A microparticle approach for non-viral gene delivery within 3D human mesenchymal stromal cell aggregates

Abstract

Three-dimensional (3D) multicellular aggregates, in comparison to two-dimensional monolayer culture, can provide tissue culture models that better recapitulate the abundant cell-cell and cell-matrix interactions found in vivo. In addition, aggregates are potentially useful building blocks for tissue engineering. However, control over the interior aggregate microenvironment is challenging due to inherent barriers for diffusion of biological mediators (e.g. growth factors) throughout the multicellular aggregates. Previous studies have shown that incorporation of biomaterials into multicellular aggregates can support cell survival and control differentiation of stem cell aggregates by delivering morphogens from within the 3D construct. In this study, we developed a highly efficient microparticle-based gene delivery approach to uniformly transfect human mesenchymal stromal cells (hMSC) within multicellular aggregates and cell sheets. We hypothesized that release of plasmid DNA (pDNA) lipoplexes from mineral-coated microparticles (MCMs) within 3D hMSC constructs would improve transfection in comparison to standard free pDNA lipoplex delivery in the media. Our approach increased transfection efficiency 5-fold over delivery of free pDNA lipoplexes in the media and resulted in homogenous distribution of transfected cells throughout the 3D constructs. Additionally, we found that MCMs improved hMSC transfection by specifically increasing macropinocytosis-mediated uptake of pDNA. Finally, we showed up to a three-fold increase of bone morphogenetic protein-2 (BMP-2) expression and enhanced calcium deposition within 3D hMSC constructs following MCM-mediated delivery of a BMP-2 encoding plasmid and culture in osteogenic medium. The technology described here provides a valuable tool for achieving efficient and homogenous transfection of 3D cell constructs and is therefore of particular value in tissue engineering and regenerative medicine applications. STATEMENT OF SIGNIFICANCE: This original research describes a materials-based approach, whereby use of mineral-coated microparticles improves the efficiency of non-viral gene delivery in three-dimensional human mesenchymal stromal cell constructs. Specifically, it demonstrates the use of mineral-coated microparticles to enable highly efficient transfection of human mesenchymal stromal cells in large, 3D culture formats. The manuscript also identifies specific endocytosis pathways that interact with the mineral coating to afford the improved transfection efficiency, as well as demonstrates the utility of this approach toward improving differentiation of large cell constructs. We feel that this manuscript will impact the current understanding and near-term development of materials for non-viral gene delivery in broad tissue engineering and biofabrication applications, and therefore be of interest to a diverse biomaterials audience.

Figure 1.. Incorporation of pDNA-laden MCMs into hMSC aggregates:

(A) Schematic of the microparticle-based gene delivery approach: pDNA was condensed using Lipofectamine2000 and the resulting complex was adsorbed to the MCM surface. The pDNA-laden MCMs were mixed with hMSCs and centrifuged to transfect the aggregates. (B) Scanning electron micrograph (SEM) of MCMs at low magnification showing MCMs of 5–8 μm in diameter. (C) High magnification SEM micrograph of MCMs showing plate-like features around 200–400 nm in size. (D) MCM binding capacity of luciferase-encoding plasmid DNA lipoplexes.

Figure 2.. Comparing transgene expression after plasmid DNA delivery into hMSC aggregates using different strategies:

A) Schematic of three pDNA delivery strategies: i) Standard: free pDNA lipoplexes added into the aggregate medium culture medium, ii) pDNA-free MCMs incorporated into hMSC aggregates, and then pDNA lipoplexes added into the aggregate culture medium, iii) pDNA lipoplexes adsorbed to the surface of MCMs, and then incorporated into hMSC aggregates. B) Luciferase activity of hMSC aggregate culture medium, 2 days after transfection using three different approaches. “*, ***, ****” represents p-value < 0.05, 0.001, and 0.0001 respectively for 2-way anova with Tukey post-hoc test. C) Luciferase activity of hMSC aggregate culture medium over time for 7 days after transfection using three different approaches. “****” represents p-value < 0.0001 for 2-way anova with Tukey post-hoc test. D) hMSC aggregates viability (cell-titer blue) after 2 days of transfection with the different approaches. “****” represents p-value < 0.0001 for 2-way anova with Tukey post-hoc test. “a” represents p-value < 0.05 for 2-way anova with Dunnet post-hoc test to hMSC only control. “ns” represents no significant difference.

