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Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor
BACKGROUND: The number of patients suffering from osteoporosis is increasing as the elderly population increases. The demand for investigating bone regeneration strategies naturally arises. One of the approaches to induce bone regeneration is somatic cell transdifferentiation. Among the transcriptio...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9281139/ https://www.ncbi.nlm.nih.gov/pubmed/35836274 http://dx.doi.org/10.1186/s40824-022-00280-8 |
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author | Kim, Seung Hyun L. Cho, Sungwoo Kim, Seoyeon Kwon, Janet Lee, Jaeyoung Koh, Rachel H. Park, Ju Hyun Lee, Hwajin Park, Tai Hyun Hwang, Nathaniel S. |
author_facet | Kim, Seung Hyun L. Cho, Sungwoo Kim, Seoyeon Kwon, Janet Lee, Jaeyoung Koh, Rachel H. Park, Ju Hyun Lee, Hwajin Park, Tai Hyun Hwang, Nathaniel S. |
author_sort | Kim, Seung Hyun L. |
collection | PubMed |
description | BACKGROUND: The number of patients suffering from osteoporosis is increasing as the elderly population increases. The demand for investigating bone regeneration strategies naturally arises. One of the approaches to induce bone regeneration is somatic cell transdifferentiation. Among the transcriptional regulators for transdifferentiation, octamer-binding transcription factor 4 (OCT4) is famous for its role in the regulation of pluripotency of stem cells. Bone morphogenetic protein 4 (BMP4) is another factor that is known to have a significant role in osteogenic differentiation. Previous studies have achieved transdifferentiation of cells into osteoblasts using viral and plasmid deliveries of these factors. Although these methods are efficient, viral and plasmid transfection have safety issues such as permanent gene incorporations and bacterial DNA insertions. Herein, we developed a cell penetrating protein-based strategy to induce transdifferentiation of endothelial cells into osteoblasts via nuclear delivery of OCT4 recombinant protein combined with the BMP4 treatment. For the nuclear delivery of OCT4 protein, we fused the protein with 30Kc19, a cell-penetrating and protein stabilizing protein derived from a silkworm hemolymph of Bombyx mori with low cytotoxic properties. This study proposes a promising cell-based therapy without any safety issues that existing transdifferentiation approaches had. METHODS: OCT4-30Kc19 protein with high penetrating activities and stability was synthesized for a protein-based osteogenic transdifferentiation system. Cells were treated with OCT4-30Kc19 and BMP4 to evaluate their cellular penetrating activity, cytotoxicity, osteogenic and angiogenic potentials in vitro. The osteogenic potential of 3D cell spheroids was also analyzed. In addition, in vivo cell delivery into subcutaneous tissue and cranial defect model was performed. RESULTS: OCT4-30Kc19 protein was produced in a soluble and stable form. OCT4-30Kc19 efficiently penetrated cells and were localized in intracellular compartments and the nucleus. Cells delivered with OCT4-30Kc19 protein combined with BMP4 showed increased osteogenesis, both in 2D and 3D culture, and showed increased angiogenesis capacity in vitro. Results from in vivo subcutaneous tissue delivery of cell-seeded scaffolds confirmed enhanced osteogenic properties of transdifferentiated HUVECs via treatment with both OCT4-30Kc19 and BMP4. In addition, in vivo mouse cranial defect experiment demonstrated successful bone regeneration of HUVECs pretreated with both OCT4-30Kc19 and BMP4. CONCLUSIONS: Using a protein-based transdifferentiation method allows an alternative approach without utilizing any genetic modification strategies, thus providing a possibility for safer use of cell-based therapies in clinical applications. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40824-022-00280-8. |
format | Online Article Text |
id | pubmed-9281139 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-92811392022-07-15 Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor Kim, Seung Hyun L. Cho, Sungwoo Kim, Seoyeon Kwon, Janet Lee, Jaeyoung Koh, Rachel H. Park, Ju Hyun Lee, Hwajin Park, Tai Hyun Hwang, Nathaniel S. Biomater Res Research Article BACKGROUND: The number of patients suffering from osteoporosis is increasing as the elderly population increases. The demand for investigating bone regeneration strategies naturally arises. One of the approaches to induce bone regeneration is somatic cell transdifferentiation. Among the transcriptional regulators for transdifferentiation, octamer-binding transcription factor 4 (OCT4) is famous for its role in the regulation of pluripotency of stem cells. Bone morphogenetic protein 4 (BMP4) is another factor that is known to have a significant role in osteogenic differentiation. Previous studies have achieved transdifferentiation of cells into osteoblasts using viral and plasmid deliveries of these factors. Although these methods are efficient, viral and plasmid transfection have safety issues such as permanent gene incorporations and bacterial DNA insertions. Herein, we developed a cell penetrating protein-based strategy to induce transdifferentiation of endothelial cells into osteoblasts via nuclear delivery of OCT4 recombinant protein combined with the BMP4 treatment. For the nuclear delivery of OCT4 protein, we fused the protein with 30Kc19, a cell-penetrating and protein stabilizing protein derived from a silkworm hemolymph of Bombyx mori with low cytotoxic properties. This study proposes a promising cell-based therapy without any safety issues that existing transdifferentiation approaches had. METHODS: OCT4-30Kc19 protein with high penetrating activities and stability was synthesized for a protein-based osteogenic transdifferentiation system. Cells were treated with OCT4-30Kc19 and BMP4 to evaluate their cellular penetrating activity, cytotoxicity, osteogenic and angiogenic potentials in vitro. The osteogenic potential of 3D cell spheroids was also analyzed. In addition, in vivo cell delivery into subcutaneous tissue and cranial defect model was performed. RESULTS: OCT4-30Kc19 protein was produced in a soluble and stable form. OCT4-30Kc19 efficiently penetrated cells and were localized in intracellular compartments and the nucleus. Cells delivered with OCT4-30Kc19 protein combined with BMP4 showed increased osteogenesis, both in 2D and 3D culture, and showed increased angiogenesis capacity in vitro. Results from in vivo subcutaneous tissue delivery of cell-seeded scaffolds confirmed enhanced osteogenic properties of transdifferentiated HUVECs via treatment with both OCT4-30Kc19 and BMP4. In addition, in vivo mouse cranial defect experiment demonstrated successful bone regeneration of HUVECs pretreated with both OCT4-30Kc19 and BMP4. CONCLUSIONS: Using a protein-based transdifferentiation method allows an alternative approach without utilizing any genetic modification strategies, thus providing a possibility for safer use of cell-based therapies in clinical applications. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40824-022-00280-8. BioMed Central 2022-07-14 /pmc/articles/PMC9281139/ /pubmed/35836274 http://dx.doi.org/10.1186/s40824-022-00280-8 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Article Kim, Seung Hyun L. Cho, Sungwoo Kim, Seoyeon Kwon, Janet Lee, Jaeyoung Koh, Rachel H. Park, Ju Hyun Lee, Hwajin Park, Tai Hyun Hwang, Nathaniel S. Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor |
title | Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor |
title_full | Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor |
title_fullStr | Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor |
title_full_unstemmed | Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor |
title_short | Cellular direct conversion by cell penetrable OCT4-30Kc19 protein and BMP4 growth factor |
title_sort | cellular direct conversion by cell penetrable oct4-30kc19 protein and bmp4 growth factor |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9281139/ https://www.ncbi.nlm.nih.gov/pubmed/35836274 http://dx.doi.org/10.1186/s40824-022-00280-8 |
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