Nanoparticle-mediated delivery of siRNAs modulates mesenchymal stem cell differentiation
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1
University of Rochester, Biomedical Engineering, United States
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2
University of Rochester Medical Center, Center for Musculoskeletal Research, United States
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3
University of Rochester, Chemical Engineering, United States
Introduction: Mesenchymal stem cells (MSC) are an attractive cell source for tissue engineering approaches due to multilineage differentiation. However, controlling MSC fate is critical for tissue regeneration. microRNAs (miRNA) are known as ‘master regulators’ of differentiation, serving to integrate the myriad of complex signals driving differentiation [1]. However, the use of small interfering RNA (siRNA), which are exogenous analogs of miRNA, to control MSC fate has been largely unexplored due to a paucity of delivery systems and poor appreciation for siRNAs necessary to achieve differentiation. Towards this end, we analyzed temporal expression of multiple miRNAs in MSCs undergoing osteogenesis in vitro. Subsequently, we used polymeric nanoparticles (NPs) previously shown to achieve successful siRNA delivery to MSCs [2] to deliver siRNA mimicking the identified miRNA while monitoring MSC differentiation.
Materials and Methods: Human bone marrow derived MSCs (MSCs) were cultured in osteogenic media for two weeks. miRNA expression was monitored at various time points via qRT-PCR. NPs were then utilized to deliver siRNAs mimicking the miRNAs we found to be upregulated during osteogenesis (e.g., miR-145, and miR-2861) in vitro at 30 nM. For dual delivery of siRNAs, individual siRNAs were delivered at 15 nM to maintain a 30 nM dose.
Results and Discussion: qRT-PCR shows miR-145 was upregulated 2-fold at days 3 and 14 of osteogenesis. Additionally, miR-2861 was upregulated 5-fold at day 14 (Figure 1).
Simply delivering NPs with siRNA mimicking the miRNAs enhanced osteogenesis compared to untreated and scrambled siRNA-treated MSCs. Specifically, NP delivered siRNA mimicking miR-145 (NP-miR-145) resulted in 2-fold increase in Runx2 expression, an osteogenic transcription factor, at day 7 while NP-miR-2861 increased Runx2 expression 3.8-fold at day 14, with no significant differences at day 7 (Figure 2). These data are consistent with previous studies, showing that miR-145 and miR-2861 enhance osteogenesis by inhibiting pluripotency genes Sox2, Oct4 and KLF4 [3] and histone deacetylase 5 (HDAC5), a negative regulator of Runx2 [4], respectively. Interestingly, dual delivery of miR-145 and miR-2861 (NP-miR-145+miR-2861) resulted in 2.6-fold increase in Runx2 at day 7, which is significantly greater than either treatment alone (Figure 2). 
These data suggest dual delivery of siRNAs that target stem cell machinery in concert with siRNAs that drive expression of osteogenic transcription factors enhances MSC osteogenesis. Ongoing experiments include verification of Sox2 and HDAC5 suppression. Further, NP chemistry is being developed to allow for incorporation of NP-siRNAs in hydrogel scaffolds to achieve localized, sustained delivery of NP-siRNAs to encapsulated MSCs.
Conclusion: Polymeric NPs can be used to exploit the effects of endogenous miRNAs via exogenous delivery of siRNA analogues. Overall, our data support the hypothesis that siRNA can be used to direct MSC differentiation. Further, NPs can simultaneously deliver multiple siRNAs to cells, resulting in synergistic modulation of MSC differentiation.
National Science Foundation (DMR-1206219); NYSTEM (IDEA N11G-035)
References:
[1] Gao, J., T. Yang, J. Han, K. Yan, X. Qiu, Y. Zhou, Q. Fan, and B. Ma, MicroRNA expression during osteogenic differentiation of human multipotent mesenchymal stromal cells from bone marrow. J Cell Biochem, 2011. 112(7): p. 1844-56.
[2] Benoit, D.S.W. and M.E. Boutin, Controlling mesenchymal stem cell gene expression using polymer-mediated delivery of siRNA. Biomacromolecules, 2012. 13(11): p. 3841-3849.
[3] Xu, N., T. Papagiannakopoulos, G. Pan, J.A. Thomson, and K.S. Kosik, MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell, 2009. 137(4): p. 647-58.
[4] Li, H., H. Xie, W. Liu, R. Hu, B. Huang, Y.F. Tan, K. Xu, Z.F. Sheng, H.D. Zhou, X.P. Wu, and X.H. Luo, A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans. J Clin Invest, 2009. 119(12): p. 3666-77.
Keywords:
stem cell,
nanoparticle,
Tissue Regeneration,
siRNA delivery
Conference:
10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016.
Presentation Type:
Poster
Topic:
Enabling RNAi with biomaterials
Citation:
Malcolm
DW and
Benoit
DS
(2016). Nanoparticle-mediated delivery of siRNAs modulates mesenchymal stem cell differentiation.
Front. Bioeng. Biotechnol.
Conference Abstract:
10th World Biomaterials Congress.
doi: 10.3389/conf.FBIOE.2016.01.01507
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Received:
27 Mar 2016;
Published Online:
30 Mar 2016.
*
Correspondence:
Dr. Dominic W Malcolm, University of Rochester, Biomedical Engineering, Rochester, NY, United States, dominic.malcolm@rochester.edu
Dr. Danielle S Benoit, University of Rochester, Biomedical Engineering, Rochester, NY, United States, danielleswbenoit@rochester.edu