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Theranostics 2018; 8(20):5548-5561. doi:10.7150/thno.29214 This issue Cite

Research Paper

Verification of Long-Term Genetic Stability of hMSCs during Subculture after Internalization of Sunflower-Type Nanoparticles (SF-NPs)

Ji Sun Park^1*, Se Won Yi^1*, Hye Jin Kim¹, Hyun Jyung Oh¹, Jung Sun Lee¹, Minyeon Go², Sung Han Shim^2✉, Keun-Hong Park ^1✉

1. Laboratory of Nano-regenerative Medical Engineering, Department of Biomedical Science, College of Life Science, CHA University, 618, CHA Biocomplex, Sampyeong-Dong, Bundang-gu, Seongnam-si, 13488, Republic of Korea.
2. Laboratory of Molecular Genetics, Department of Biomedical Science, College of Life Science, CHA University, 629, CHA Biocomplex, Sampyeong-Dong, Bundang-gu, Seongnam-si, 13488, Republic of Korea.
*These authors contributed equally to this work.

✉ Corresponding authors: Prof. Sung Han Shim (shshimac.kr) and Prof. Keun-Hong Park (pkh0410ac.kr)

Abstract

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.

Keywords: quantum dot, Sunflower-type nanoparticle, genomic stability, single-nucleotide polymorphism, karyotyping, mRNA/DNA profile

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