Figure 3.. MCM-mediated delivery increases transfection efficiency and homogeneity of hMSC aggregates.

hMSC aggregates (0.25×10⁶ cell/Agg), transfected with (A) standard approach, (B) 100 μg pDNA-free MCMs and free pDNA lipoplexes suspended in the medium, and (C) the pDNA/MCM method, examined for distribution of EGFP-positive cells within epifluorescence micrographs of cryosectioned aggregates. (D) Examination of distribution of EGFP-positive cell in hMSC aggregates after transfected with increasing amounts of pEGFP with 100 μg MCMs for each aggregate. (E) Quantification of cellular uptake using flow cytometry and rhodamine-labeled pEGFP. Plasmid uptake increased in response to both the amount of plasmid and MCM delivered. “*, **, ***” represents p-value < 0.05, 0.01, and 0.001 respectively for 2-way anova with Tukey post-hoc test. “ns” represents no significant difference. (F) Optimization of pDNA/MCM method by varying pEGFP and MCMs amounts. hMSC transfection efficiencies increased with higher amounts of plasmid delivered for the 50 and 100 μg MCMs/Agg. All pDNA/MCM aggregate transfection efficiencies were higher than standard transfection in 2D except 2 μg pEGFP/50 μg MCM. “****” represents p-value < 0.0001 for 2-way anova with Tukey post-hoc test. (G) BMP-2 expression from hMSC aggregates transfected with pBMP-2/MCMs, measured via ELISA. “**” represents p-value < 0.01 for 2-way anova with Tukey post-hoc test. “ns” represents no significant difference.

Figure 4.. MCMs increase endocytosis in hMSCs and improve non-viral transfection through induction of macropinocytosis.

(A) Representative epifluorescence micrographs of hMSCs cultured in 2D with MCMs and Alexa Fluor 564-labeled 10k MW dextran. scale bar = 250μm (B) Quantification of increase in endocytosis in response to increasing MCM concentration. Endocytosis measured as total red fluorescence area. “**” represents p-value < 0.01 1-way anova with linear trend post-hoc test for dose-dependent response. (C) Table of small molecule inhibitors and specific endocytosis pathway inhibited. (D) Quantification of endocytosis in presence to small molecule inhibitors without MCMs. Endocytosis measured as mean red fluorescence intensity. “*” represents p-value < 0.05 1-way anova with Dunnet post-hoc test compared to no treatment (E) Quantification of endocytosis in presence to small molecule inhibitors with and without MCMs. Endocytosis measured as mean red fluorescence intensity. “**, ****” represents p-value < 0.01, 0.001 t-test.

Figure 5.. MCMs improve transfection and calcium production during osteogenic differentiation of 10 mm hMSC sheets.

(A) Epifluorescence micrographs of 10 mm hMSC sheets (2×10⁶ cells per sheet) transfected and formed in 12-well transwells. Scale bar = 2.5 mm (B) Quantification of pEGFP hMSC sheet transfection via flow cytometry using the optimized pDNA/MCM condition. “****” represents p-value < 0.0001 1-way anova with linear trend post-hoc test for dose-dependent response. (C) hMSC sheet calcium production, measured Arsenazo III colorimetric detection after EDTA chelation of cell sheets transfected and cultured in osteogenic medium for 5 weeks. “*, ****” represents p-value < 0.05, and 0.0001 respectively for 2-way anova with Tukey post-hoc test.