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Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins
BACKGROUND: Ever since their discovery, induced pluripotent stem cells (iPSCs) have been extensively differentiated into a large variety of cell types. However, a limited amount of work has been dedicated to differentiating iPSCs into osteoclasts. While several differentiation protocols have been pu...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Journal Experts
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10350192/ https://www.ncbi.nlm.nih.gov/pubmed/37461708 http://dx.doi.org/10.21203/rs.3.rs-3089289/v1 |
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author | Blümke, Alexander Ijeoma, Erica Simon, Jessica Wellington, Rachel Purwaningrum, Medania Doulatov, Sergei Leber, Elizabeth Scatena, Marta Giachelli, Cecilia M |
author_facet | Blümke, Alexander Ijeoma, Erica Simon, Jessica Wellington, Rachel Purwaningrum, Medania Doulatov, Sergei Leber, Elizabeth Scatena, Marta Giachelli, Cecilia M |
author_sort | Blümke, Alexander |
collection | PubMed |
description | BACKGROUND: Ever since their discovery, induced pluripotent stem cells (iPSCs) have been extensively differentiated into a large variety of cell types. However, a limited amount of work has been dedicated to differentiating iPSCs into osteoclasts. While several differentiation protocols have been published, it remains unclear which protocols or differentiation methods are preferrable regarding the differentiation of osteoclasts. METHODS: In this study we compare the osteoclastogenesis capacity of a peripheral blood mononuclear cell (PBMC)-derived iPSC line to a fibroblast-derived iPSC line in conjunction with either embryoid body-based or monolayer-based differentiation strategies. Both cell lines and differentiation protocols were investigated regarding their ability to generate osteoclasts and their inherent robustness and ease of use. The ability of both cell lines to remain undifferentiated while propagating using a feeder-free system was assessed using alkaline phosphatase staining. This was followed by evaluating mesodermal differentiation and the characterization of hematopoietic progenitor cells using flow cytometry. Finally, osteoclast yield and functionality based on resorptive activity, Cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression were assessed. Results were validated using qRT-PCR throughout the differentiation stages. RESULTS: Embryoid-body based differentiation yielded CD45(+), CD14(+), CD11b(+) subpopulations which in turn differentiated into osteoclasts which demonstrated TRAP positivity, Cathepsin K expression and mineral resorptive capabilities. This was regardless of which iPSC line was used. Monolayer-based differentiation yielded lower quantities of hematopoietic cells that were mostly CD34(+) and did not subsequently differentiate into osteoclasts. CONCLUSIONS: The outcome of this study demonstrates the successful differentiation of osteoclasts from iPSCs in conjunction with the embryoid-based differentiation method, while the monolayer-based method did not yield osteoclasts. No differences were observed regarding osteoclast differentiation between the PBMC and fibroblast-derived iPSC lines. |
format | Online Article Text |
id | pubmed-10350192 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Journal Experts |
record_format | MEDLINE/PubMed |
spelling | pubmed-103501922023-07-17 Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins Blümke, Alexander Ijeoma, Erica Simon, Jessica Wellington, Rachel Purwaningrum, Medania Doulatov, Sergei Leber, Elizabeth Scatena, Marta Giachelli, Cecilia M Res Sq Article BACKGROUND: Ever since their discovery, induced pluripotent stem cells (iPSCs) have been extensively differentiated into a large variety of cell types. However, a limited amount of work has been dedicated to differentiating iPSCs into osteoclasts. While several differentiation protocols have been published, it remains unclear which protocols or differentiation methods are preferrable regarding the differentiation of osteoclasts. METHODS: In this study we compare the osteoclastogenesis capacity of a peripheral blood mononuclear cell (PBMC)-derived iPSC line to a fibroblast-derived iPSC line in conjunction with either embryoid body-based or monolayer-based differentiation strategies. Both cell lines and differentiation protocols were investigated regarding their ability to generate osteoclasts and their inherent robustness and ease of use. The ability of both cell lines to remain undifferentiated while propagating using a feeder-free system was assessed using alkaline phosphatase staining. This was followed by evaluating mesodermal differentiation and the characterization of hematopoietic progenitor cells using flow cytometry. Finally, osteoclast yield and functionality based on resorptive activity, Cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression were assessed. Results were validated using qRT-PCR throughout the differentiation stages. RESULTS: Embryoid-body based differentiation yielded CD45(+), CD14(+), CD11b(+) subpopulations which in turn differentiated into osteoclasts which demonstrated TRAP positivity, Cathepsin K expression and mineral resorptive capabilities. This was regardless of which iPSC line was used. Monolayer-based differentiation yielded lower quantities of hematopoietic cells that were mostly CD34(+) and did not subsequently differentiate into osteoclasts. CONCLUSIONS: The outcome of this study demonstrates the successful differentiation of osteoclasts from iPSCs in conjunction with the embryoid-based differentiation method, while the monolayer-based method did not yield osteoclasts. No differences were observed regarding osteoclast differentiation between the PBMC and fibroblast-derived iPSC lines. American Journal Experts 2023-07-07 /pmc/articles/PMC10350192/ /pubmed/37461708 http://dx.doi.org/10.21203/rs.3.rs-3089289/v1 Text en https://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) , which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. |
spellingShingle | Article Blümke, Alexander Ijeoma, Erica Simon, Jessica Wellington, Rachel Purwaningrum, Medania Doulatov, Sergei Leber, Elizabeth Scatena, Marta Giachelli, Cecilia M Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins |
title | Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins |
title_full | Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins |
title_fullStr | Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins |
title_full_unstemmed | Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins |
title_short | Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins |
title_sort | comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10350192/ https://www.ncbi.nlm.nih.gov/pubmed/37461708 http://dx.doi.org/10.21203/rs.3.rs-3089289/v1 |
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