original article
© The American Society of Gene & Cell Therapy
siRNA Nanoparticle Functionalization of Nanostructured Scaffolds Enables Controlled Multilineage Differentiation of Stem Cells Morten Ø Andersen1,2, Jens V Nygaard1, Jorge S Burns5, Merete K Raarup3, Jens R Nyengaard3, Cody Bünger4, Flemming Besenbacher1, Kenneth A Howard1,2, Moustapha Kassem5,6 and Jørgen Kjems1,2 Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus C, Denmark; 2Department of Molecular Biology, Aarhus University, Aarhus C, Denmark; 3Stereology and Electron Microscopy Laboratory and MIND Center, Aarhus University, Aarhus C, Denmark; 4 Orthopaedic Research Laboratory, Aarhus University, Aarhus C, Denmark; 5Department of Endocrinology and Metabolism, University Hospital of Odense, Odense C, Denmark; 6Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia 1
The creation of complex tissues and organs is the ultimate goal in tissue engineering. Engineered morphogenesis necessitates spatially controlled development of multiple cell types within a scaffold implant. We present a novel method to achieve this by adhering nanoparticles containing different small-interfering RNAs (siRNAs) into nanostructured scaffolds. This allows spatial retention of the RNAs within nanopores until their cellular delivery. The released siRNAs were capable of gene silencing BCL2L2 and TRIB2, in mesenchymal stem cells (MSCs), enhancing osteogenic and adipogenic differentiation, respectively. This approach for enhancing a single type of differentiation is immediately applicable to all areas of tissue engineering. Different nanoparticles localized to spatially distinct locations within a single implant allowed two different tissue types to develop in controllable areas of an implant. As a consequence of this, we predict that complex tissues and organs can be engineered by the in situ development of multiple cell types guided by spatially restricted nanoparticles. Received 18 March 2010; accepted 10 July 2010; published online 31 August 2010. doi:10.1038/mt.2010.166
Introduction Tissue engineering has the potential to alleviate disease by producing abundant and tolerated replacement organs.1 The standard approach is to seed patient-derived terminally differentiated cells on porous three-dimensional cell supports (scaffolds). When it comes to generating tissues with multiple cell types, however, this method is limited to scaffold structures that can be loaded with cells in physically separated locations such as spheres (bladders2) and tubes (larynx3). A similar rapid prototyping approach is cell printing where different cell populations are deposited into three-dimensional shapes.4,5 Unfortunately, this approach presents limitations6 including cell alterations from induced mechanotransduction during processing. Moreover,
reconstruction with a priori differentiated cells is problematic as cells perform important functions such as preparing extracellular matrix while undergoing differentiation and loose this ability when fully differentiated.7 Consequently, we explored the seeding of stem cells onto scaffolds before cell specialization. A limitation with this approach is that conventional global provision of differentiation cues fails to differentiate stem cells into multiple cell types in discrete locations. Here, we present a novel strategy where nanostructured scaffolds are coated with different nanoparticles in spatially discrete parts directing the differentiation pathway of one homogenously seeded stem cell population into multiple cell types in situ. Several different drugs have been used to steer differentiation on scaffolds including proteins,8 plasmids,9,10 and viruses.11 We believe a more versatile drug type to be small-interfering RNA (siRNA). Once introduced into cells, siRNAs can silence synthesis of a specific protein by base pairing with its mRNA sequence.12 When applied to cultured monolayers of mesenchymal stem cells (hMSCs), commonly used in tissue engineering,13 siRNAs were able to enhance their differentiation into bone,14 cartilage,15 fat,16 muscle,17 liver,18 and nerve cells.19 A nanoparticle-based implant coating, capable of delivering siRNAs into stem cells situated in a scaffold, therefore, has broad applications in tissue engineering.20,21 Here, we present a novel technology, siRNA-enhanced scaffolds (siRESs), composed of biodegradable nanostructured polyε-caprolactone (PCL) scaffolds functionalized with a lyophilized polymer/lipid-based nanoparticulate siRNA delivery system.22 We investigate, the particle localization and retention properties as well as the silencing and in vitro and in vivo enhancement of differentiation of the system, using hMSCs as progenitor cells and siRNAs targeted to enhanced green fluorescent protein (EGFP), tribbles homolog 2 (TRIB2, also known as TRB2), and BCL2 like 2 (BCL2L2, also known as BCL-w). We demonstrate successful enhancement of differentiation, and importantly, that tailored cell specialization can be affected differently in discrete locations within a composite scaffold by controlled deposition of BCL2L2 siRNA and TRIB2 siRNA containing nanoparticles.
