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Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs)
Background: For many years, researchers have sought to overcome major challenges in the use of nanoparticles as therapeutics, including issues related to intracellular delivery, biocompatibility, and activation. In particular, the genetic stability of cells treated with nanoparticles has become incr...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Ivyspring International Publisher
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276296/ https://www.ncbi.nlm.nih.gov/pubmed/30555563 http://dx.doi.org/10.7150/thno.29214 |
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author | Park, Ji Sun Yi, Se Won Kim, Hye Jin Oh, Hyun Jyung Lee, Jung Sun Go, Minyeon Shim, Sung Han Park, Keun-Hong |
author_facet | Park, Ji Sun Yi, Se Won Kim, Hye Jin Oh, Hyun Jyung Lee, Jung Sun Go, Minyeon Shim, Sung Han Park, Keun-Hong |
author_sort | Park, Ji Sun |
collection | PubMed |
description | Background: For many years, researchers have sought to overcome major challenges in the use of nanoparticles as therapeutics, including issues related to intracellular delivery, biocompatibility, and activation. In particular, the genetic stability of cells treated with nanoparticles has become increasingly important in the context of stem cell therapy. Methods: Functional nanoparticles (Sunflower typed nanoparticles; SF-NPs) were fabricated by coating heparin pluronic F127 gels with quantum dot nanoparticles (QDs), and then bound the SOX9 gene to the QD nanogels. The resultant nanoparticles were transferred into stem cells, and the effect on genetic stability was monitored. To determinate gene delivery efficacy and long-term genomic stability of cells transfected with QD nanogels, hMSCs were transfected with nanogels at passage 4 (T1; Transfected cells 1) and then sub-cultured to passage of (T4). Following transplantation of transfected T1-T4 cells, the cells were monitored by in vivo imaging. The genetic stability of cells treated with nanoparticles was confirmed by chromosomal analysis, copy number variation (CNV) analysis, and mRNA profiling. Results: After 21 days of pellet culture after sub-culture from T1 to T4, hMSCs treated with QD nanogels complexed with SOX9 plasmid DNA (pDNA) significantly increased expression of specific extracellular matrix (ECM) polysaccharides and glycoproteins, as determined by Safranin O and Alcian blue staining. Moreover, the T4 hMSCs expressed higher levels of specific proteins, including collagen type II (COLII) and SOX9, than P4 hMSCs, with no evidence of DNA damage or genomic malfunction. Microarray analysis confirmed expression of genes specific to matured chondrocytes. Stem cells that internalized nanoparticles at the early stage retained genetic stability, even after passage. In in vivo studies in rats, neuronal cartilage formation was observed in damaged lesions 6 weeks after transplantation of T1 and T4 cells. The degree of differentiation into chondrocytes in the cartilage defect area, as determined by mRNA and protein expression of COLII and SOX9, was higher in rats treated with SF-NPs. Conclusion: The QD nanogels used in this study, did not affect genome integrity during long-term subculture, and are thus suitable for multiple theranostic applications. |
format | Online Article Text |
id | pubmed-6276296 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Ivyspring International Publisher |
record_format | MEDLINE/PubMed |
spelling | pubmed-62762962018-12-14 Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs) Park, Ji Sun Yi, Se Won Kim, Hye Jin Oh, Hyun Jyung Lee, Jung Sun Go, Minyeon Shim, Sung Han Park, Keun-Hong Theranostics Research Paper Background: For many years, researchers have sought to overcome major challenges in the use of nanoparticles as therapeutics, including issues related to intracellular delivery, biocompatibility, and activation. In particular, the genetic stability of cells treated with nanoparticles has become increasingly important in the context of stem cell therapy. Methods: Functional nanoparticles (Sunflower typed nanoparticles; SF-NPs) were fabricated by coating heparin pluronic F127 gels with quantum dot nanoparticles (QDs), and then bound the SOX9 gene to the QD nanogels. The resultant nanoparticles were transferred into stem cells, and the effect on genetic stability was monitored. To determinate gene delivery efficacy and long-term genomic stability of cells transfected with QD nanogels, hMSCs were transfected with nanogels at passage 4 (T1; Transfected cells 1) and then sub-cultured to passage of (T4). Following transplantation of transfected T1-T4 cells, the cells were monitored by in vivo imaging. The genetic stability of cells treated with nanoparticles was confirmed by chromosomal analysis, copy number variation (CNV) analysis, and mRNA profiling. Results: After 21 days of pellet culture after sub-culture from T1 to T4, hMSCs treated with QD nanogels complexed with SOX9 plasmid DNA (pDNA) significantly increased expression of specific extracellular matrix (ECM) polysaccharides and glycoproteins, as determined by Safranin O and Alcian blue staining. Moreover, the T4 hMSCs expressed higher levels of specific proteins, including collagen type II (COLII) and SOX9, than P4 hMSCs, with no evidence of DNA damage or genomic malfunction. Microarray analysis confirmed expression of genes specific to matured chondrocytes. Stem cells that internalized nanoparticles at the early stage retained genetic stability, even after passage. In in vivo studies in rats, neuronal cartilage formation was observed in damaged lesions 6 weeks after transplantation of T1 and T4 cells. The degree of differentiation into chondrocytes in the cartilage defect area, as determined by mRNA and protein expression of COLII and SOX9, was higher in rats treated with SF-NPs. Conclusion: The QD nanogels used in this study, did not affect genome integrity during long-term subculture, and are thus suitable for multiple theranostic applications. Ivyspring International Publisher 2018-11-05 /pmc/articles/PMC6276296/ /pubmed/30555563 http://dx.doi.org/10.7150/thno.29214 Text en © Ivyspring International Publisher This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions. |
spellingShingle | Research Paper Park, Ji Sun Yi, Se Won Kim, Hye Jin Oh, Hyun Jyung Lee, Jung Sun Go, Minyeon Shim, Sung Han Park, Keun-Hong Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs) |
title | Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs) |
title_full | Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs) |
title_fullStr | Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs) |
title_full_unstemmed | Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs) |
title_short | Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs) |
title_sort | verification of long-term genetic stability of hmscs during subculture after internalization of sunflower-type nanoparticles (sf-nps) |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276296/ https://www.ncbi.nlm.nih.gov/pubmed/30555563 http://dx.doi.org/10.7150/thno.29214 |
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