Spatial control of gene expression within a scaffold by localized inducer release

Abstract

Gene expression can be controlled in genetically modified cells by employing an inducer/promoter system where presence of the inducer molecule regulates the timing and level of gene expression. By applying the principles of controlled release, it should be possible to control gene expression on a biomaterial surface by the presence or absence of inducer release from the underlying material matrix, thus avoiding alternative techniques that rely upon uptake of relatively labile DNA from material surfaces. To evaluate this concept, a modified ecdysone-responsive gene expression system was transfected into B16 murine cells and the ability of an inducer ligand, which was released from elastomeric poly(ester urethane) urea (PEUU), to initiate gene expression was studied. The synthetic inducer ligand was first loaded into PEUU to demonstrate extended release of the bioactive molecule at various loading densities over a one year period in vitro. Patterning films of PEUU variably-loaded with inducer resulted in spatially controlled cell expression of the gene product (green fluorescent protein, GFP). In porous scaffolds made from PEUU by salt leaching, where the central region was exclusively loaded with inducer, cells expressed GFP predominately in the loaded central regions whereas expression was minimal in outer regions where ligand was omitted. This scaffold system may ultimately provide a means to precisely control progenitor cell commitment in a spatially-defined manner in vivo for soft tissue repair and regeneration.

Figure 1

Modified ecdysone-responsive gene expression (RheoSwitch) system (a) and chemical structure of the RSL1 inducer ligand (b). Plasmid pNEBR-R1 uses ubiquitin B (UbB) and ubiquitin C (UbC) promoters to constitutively express proteins RheoActivator (RA) and RheoReceptor-1 (RR1), respectively. RR1and RA form a bipartite holoreceptor that binds the 5X RE promoter sequence on plasmid pNEBR-X1, which contains the gene of interest (green fluorescent protein, GFP). When bound to inducer ligand RSL1, the holoreceptor exchanges bound negative regulatory cofactors for positive cofactors, allowing high transcription rates of GFP.

Figure 2

Gene expression control by culture medium manipulation. At 48 hrs post-RSL1 administration, cells in growth medium without ligand did not express GFP (a), while cells in growth medium containing 1, 2, 3, and 5 μM ligand (b-d, respectively) expressed GFP with increasing frequency in a linear fashion (f). Scale bar = 200 μm.

Figure 3

Release of RSL1 from PEUU films in vitro. Films had initial loading concentrations of 25, 75, and 150 μM RSL1.

Figure 4

Gene expression in cells treated with release medium from PEUU films containing (a) 0 μM, (b) 1 μM, (c) 2 μM, (d) 3 μM, (e) 4 μM, and (f) 5 μM RSL1. Scale bar = 500 μm.

Figure 5

Long-term bioactivity of released RSL1. Release fluid from films loaded with 25, 75, or 150 μM RSL1 at designated time points was able to cause GFP expression in cultured B16 cells. * denotes p<.001 between each of the 3 concentrations.

Figure 6

Gene expression on PEUU films containing (a) 0 μM, (b) 1 μM, (c) 2 μM, (d) 3 μM, (e) 4 μM, and (f) 5 μM RSL1. (g) Dose-dependent GFP expression is preserved when cells are maintained on polymer films. Scale bar = 500 μm.

Figure 7

Spatial control of gene expression on PEUU films. (a) Typical slide layout for 2-dimensional spatial control experiments. (b) Cells on all regions of slides containing PEUU without ligand (grey) did not express GFP (left), whereas cells on all regions of slides containing PEUU loaded with RSL1 (black) clearly expressed GFP (right), demonstrating spatial control of gene expression on 2-dimensional PEUU films. Scale bar = 500 μm

Figure 8

Macroscopic and scanning electron micrograph of porous scaffold differentially loaded with RSL1. (a) RSL1 was added to a center core of the material whereas no RSL1 was present in the outer ring, (b) Scanning electron micrograph of the boundary between RSL1-containing core and RSL1-free outer ring. White arrows delineate boundary between regions. (c) Mechanical stretching of the scaffold seen in (a) reveals continuity between concentric regions (Also demonstrated in supplementary video). Scale bar = 1 mm.

Figure 9

Spatial control of gene expression within 3D PEUU scaffolds. GFP expression was concentrated in the inner region of the scaffold where RSL1 inducer was present. (a) Blue - DAPI nuclear staining only, (b) green - GFP expression only, and (c) combined image showing localization of both cells and GFP expression. Dashed white circle represents boundary between inner and outer scaffold regions. Scale bar = 1 mm. (d) Ratio of green to blue (G/B) fluorescence signal with respect to location across center of scaffold, demonstrating spatial control of gene expression.