Technical advantage of recombinant collagenase for isolation of muscle stem cells
Background: Muscle satellite cells are resident skeletal muscle stem cells responsible for muscle regeneration. Isolation of satellite cells is a critical process for clinical application such as drug screening and cell transplantation. Fluorescence-activated cell sorting (FACS) enables the direct i...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2017-12-01
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| Series: | Regenerative Therapy |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2352320417300123 |
| _version_ | 1818481660809707520 |
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| author | Kana Ishii Nobuharu Suzuki Yo Mabuchi Ichiro Sekiya Chihiro Akazawa |
| author_facet | Kana Ishii Nobuharu Suzuki Yo Mabuchi Ichiro Sekiya Chihiro Akazawa |
| author_sort | Kana Ishii |
| collection | DOAJ |
| description | Background: Muscle satellite cells are resident skeletal muscle stem cells responsible for muscle regeneration. Isolation of satellite cells is a critical process for clinical application such as drug screening and cell transplantation. Fluorescence-activated cell sorting (FACS) enables the direct isolation of satellite cells from muscle tissue. During the process used to isolate satellite cells from skeletal muscle, enzymatic digestion is the first step. Therefore, the evaluation and standardization of enzymes is important not only for reproducibility of cellular yield and viability, but also for traceability of material used in protocols.
Methods: The comparison of muscle digestion was performed either by a mixture of recombinant collagenase G (ColG) and collagenase H (ColH) or by a conventional collagenase II. The degree of cell damage and surface antigen expression upon collagenase treatment were analyzed by FACS. To investigate whether satellite cells isolated using recombinant collagenase can regenerate injured muscle, satellite cells were cultured, transplanted into injured muscles, and analyzed by immunostaining.
Results: We show that ColG and ColH were efficient to isolate satellite cells from mouse skeletal muscle tissue. Digestion with a combination of ColG and ColH enriched satellite cells with intact surface antigens such as α7 and β1 integrins. Furthermore, satellite cells isolated using ColG and ColH dramatically proliferated and remained undifferentiated in vitro. When transplanted, satellite cells isolated using ColG and ColH enhanced the therapeutic efficacy in vivo.
Conclusions: Our results provide an efficient method of satellite cell preparation using recombinant collagenases with a high cell yield, viability of cells, and regeneration potency to fit the biological raw material criteria. |
| first_indexed | 2024-12-10T11:37:54Z |
| format | Article |
| id | doaj.art-dae4efde53ba484ab839d807681b5a66 |
| institution | Directory Open Access Journal |
| issn | 2352-3204 |
| language | English |
| last_indexed | 2024-12-10T11:37:54Z |
| publishDate | 2017-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Regenerative Therapy |
| spelling | doaj.art-dae4efde53ba484ab839d807681b5a662022-12-22T01:50:21ZengElsevierRegenerative Therapy2352-32042017-12-017C1710.1016/j.reth.2017.06.001Technical advantage of recombinant collagenase for isolation of muscle stem cellsKana Ishii0Nobuharu Suzuki1Yo Mabuchi2Ichiro Sekiya3Chihiro Akazawa4Department of Biochemistry and Biophysics, Graduate School of Health Care Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, JapanDepartment of Biochemistry and Biophysics, Graduate School of Health Care Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, JapanDepartment of Biochemistry and Biophysics, Graduate School of Health Care Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, JapanCenter for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, JapanDepartment of Biochemistry and Biophysics, Graduate School of Health Care Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, JapanBackground: Muscle satellite cells are resident skeletal muscle stem cells responsible for muscle regeneration. Isolation of satellite cells is a critical process for clinical application such as drug screening and cell transplantation. Fluorescence-activated cell sorting (FACS) enables the direct isolation of satellite cells from muscle tissue. During the process used to isolate satellite cells from skeletal muscle, enzymatic digestion is the first step. Therefore, the evaluation and standardization of enzymes is important not only for reproducibility of cellular yield and viability, but also for traceability of material used in protocols. Methods: The comparison of muscle digestion was performed either by a mixture of recombinant collagenase G (ColG) and collagenase H (ColH) or by a conventional collagenase II. The degree of cell damage and surface antigen expression upon collagenase treatment were analyzed by FACS. To investigate whether satellite cells isolated using recombinant collagenase can regenerate injured muscle, satellite cells were cultured, transplanted into injured muscles, and analyzed by immunostaining. Results: We show that ColG and ColH were efficient to isolate satellite cells from mouse skeletal muscle tissue. Digestion with a combination of ColG and ColH enriched satellite cells with intact surface antigens such as α7 and β1 integrins. Furthermore, satellite cells isolated using ColG and ColH dramatically proliferated and remained undifferentiated in vitro. When transplanted, satellite cells isolated using ColG and ColH enhanced the therapeutic efficacy in vivo. Conclusions: Our results provide an efficient method of satellite cell preparation using recombinant collagenases with a high cell yield, viability of cells, and regeneration potency to fit the biological raw material criteria.http://www.sciencedirect.com/science/article/pii/S2352320417300123Satellite cellMuscle stem cellTransplantationRegenerationCollagenase |
| spellingShingle | Kana Ishii Nobuharu Suzuki Yo Mabuchi Ichiro Sekiya Chihiro Akazawa Technical advantage of recombinant collagenase for isolation of muscle stem cells Regenerative Therapy Satellite cell Muscle stem cell Transplantation Regeneration Collagenase |
| title | Technical advantage of recombinant collagenase for isolation of muscle stem cells |
| title_full | Technical advantage of recombinant collagenase for isolation of muscle stem cells |
| title_fullStr | Technical advantage of recombinant collagenase for isolation of muscle stem cells |
| title_full_unstemmed | Technical advantage of recombinant collagenase for isolation of muscle stem cells |
| title_short | Technical advantage of recombinant collagenase for isolation of muscle stem cells |
| title_sort | technical advantage of recombinant collagenase for isolation of muscle stem cells |
| topic | Satellite cell Muscle stem cell Transplantation Regeneration Collagenase |
| url | http://www.sciencedirect.com/science/article/pii/S2352320417300123 |
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