Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter
Abstract Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy’s success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tool...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-06-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-12956-1 |
| _version_ | 1818237441517027328 |
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| author | Anita Fehér Andrea Schnúr Suchitra Muenthaisong Tamás Bellák Ferhan Ayaydin György Várady Elisabeth Kemter Eckhard Wolf András Dinnyés |
| author_facet | Anita Fehér Andrea Schnúr Suchitra Muenthaisong Tamás Bellák Ferhan Ayaydin György Várady Elisabeth Kemter Eckhard Wolf András Dinnyés |
| author_sort | Anita Fehér |
| collection | DOAJ |
| description | Abstract Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy’s success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications. |
| first_indexed | 2024-12-12T12:25:49Z |
| format | Article |
| id | doaj.art-ef9de7a5d9d6400abedfcdb22e4b59c8 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-12T12:25:49Z |
| publishDate | 2022-06-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-ef9de7a5d9d6400abedfcdb22e4b59c82022-12-22T00:24:33ZengNature PortfolioScientific Reports2045-23222022-06-0112111310.1038/s41598-022-12956-1Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporterAnita Fehér0Andrea Schnúr1Suchitra Muenthaisong2Tamás Bellák3Ferhan Ayaydin4György Várady5Elisabeth Kemter6Eckhard Wolf7András Dinnyés8BioTalentum LtdBioTalentum LtdBioTalentum LtdBioTalentum LtdFunctional Cell Biology and Immunology Advanced Core Facility, Hungarian Centre of Excellence for Molecular Medicine, University of Szeged (HCEMM-USZ)Research Centre for Natural Sciences, Institute of EnzymologyChair for Molecular Animal Breeding and Biotechnology, Gene Centre and Department of Veterinary Sciences, LMU MunichChair for Molecular Animal Breeding and Biotechnology, Gene Centre and Department of Veterinary Sciences, LMU MunichBioTalentum LtdAbstract Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy’s success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications.https://doi.org/10.1038/s41598-022-12956-1 |
| spellingShingle | Anita Fehér Andrea Schnúr Suchitra Muenthaisong Tamás Bellák Ferhan Ayaydin György Várady Elisabeth Kemter Eckhard Wolf András Dinnyés Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter Scientific Reports |
| title | Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter |
| title_full | Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter |
| title_fullStr | Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter |
| title_full_unstemmed | Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter |
| title_short | Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter |
| title_sort | establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the irfp720 reporter |
| url | https://doi.org/10.1038/s41598-022-12956-1 |
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