EGFP reporter. We show that human embryonic stem cells (and induced pluripotent stem cells) transduced with the NCX1cp-EGFP reporter cassette exhibit enhanced green fluorescent protein (EGFP) expression in cardiac progenitors from 5 days into the directed cardiac hPSC differentiation protocol, with all reporter-positive cells transitioning to spontaneously contracting foci 3 days later. In subsequent stages of cardiomyocyte maturation,
NCX1cp
-EGFP expression was exclusively limited to contractile cells expressing high levels of cardiac troponin T (CTNT), MLC2a/v, and α-actinin proteins, and was not present in CD90/THY1
+
cardiac stromal cells or CD31/PECAM
+
endothelial cells. Flow-assisted cytometrically sorted EGFP
+
fractions of differentiated cultures were highly enriched in both early (
NKX2.5
and
TBX5
) and late (CTNT/
TNNI2
,
MYH6
,
MYH7
,
NPPA
, and
MYL2
) cardiomyocyte markers, with a significant proportion of cells displaying a ventricular-like action potential pattern in patch-clamp recordings. We conclude that the use of the cardiac-specific promoter of the human
SLC8A1
(
NCX1
) gene is an effective strategy to isolate contractile cardiac cells and their progenitors from hPSC-derived cardiomyogenic cultures.</abstract><cop>United States</cop><pub>Mary Ann Liebert, Inc</pub><pmid>25075536</pmid><doi>10.1089/scd.2014.0195</doi><tpages>10</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1547-3287 |
| ispartof | Stem cells and development, 2015-01, Vol.24 (1), p.11-20 |
| issn | 1547-3287 1557-8534 |
| language | eng |
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| source | MEDLINE; Alma/SFX Local Collection |
| subjects | Cell Culture Techniques Cell Line Flow Cytometry Genes, Reporter Green Fluorescent Proteins - biosynthesis Green Fluorescent Proteins - genetics Humans Induced Pluripotent Stem Cells - cytology Induced Pluripotent Stem Cells - metabolism Myocardial Contraction Myocytes, Cardiac - cytology Myocytes, Cardiac - metabolism Original Research Reports Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - genetics Sodium-Calcium Exchanger - biosynthesis Sodium-Calcium Exchanger - genetics |
| title | Isolation of Contractile Cardiomyocytes from Human Pluripotent Stem-Cell-Derived Cardiomyogenic Cultures Using a Human NCX1-EGFP Reporter |
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