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Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells
Published on: March 8, 2019
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Efficient gene silencing in mesenchymal stem cells by substrate-mediated RNA interference
Shan-Hui Hsu1, Guo-Shiang Huang, Tung-Tso Ho
11 Institute of Polymer Science and Engineering, National Taiwan University , Taipei, Taiwan, Republic of China .
Tissue Engineering. Part C, Methods
|March 15, 2014
Summary
Human gingival fibroblasts (HGFs) exhibit multipotency linked to neural crest marker FoxD3 expression. A novel chitosan substrate enhances RNA interference delivery for gene silencing, improving differentiation studies.
Area of Science:
- Biotechnology
- Stem Cell Biology
- Regenerative Medicine
Background:
- Human gingival fibroblasts (HGFs) possess multipotency, with potential for neural and chondrogenic differentiation.
- Neural crest marker gene Forkhead box D3 (FoxD3) expression is implicated in HGF multipotency.
- Conventional delivery vehicles for RNA interference (RNAi) can exhibit toxicity.
Purpose of the Study:
- To develop and evaluate a novel substrate-mediated RNA interference (RNAi) technology for gene silencing in HGFs.
- To investigate the role of neural crest marker FoxD3 in HGF multipotency using the new technology.
- To assess the efficiency and safety of chitosan substrate-mediated gene delivery.
Main Methods:
- Utilized a novel chitosan substrate for delivering short-hairpin RNA (shRNA) constructs to HGFs.
- Employed RNA interference (RNAi) to knockdown the Forkhead box D3 (FoxD3) gene in HGFs.
- Measured delivery efficiency via flow cytometry and assessed cell viability.
Main Results:
- Chitosan substrate-mediated delivery showed a 10-fold increase in efficiency compared to conventional methods, with >95% cell viability.
- HGFs with FoxD3 gene knockdown on chitosan did not form spheroids, indicating functional gene silencing.
- Nonsilenced HGFs exhibited significantly higher neural differentiation (40-fold increase in nestin expression) compared to FoxD3-silenced cells.
Conclusions:
- The novel chitosan substrate-mediated RNAi platform offers efficient and non-toxic gene delivery for HGFs.
- This technology facilitates the investigation of gene functions, such as FoxD3, in cellular multipotency and differentiation.
- The platform holds promise for advancing stem cell research and regenerative medicine applications.
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