Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs.
Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive t...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2010-07-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC2911382?pdf=render |
| _version_ | 1828395772702359552 |
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| author | Masakazu Kamata Min Liang Shirley Liu Yoshiko Nagaoka Irvin S Y Chen |
| author_facet | Masakazu Kamata Min Liang Shirley Liu Yoshiko Nagaoka Irvin S Y Chen |
| author_sort | Masakazu Kamata |
| collection | DOAJ |
| description | Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs (miR-302a and miR-302d) and mCherry conjugated to the targets of differentiated cells specific miRNAs (miR-142-3p, miR-155, and miR-223). The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors (OCT4, SOX2, KLF4 and cMYC) were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells. |
| first_indexed | 2024-12-10T08:18:56Z |
| format | Article |
| id | doaj.art-63ffac6c9df94677a8ed390175ef6349 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-10T08:18:56Z |
| publishDate | 2010-07-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-63ffac6c9df94677a8ed390175ef63492022-12-22T01:56:23ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-07-0157e1183410.1371/journal.pone.0011834Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs.Masakazu KamataMin LiangShirley LiuYoshiko NagaokaIrvin S Y ChenHuman induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs (miR-302a and miR-302d) and mCherry conjugated to the targets of differentiated cells specific miRNAs (miR-142-3p, miR-155, and miR-223). The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors (OCT4, SOX2, KLF4 and cMYC) were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells.http://europepmc.org/articles/PMC2911382?pdf=render |
| spellingShingle | Masakazu Kamata Min Liang Shirley Liu Yoshiko Nagaoka Irvin S Y Chen Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs. PLoS ONE |
| title | Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs. |
| title_full | Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs. |
| title_fullStr | Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs. |
| title_full_unstemmed | Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs. |
| title_short | Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs. |
| title_sort | live cell monitoring of hipsc generation and differentiation using differential expression of endogenous micrornas |
| url | http://europepmc.org/articles/PMC2911382?pdf=render |
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