New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies
Abstract Background Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis...
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| Format: | Article |
| Language: | English |
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BMC
2023-12-01
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| Series: | Stem Cell Research & Therapy |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13287-023-03586-z |
| _version_ | 1797388455430324224 |
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| author | Marija Zekušić Marina Bujić Mihica Marija Skoko Kruno Vukušić Patrik Risteski Jelena Martinčić Iva M. Tolić Krešo Bendelja Snježana Ramić Tamara Dolenec Ivana Vrgoč Zimić Dominik Puljić Ivanka Petric Vicković Renata Iveković Ivanka Batarilo Uršula Prosenc Zmrzljak Alan Hoffmeister Tiha Vučemilo |
| author_facet | Marija Zekušić Marina Bujić Mihica Marija Skoko Kruno Vukušić Patrik Risteski Jelena Martinčić Iva M. Tolić Krešo Bendelja Snježana Ramić Tamara Dolenec Ivana Vrgoč Zimić Dominik Puljić Ivanka Petric Vicković Renata Iveković Ivanka Batarilo Uršula Prosenc Zmrzljak Alan Hoffmeister Tiha Vučemilo |
| author_sort | Marija Zekušić |
| collection | DOAJ |
| description | Abstract Background Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. Methods After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. Results Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype. Conclusion This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans. |
| first_indexed | 2024-03-08T22:41:03Z |
| format | Article |
| id | doaj.art-1ea97ef887f741b2bd77b5d00bf8449d |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-03-08T22:41:03Z |
| publishDate | 2023-12-01 |
| publisher | BMC |
| record_format | Article |
| series | Stem Cell Research & Therapy |
| spelling | doaj.art-1ea97ef887f741b2bd77b5d00bf8449d2023-12-17T12:08:53ZengBMCStem Cell Research & Therapy1757-65122023-12-0114112610.1186/s13287-023-03586-zNew characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologiesMarija Zekušić0Marina Bujić Mihica1Marija Skoko2Kruno Vukušić3Patrik Risteski4Jelena Martinčić5Iva M. Tolić6Krešo Bendelja7Snježana Ramić8Tamara Dolenec9Ivana Vrgoč Zimić10Dominik Puljić11Ivanka Petric Vicković12Renata Iveković13Ivanka Batarilo14Uršula Prosenc Zmrzljak15Alan Hoffmeister16Tiha Vučemilo17Department of Transfusion and Regenerative Medicine, Sestre milosrdnice University Hospital CenterDepartment of Transfusion and Regenerative Medicine, Sestre milosrdnice University Hospital CenterDepartment of Transfusion and Regenerative Medicine, Sestre milosrdnice University Hospital CenterDivision of Molecular Biology, Ruđer Bošković InstituteDivision of Molecular Biology, Ruđer Bošković InstituteDivision of Molecular Biology, Ruđer Bošković InstituteDivision of Molecular Biology, Ruđer Bošković InstituteCenter for Research and Knowledge Transfer in Biotechnology, Laboratory of Immunology, University of ZagrebDepartment of Oncological Pathology and Clinical Cytology ‘Ljudevit Jurak’, University Hospital Center Sestre MilosrdniceDepartment of Transfusion and Regenerative Medicine, Sestre milosrdnice University Hospital CenterDepartment of Transfusion and Regenerative Medicine, Sestre milosrdnice University Hospital CenterDepartment of Transfusion and Regenerative Medicine, Sestre milosrdnice University Hospital CenterClinical Department of Ophthalmology, Sestre milosrdnice University Hospital CenterClinical Department of Ophthalmology, Sestre milosrdnice University Hospital CenterDepartment of Microbiology, Croatian Institute of Transfusion MedicineMolecular Biology Department, BIA Separations CROCharles River LaboratoriesDepartment of Transfusion and Regenerative Medicine, Sestre milosrdnice University Hospital CenterAbstract Background Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. Methods After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. Results Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype. Conclusion This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.https://doi.org/10.1186/s13287-023-03586-zLimbal stem cellLimbal stem cell deficiencyLimbal graftAdvanced therapy medicinal productsp63 |
| spellingShingle | Marija Zekušić Marina Bujić Mihica Marija Skoko Kruno Vukušić Patrik Risteski Jelena Martinčić Iva M. Tolić Krešo Bendelja Snježana Ramić Tamara Dolenec Ivana Vrgoč Zimić Dominik Puljić Ivanka Petric Vicković Renata Iveković Ivanka Batarilo Uršula Prosenc Zmrzljak Alan Hoffmeister Tiha Vučemilo New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies Stem Cell Research & Therapy Limbal stem cell Limbal stem cell deficiency Limbal graft Advanced therapy medicinal products p63 |
| title | New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies |
| title_full | New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies |
| title_fullStr | New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies |
| title_full_unstemmed | New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies |
| title_short | New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies |
| title_sort | new characterization and safety evaluation of human limbal stem cells used in clinical application fidelity of mitotic process and mitotic spindle morphologies |
| topic | Limbal stem cell Limbal stem cell deficiency Limbal graft Advanced therapy medicinal products p63 |
| url | https://doi.org/10.1186/s13287-023-03586-z |
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