Targeted gene silencing in human embryonic stem cells using cell-penetrating peptide PepFect 14.
B2M
Cell-penetrating peptide
Human embryonic stem cells
OCT4
PepFect 14
siRNA transfection
Journal
Stem cell research & therapy
ISSN: 1757-6512
Titre abrégé: Stem Cell Res Ther
Pays: England
ID NLM: 101527581
Informations de publication
Date de publication:
24 01 2019
24 01 2019
Historique:
received:
26
09
2018
accepted:
10
01
2019
revised:
29
12
2018
entrez:
26
1
2019
pubmed:
27
1
2019
medline:
9
4
2020
Statut:
epublish
Résumé
Human embryonic stem (hES) cells serve as an invaluable tool for research and future medicine, but their transfection often leads to unwanted side effects as the method itself may induce differentiation. On the other hand, RNA interference (RNAi)-based targeted gene silencing is a quick, cost-effective, and easy-to-perform method to address questions regarding the function of genes, especially when hypomorphic knockdowns are needed. Therefore, effective transfection method with minimal side effects is essential for applying RNAi to hES cells. Here, we report a highly promising approach for targeted gene silencing in hES cells with siRNA complexed with cell-penetrating peptide PepFect 14 (PF14). This strategy provides researchers with efficient tool for unraveling the functions of genes or addressing the differentiation of pluripotent stem cells. We present a method for delivery of siRNA into hES cells with cell-penetrating peptide PF14. Accordingly, hES cells were transfected in ROCK inhibitor containing medium for 24 h right after EDTA passaging as small cell clumps. Fluorescently labeled siRNA and siRNAs targeting OCT4 or beta-2-microglobulin (B2M) mRNA sequences were used to evaluate the efficiency of transfection and silencing. Analyses were performed at various time points by flow cytometry, RT-qPCR, and immunofluorescence microscopy. Effective downregulation of OCT4 in 70% of treated hES cells at protein level was achieved, along with 90% reduction at mRNA level in bulk population of cells. The applicability of this low-cost and easy-to-perform method was confirmed by inducing silencing of another target not associated with hES cell pluripotency (B2M). Furthermore, we discovered that downregulation of OCT4 induces neuroectodermal differentiation accompanied by reduced expression of B2M during early stage of this lineage. The results demonstrate PF14 as a promising tool for studying gene function and regulatory networks in hES cells by using RNAi.
Sections du résumé
BACKGROUND
Human embryonic stem (hES) cells serve as an invaluable tool for research and future medicine, but their transfection often leads to unwanted side effects as the method itself may induce differentiation. On the other hand, RNA interference (RNAi)-based targeted gene silencing is a quick, cost-effective, and easy-to-perform method to address questions regarding the function of genes, especially when hypomorphic knockdowns are needed. Therefore, effective transfection method with minimal side effects is essential for applying RNAi to hES cells. Here, we report a highly promising approach for targeted gene silencing in hES cells with siRNA complexed with cell-penetrating peptide PepFect 14 (PF14). This strategy provides researchers with efficient tool for unraveling the functions of genes or addressing the differentiation of pluripotent stem cells.
METHODS
We present a method for delivery of siRNA into hES cells with cell-penetrating peptide PF14. Accordingly, hES cells were transfected in ROCK inhibitor containing medium for 24 h right after EDTA passaging as small cell clumps. Fluorescently labeled siRNA and siRNAs targeting OCT4 or beta-2-microglobulin (B2M) mRNA sequences were used to evaluate the efficiency of transfection and silencing. Analyses were performed at various time points by flow cytometry, RT-qPCR, and immunofluorescence microscopy.
RESULTS
Effective downregulation of OCT4 in 70% of treated hES cells at protein level was achieved, along with 90% reduction at mRNA level in bulk population of cells. The applicability of this low-cost and easy-to-perform method was confirmed by inducing silencing of another target not associated with hES cell pluripotency (B2M). Furthermore, we discovered that downregulation of OCT4 induces neuroectodermal differentiation accompanied by reduced expression of B2M during early stage of this lineage.
CONCLUSIONS
The results demonstrate PF14 as a promising tool for studying gene function and regulatory networks in hES cells by using RNAi.
Identifiants
pubmed: 30678718
doi: 10.1186/s13287-019-1144-x
pii: 10.1186/s13287-019-1144-x
pmc: PMC6345057
doi:
Substances chimiques
Cell-Penetrating Peptides
0
Lipopeptides
0
PepFect14 peptide
0
RNA, Small Interfering
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
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