Efficient culturing and genetic manipulation of human pluripotent stem cells
Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a p...
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| Veröffentlicht in: | PloS one 2011-12, Vol.6 (12), p.e27495 |
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| creator | Schinzel, Robert T Ahfeldt, Tim Lau, Frank H Lee, Youn-Kyoung Cowley, Alicia Shen, Tony Peters, Derek Lum, David H Cowan, Chad A |
| description | Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs. |
| doi_str_mv | 10.1371/journal.pone.0027495 |
| format | Article |
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However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0027495</identifier><identifier>PMID: 22194785</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Analysis ; Animals ; Antibiotics ; Biology ; Cell culture ; Cell Culture Techniques - methods ; Cells, Cultured ; Cloning ; Colonies ; Dissociation ; Electroporation ; Embryos ; Freeze-thawing ; Genetic engineering ; Genetic Techniques ; HEK293 Cells ; Homologous recombination ; Homology ; Humans ; Islet-1 protein ; Medicine ; Mice ; Pluripotency ; Pluripotent Stem Cells - cytology ; Pluripotent Stem Cells - metabolism ; Rodents ; Stem cell research ; Stem cells ; Transplantation ; Viability</subject><ispartof>PloS one, 2011-12, Vol.6 (12), p.e27495</ispartof><rights>COPYRIGHT 2011 Public Library of Science</rights><rights>2011 Schinzel 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>Schinzel et al. 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c725t-2b79c510ae401fd91409693527cf03d29151c603093e4ce99b4792974dd28f293</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3240614/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3240614/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22194785$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Pera, Martin</contributor><creatorcontrib>Schinzel, Robert T</creatorcontrib><creatorcontrib>Ahfeldt, Tim</creatorcontrib><creatorcontrib>Lau, Frank H</creatorcontrib><creatorcontrib>Lee, Youn-Kyoung</creatorcontrib><creatorcontrib>Cowley, Alicia</creatorcontrib><creatorcontrib>Shen, Tony</creatorcontrib><creatorcontrib>Peters, Derek</creatorcontrib><creatorcontrib>Lum, David H</creatorcontrib><creatorcontrib>Cowan, Chad A</creatorcontrib><title>Efficient culturing and genetic manipulation of human pluripotent stem cells</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. 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However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. 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| subjects | Analysis Animals Antibiotics Biology Cell culture Cell Culture Techniques - methods Cells, Cultured Cloning Colonies Dissociation Electroporation Embryos Freeze-thawing Genetic engineering Genetic Techniques HEK293 Cells Homologous recombination Homology Humans Islet-1 protein Medicine Mice Pluripotency Pluripotent Stem Cells - cytology Pluripotent Stem Cells - metabolism Rodents Stem cell research Stem cells Transplantation Viability |
| title | Efficient culturing and genetic manipulation of human pluripotent stem cells |
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