Induction of osteogenic differentiation of MSCs by GSK3β knockdown through GSK3β siRNAs transfection
Elena V. Galitsyna, Anastasiia A. Buianova, Tatiana B. Bukharova, Irina A. Krivosheeva, Mikhail Yu. Skoblov, Dmitriy V. Goldshtein
Abstract
The development of effective strategies for treating bone defects can be based on gene therapy methods aimed at regulating the differentiation of osteoprogenitor cells. One of the approaches is to use siRNA molecules in knockdown systems for genes inhibiting osteogenic cell differentiation. In this work, we aimed at developingapproaches to induce osteogenic differentiation of mesenchymal stem cells (MSCs) by siRNAs-mediated knockdown of GSK3β siRNAs in cultures of MSCs derived from human adipose tissue (AD-MSCs).
Introduction
The use of small interfering RNA (siRNA) molecules for organotypic bone tissue regeneration represents a promising approach in regenerative medicine. Mesenchymal stem cells (MSCs) are among the principal in vitro models for the development of gene–cell technologies due to their accessibility from different tissues and their high proliferative and multilineage differentiation potential [1].
Materials and method
Cell cultures
The HEK 293 cell line, primary human adipose tissue-derived MSCs (AD-MSCs), and primary MSCs derived from human exfoliated deciduous teeth (SHED) cultures at passages 2–3 were used in this study. MSC cultures were obtained from tissues of healthy donors recruited between 18 December 2020 and 19 April 2022. All donors provided written informed consent prior to tissue collection. In the case of minors, written informed consent was obtained from their parents or legal guardians. The study was approved by the local Ethics Committee of the Research Centre for Medical Genetics (RCMG), Moscow, Russia (Protocol No. 8/3 dated 14.12.2020).
Results
siRNA design
To date, there is no uniform algorithm for siRNA sequence design [42]. Freeware programs are based on different approaches (empirical rules, BLAST data, neural networks) and may produce varying results when designing siRNAs even for the same target sequence [43–45]. To develop our own software for designing an effective sequence of siRNA molecules, we took the empirical rules described in the review article [37] summarizing a large amount of experimental data. The siRNAfit software analyzes a given mRNA sequence of 19–21 nucleotides, assigning scores to each oligonucleotide according to empirical rules described by Laganà et al. (2015) [37].
Discussion
The present study provides a systematic methodological framework for siRNA-mediated GSK3β knockdown in primary AD-MSCs, addressing both siRNA delivery optimization and functional osteogenic outcomes. While the negative regulatory role of GSK3β in osteogenic signaling pathways has been established through pharmacological and genetic approaches [23–25,29,30], the application of siRNA-based GSK3β silencing strategies to primary human MSC cultures remains underexplored, with existing reports yielding conflicting results depending on cell source and experimental conditions [7–9,31].
Conclusion
siRNA molecules, acting via the RNA interference mechanism, represent a highly precise tool for genetic silencing of target mRNA transcripts without directly effecting the genome. However, the chemical transfection vectors utilized for their delivery often exhibit inherent cytotoxicity [52,53]. In the course of this work, we identified PEI as the most effective transfection agent for MSCs and determined the optimal concentration of siRNA molecules in polyplexes to be 50 pmol/mL.
Acknowledgments
The authors are grateful for the help with the translation of this article into English to Dr. Andrey Marakhonov, a Senior Researcher of the Laboratory of functional genomics of the RCMG.
Citation: Galitsyna EV, Buianova AA, Bukharova TB, Krivosheeva IA, Skoblov MY, Goldshtein DV (2026) Induction of osteogenic differentiation of MSCs by GSK3β knockdown through GSK3β siRNAs transfection. PLoS One 21(8): e0355286. https://doi.org/10.1371/journal.pone.0355286
Editor: Nazmul Haque, TotiCell Limited, Bangladesh, BANGLADESH
Received: July 13, 2025; Accepted: July 20, 2026; Published: August 7, 2026
Copyright: © 2026 Galitsyna et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This research was carried out within the state assignment of the Ministry of Science and Higher Education of the Russian Federation for the Research Centre for Medical Genetics (RCMG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
