Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro
Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets <i>in vitro</i> and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications....
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MDPI AG
2023-01-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/12/3/346 |
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| author | Vignesh Jayarajan George T. Hall Theodoros Xenakis Neil Bulstrode Dale Moulding Sergi Castellano Wei-Li Di |
| author_facet | Vignesh Jayarajan George T. Hall Theodoros Xenakis Neil Bulstrode Dale Moulding Sergi Castellano Wei-Li Di |
| author_sort | Vignesh Jayarajan |
| collection | DOAJ |
| description | Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets <i>in vitro</i> and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications. |
| first_indexed | 2024-03-11T09:49:42Z |
| format | Article |
| id | doaj.art-0305322fe49c4ab8a545bf67ed65af03 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-11T09:49:42Z |
| publishDate | 2023-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-0305322fe49c4ab8a545bf67ed65af032023-11-16T16:20:19ZengMDPI AGCells2073-44092023-01-0112334610.3390/cells12030346Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In VitroVignesh Jayarajan0George T. Hall1Theodoros Xenakis2Neil Bulstrode3Dale Moulding4Sergi Castellano5Wei-Li Di6Infection, Immunity and Inflammation Research & Teaching Department, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UKGenetics and Genomic Medicine Research & Teaching Department, UCL Great Ormond Street Institute of Child Health, 20 Guilford Street, London WC1N 1DZ, UKGenetics and Genomic Medicine Research & Teaching Department, UCL Great Ormond Street Institute of Child Health, 20 Guilford Street, London WC1N 1DZ, UKDepartment of Plastic Surgery, Great Ormond Street Hospital for Children, Great Ormond Street, London WC1N 3JH, UKLight Microscopy Core Facility, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UKGenetics and Genomic Medicine Research & Teaching Department, UCL Great Ormond Street Institute of Child Health, 20 Guilford Street, London WC1N 1DZ, UKInfection, Immunity and Inflammation Research & Teaching Department, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UKPrimary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets <i>in vitro</i> and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.https://www.mdpi.com/2073-4409/12/3/346keratinocyte stem cellsROCK inhibitorsingle-cell RNA sequencinggene therapyY-27632 |
| spellingShingle | Vignesh Jayarajan George T. Hall Theodoros Xenakis Neil Bulstrode Dale Moulding Sergi Castellano Wei-Li Di Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro Cells keratinocyte stem cells ROCK inhibitor single-cell RNA sequencing gene therapy Y-27632 |
| title | Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro |
| title_full | Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro |
| title_fullStr | Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro |
| title_full_unstemmed | Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro |
| title_short | Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro |
| title_sort | short term treatment with rho associated kinase inhibitor preserves keratinocyte stem cell characteristics in vitro |
| topic | keratinocyte stem cells ROCK inhibitor single-cell RNA sequencing gene therapy Y-27632 |
| url | https://www.mdpi.com/2073-4409/12/3/346 |
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