Correspondence: Jørgen Kjems, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, C.F. Møllers Allé 3 (Building 1130), Aarhus C 8000, Denmark. E-mail:
[email protected]
2018
www.moleculartherapy.org vol. 18 no. 11, 2018–2027 nov. 2010
© The American Society of Gene & Cell Therapy
siRNA Functionalized Scaffolds Enable Multilineage Differentiation
Results Monolayer culture
had an equal reduction in EGFP approximately corresponding to the average decline in EGFP (Supplementary Figure S1). The specificity of the siRNAs was investigated using siRNAs targeting different regions of TRIB2 and BCL2L2 and by scrambling part of the seed sequences (Figure 1d,e). Targeting a different region of the mRNA resulted in the same degree of knockdown, whereas partial scrambling of the siRNA seed sequence led to a significant decrease in knockdown. The influence of the siRNA transfection on cell viability was studied by growing hMSCs for 2 days on siRNA-coated plates in maintenance medium followed by 12 days in various differentiation mediums (Figure 1f). Transfected cell viability was slightly reduced (~30, ~40, and ~45% reduction in viability for EGFP, TRIB2, and BCL2L2 siRNA) in maintenance medium. This reduction was comparable to that induced by differentiation medium. To confirm that osteogenic and adipogenic
The potential of reverse transfecting hMSCs with siRNA was initially studied in monolayer culture. Tissue culture plates coated by a lyophilization process with TransIT-TKO/siRNA particles with hydrodynamic diameter (259 ± 14 nm) and ζ potential (12.6 ± 0.5 mV) were seeded with telomerase-immortalized hMSCs.23 siRNA targeting EGFP (EGFP-expressing hMSCs were used in this case24), BCL2L2, and TRIB2 (Figure 1a–c, respectively) were used. Flow cytometry and quantitative PCR (qPCR) revealed that the delivery system was capable of reducing expression of all siRNA targeted genes by at least 50% after 2 days. EGFP protein levels were reduced by over 95% 7 days post-transfection. Histograms of cellular EGFP fluorescence with or without EGFP knockdown showed that the majority of the EGFP silenced cells
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Figure 1 Monolayer transfection of human mesenchymal stem cells (hMSCs) grown on tissue culture polystyrene plates, either noncoated (NC) or coated with freeze-dried TransIT-TKO nanoparticles containing EGFP-targeted siRNA (siEGFP), EGFP mismatch siRNA (siMM), TRIB2targeted siRNA (siTRIB2 #1), or BCL2L2-targeted siRNA (siBCL2L2 #1). (a) The relative fluorescence level of EGFP in hMSCs (EGFP+) after 2 and 7 days of reverse transfection was determined by flow cytometry. Displayed is the average geometric mean fluorescence normalized to nontransfected cells which were set to 100%, the standard deviation is indicated by error bar (N = 3). (b) The relative expression level of BCL2L2 mRNA normalized to GAPDH mRNA after 48 hours of reverse transfection of hMSCs (EGFP−). The graph shows average normalized to untransfected cells which were set to 100%, the standard deviation is indicated by error bars (N = 3). (c) The relative expression level of TRIB2 mRNA normalized to GAPDH mRNA after 48 hours of reverse transfection of hMSCs (EGFP−). The graph shows average normalized to untransfected cells which were set to 100%, the standard deviation is indicated by error bars (N = 3). (d,e) The relative expression level of BCL2L2 mRNA and TRIB2 mRNA normalized to GAPDH mRNA after 48 hours of reverse transfection of hMSCs (EGFP−). The graph shows average normalized to untransfected cells which were set to 100%, the standard deviation is indicated by error bars (N = 5). siRNA #1 corresponds to the same sequence as in graph b and c, although a different siRNA supplier was used. siRNAs #2 targeted different regions of the genes representing a completely different sequence. SScr denotes siRNA #1 where the sequence of four nucleic acids in the seed sequence (position 3–6) has been scrambled. (f) Displays viability after hMSCs (EGFP−) were cultured for 2 days in maintenance medium followed by 12 days in the indicated mediums (M, maintenance medium; O, osteogenic medium without vitamin D3; A, adipogenic medium; C, complex medium; D, osteogenic medium with vitamin D3). The graph shows average normalized to nontransfected cells in maintenance media which were set to 100%, the standard deviation is indicated by error bars (N = 4). EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Molecular Therapy vol. 18 no. 11 nov. 2010
2019
© The American Society of Gene & Cell Therapy
siRNA Functionalized Scaffolds Enable Multilineage Differentiation
a
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Figure 2 Scanning electron microscopy of scaffolds. (a) Scaffold before coating with nanoparticles. (b) High magnification of scaffold structure indicated in a. (c) Scaffold after coating with TransIT-TKO/ BCL2L2 siRNA nanoparticles. (d) High magnification of scaffold structure indicated in c.
differentiation could take place in the presence of siRNA particles, we performed alkaline phosphatase (ALP), alizarin red, and oil red O staining after transfection in maintenance medium and culturing in differentiation medium (Supplementary Figure S2), the stains showed that the transfection process did not adversely affect the ability of the stem cells to differentiate. In conclusion, freezedried TransIT-TKO/siRNA particles were an effective transfection agent for hMSCs in monolayer culture.
siRNA coating of scaffolds Hierarchically organized scaffolds were produced through thermally induced phase separation of PCL in 1,4-dioxane and H2O (ref. 25) and a nanoroughened hydrophilic surface was subsequently introduced by partial chemical degradation.26 When visualized with scanning electron microscopy (Figure 2a,b), the scaffolds appeared bimodal in pore size distribution. Large pores (diameter >50 µm) were separated by walls (thickness 10–40 µm) containing smaller cavities of decreasing size (the smallest structural features extended
