High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model
Abstract Background Mesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration. However, the function of MSC can vary according to cell culture conditions, donor age, and heterogeneity of the MSC population, resulting in unregulated MSC quality control. To overcome these li...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
American Association for the Advancement of Science (AAAS)
2023-05-01
|
| Series: | Biomaterials Research |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s40824-023-00398-3 |
| _version_ | 1797280698296434688 |
|---|---|
| author | Gun Hee Cho Hyun Cheol Bae Won Young Cho Eui Man Jeong Hee Jung Park Ha Ru Yang Sun Young Wang You Jung Kim Dong Myung Shin Hyung Min Chung In Gyu Kim Hyuk-Soo Han |
| author_facet | Gun Hee Cho Hyun Cheol Bae Won Young Cho Eui Man Jeong Hee Jung Park Ha Ru Yang Sun Young Wang You Jung Kim Dong Myung Shin Hyung Min Chung In Gyu Kim Hyuk-Soo Han |
| author_sort | Gun Hee Cho |
| collection | DOAJ |
| description | Abstract Background Mesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration. However, the function of MSC can vary according to cell culture conditions, donor age, and heterogeneity of the MSC population, resulting in unregulated MSC quality control. To overcome these limitations, we previously developed a fluorescent real-time thiol tracer (FreSHtracer) that monitors cellular levels of glutathione (GSH), which are known to be closely associated with stem cell function. In this study, we investigated whether using FreSHtracer could selectively separate high-functioning MSCs based on GSH levels and evaluated the chondrogenic potential of MSCs with high GSH levels to repair cartilage defects in vivo. Methods Flow cytometry was conducted on FreSHtracer-loaded MSCs to select cells according to their GSH levels. To determine the function of FreSHtracer-isolated MSCs, mRNA expression, migration, and CFU assays were conducted. The MSCs underwent chondrogenic differentiation, followed by analysis of chondrogenic-related gene expression. For in vivo assessment, MSCs with different cellular GSH levels or cell culture densities were injected in a rabbit chondral defect model, followed by histological analysis of cartilage-regenerated defect sites. Results FreSHtracer successfully isolated MSCs according to GSH levels. MSCs with high cellular GSH levels showed enhanced MSC function, including stem cell marker mRNA expression, migration, CFU, and oxidant resistance. Regardless of the stem cell tissue source, FreSHtracer selectively isolated MSCs with high GSH levels and high functionality. The in vitro chondrogenic potential was the highest in pellets generated by MSCs with high GSH levels, with increased ECM formation and chondrogenic marker expression. Furthermore, the MSCs’ function was dependent on cell culture conditions, with relatively higher cell culture densities resulting in higher GSH levels. In vivo, improved cartilage repair was achieved by articular injection of MSCs with high levels of cellular GSH and MSCs cultured under high-density conditions, as confirmed by Collagen type 2 IHC, Safranin-O staining and O’Driscoll scores showing that more hyaline cartilage was formed on the defects. Conclusion FreSHtracer selectively isolates highly functional MSCs that have enhanced in vitro chondrogenesis and in vivo hyaline cartilage regeneration, which can ultimately overcome the current limitations of MSC therapy. Graphical Abstract |
| first_indexed | 2024-03-07T16:43:59Z |
| format | Article |
| id | doaj.art-1a5f5c681d104913887a562a2e6329c1 |
| institution | Directory Open Access Journal |
| issn | 2055-7124 |
| language | English |
| last_indexed | 2024-03-07T16:43:59Z |
| publishDate | 2023-05-01 |
| publisher | American Association for the Advancement of Science (AAAS) |
| record_format | Article |
| series | Biomaterials Research |
| spelling | doaj.art-1a5f5c681d104913887a562a2e6329c12024-03-03T06:55:38ZengAmerican Association for the Advancement of Science (AAAS)Biomaterials Research2055-71242023-05-0127111410.1186/s40824-023-00398-3High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect modelGun Hee Cho0Hyun Cheol Bae1Won Young Cho2Eui Man Jeong3Hee Jung Park4Ha Ru Yang5Sun Young Wang6You Jung Kim7Dong Myung Shin8Hyung Min Chung9In Gyu Kim10Hyuk-Soo Han11Department of Orthopedic Surgery, College of Medicine, Seoul National UniversityDepartment of Orthopedic Surgery, Seoul National University HospitalDepartment of Orthopedic Surgery, Seoul National University HospitalDepartment of Pharmacy, College of Pharmacy, Jeju National University, Jeju Special Self-Governing ProvinceDepartment of Orthopedic Surgery, Seoul National University HospitalDepartment of Orthopedic Surgery, Seoul National University HospitalDepartment of Orthopedic Surgery, Seoul National University HospitalDepartment of Orthopedic Surgery, Seoul National University HospitalDepartment of Biomedical Sciences, Asan Medical Center, University of Ulsan College of MedicineDepartment of Stem Cell Biology, School of Medicine, Konkuk UniversityLaboratory for Cellular Response to Oxidative Stress, Cell2in, IncDepartment of Orthopedic Surgery, College of Medicine, Seoul National UniversityAbstract Background Mesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration. However, the function of MSC can vary according to cell culture conditions, donor age, and heterogeneity of the MSC population, resulting in unregulated MSC quality control. To overcome these limitations, we previously developed a fluorescent real-time thiol tracer (FreSHtracer) that monitors cellular levels of glutathione (GSH), which are known to be closely associated with stem cell function. In this study, we investigated whether using FreSHtracer could selectively separate high-functioning MSCs based on GSH levels and evaluated the chondrogenic potential of MSCs with high GSH levels to repair cartilage defects in vivo. Methods Flow cytometry was conducted on FreSHtracer-loaded MSCs to select cells according to their GSH levels. To determine the function of FreSHtracer-isolated MSCs, mRNA expression, migration, and CFU assays were conducted. The MSCs underwent chondrogenic differentiation, followed by analysis of chondrogenic-related gene expression. For in vivo assessment, MSCs with different cellular GSH levels or cell culture densities were injected in a rabbit chondral defect model, followed by histological analysis of cartilage-regenerated defect sites. Results FreSHtracer successfully isolated MSCs according to GSH levels. MSCs with high cellular GSH levels showed enhanced MSC function, including stem cell marker mRNA expression, migration, CFU, and oxidant resistance. Regardless of the stem cell tissue source, FreSHtracer selectively isolated MSCs with high GSH levels and high functionality. The in vitro chondrogenic potential was the highest in pellets generated by MSCs with high GSH levels, with increased ECM formation and chondrogenic marker expression. Furthermore, the MSCs’ function was dependent on cell culture conditions, with relatively higher cell culture densities resulting in higher GSH levels. In vivo, improved cartilage repair was achieved by articular injection of MSCs with high levels of cellular GSH and MSCs cultured under high-density conditions, as confirmed by Collagen type 2 IHC, Safranin-O staining and O’Driscoll scores showing that more hyaline cartilage was formed on the defects. Conclusion FreSHtracer selectively isolates highly functional MSCs that have enhanced in vitro chondrogenesis and in vivo hyaline cartilage regeneration, which can ultimately overcome the current limitations of MSC therapy. Graphical Abstracthttps://doi.org/10.1186/s40824-023-00398-3FreSHtracerGlutathioneMesenchymal stem cellsCartilage regenerationChondral defect |
| spellingShingle | Gun Hee Cho Hyun Cheol Bae Won Young Cho Eui Man Jeong Hee Jung Park Ha Ru Yang Sun Young Wang You Jung Kim Dong Myung Shin Hyung Min Chung In Gyu Kim Hyuk-Soo Han High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model Biomaterials Research FreSHtracer Glutathione Mesenchymal stem cells Cartilage regeneration Chondral defect |
| title | High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model |
| title_full | High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model |
| title_fullStr | High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model |
| title_full_unstemmed | High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model |
| title_short | High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model |
| title_sort | high glutathione mesenchymal stem cells isolated using the freshtracer probe enhance cartilage regeneration in a rabbit chondral defect model |
| topic | FreSHtracer Glutathione Mesenchymal stem cells Cartilage regeneration Chondral defect |
| url | https://doi.org/10.1186/s40824-023-00398-3 |
| work_keys_str_mv | AT gunheecho highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT hyuncheolbae highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT wonyoungcho highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT euimanjeong highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT heejungpark highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT haruyang highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT sunyoungwang highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT youjungkim highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT dongmyungshin highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT hyungminchung highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT ingyukim highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel AT hyuksoohan highglutathionemesenchymalstemcellsisolatedusingthefreshtracerprobeenhancecartilageregenerationinarabbitchondraldefectmodel |