17 research outputs found
Organizational metrics of interchromatin speckle factor domains: integrative classifier for stem cell adhesion & lineage signaling.
Stem cell fates on biomaterials are influenced by the complex confluence of microenvironmental cues emanating from soluble growth factors, cell-to-cell contacts, and biomaterial properties. Cell-microenvironment interactions influence the cell fate by initiating a series of outside-in signaling events that traverse from the focal adhesions to the nucleus via the cytoskeleton and modulate the sub-nuclear protein organization and gene expression. Here, we report a novel imaging-based framework that highlights the spatial organization of sub-nuclear proteins, specifically the splicing factor SC-35 in the nucleoplasm, as an integrative marker to distinguish between minute differences of stem cell lineage pathways in response to stimulatory soluble factors, surface topologies, and microscale topographies. This framework involves the high resolution image acquisition of SC-35 domains and imaging-based feature extraction to obtain quantitative nuclear metrics in tandem with machine learning approaches to generate a predictive cell state classification model. The acquired SC-35 metrics led to \u3e 90% correct classification of emergent human mesenchymal stem cell (hMSC) phenotypes in populations of hMSCs exposed for merely 3 days to basal, adipogenic, or osteogenic soluble cues, as well as varying levels of dexamethasone-induced alkaline phosphatase (ALP) expression. Early osteogenic cellular responses across a series of surface patterns, fibrous scaffolds, and micropillars were also detected and classified using this imaging-based methodology. Complex cell states resulting from inhibition of RhoGTPase, β-catenin, and FAK could be classified with \u3e 90% sensitivity on the basis of differences in the SC-35 organizational metrics. This indicates that SC-35 organization is sensitively impacted by adhesion-related signaling molecules that regulate osteogenic differentiation. Our results show that diverse microenvironment cues affect different attributes of the SC-35 organizational metrics and lead to distinct emergent organizational patterns. Taken together, these studies demonstrate that the early organization of SC-35 domains could serve as a “fingerprint” of the intracellular mechanotransductive signaling that governs growth factor- and topography-responsive stem cell states
Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases
Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer
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Role of cellular microenvironment in non-viral gene transfer
Gene delivery has widespread application in tissue regeneration and gene therapy. While non-viral gene delivery is less immunogenic as compared to viral gene delivery, it is hindered by its lack of efficiency. Studies aiming to improve the efficiency of non-viral gene delivery have mostly focused on improving the vector system. However, the cellular microenvironment where the cells reside is only beginning to be exploited as a means to enhance gene transfer. In this dissertation, the effect of different densities of extracellular matrix proteins namely collagen I (C I), vitronectin (Vt), laminin (Lm), collagen IV (C IV), fibronectin (Fn) and ECM gel (ECMg), and their combinations on gene transfer to mouse mesenchymal stem cells (mMSCs) were studied. Protein coatings that resulted in well spread cells such as Fn, ECMg and C IV, resulted in 14.6-, 7- and 6.1- fold increase in transgene expression, respectively, when compared to uncoated surfaces. The transgene expression was up to 90% inhibited on C I coated surface, which led to less spread cells. Interestingly, the same trend was not observed for polyplex internalization, where protein coats that resulted in less spread cells, such as C I and Vt, resulted in higher polyplex internalization. Subsequently, decreased transgene expression corresponded with inhibited trafficking of the internalized complexes to the nucleus. The effect of combining multiple ECM proteins on non-viral gene transfer was also investigated. Surfaces coated with combination including C I resulted in inhibition of transgene expression. In addition, surfaces coated with combination of Vt, C IV and Lm resulted in a statistically similar enhancement in transgene expression as compared to fibronectin. For all ECM combinations analyzed, the extent of cell spreading mediated by the ECM protein had a 70% correlation with the extent of overall gene transfer observed. The role of different endocytic pathways and cytoskeletal components on gene transfer was later analyzed on collagen I (C I) and fibronectin (Fn), which had opposite influence on gene transfer. The mechanism by which these ECM proteins affect non-viral gene transfer involved the endocytosis pathway used for polyplex uptake and intracellular tension. Fn was found to promote internalization through clathrin-mediated endocytosis and this pathway brought about more efficient transfection than caveolae-mediated endocytosis and macropinocytosis. Likewise, the disruption of actin-myosin interactions resulted in an enhancement of gene transfer for cells plated on Fn coated surfaces, but not for cells plated on C I. Furthermore, the molecular mechanism by which these ECM molecules affect the process of gene transfer is not completely understood. The Rho subfamily of GTPases regulates a number of cellular processes including cell morphology, cytoskeletal dynamics and uptake pathways and they are activated to different extents by different ECM proteins. The involvement of RhoGTPases in the ECM protein-mediated enhancement of non-viral gene transfer was studied. Fibronectin was used as the substrate for these studies. The interaction of mouse mesenchymal stem cells (mMSCs) with fibronectin was found to activate RhoGTPases (RhoA, Rac, and Cdc42). Inactivation of RhoGTPases using chemical inhibitor or expression of dominant negative genes resulted in significantly reduced transgene expression. However, the activation of RhoGTPases using chemical activators or expressing constitutively active genes did not further enhance transgene expression for cells plated on fibronectin. But, for cells plated on C I, which did not result in RhoGTPase activation, expression of constitutively active RhoA, Rac, Cdc42 genes resulted in enhanced non-viral gene transfer. Cells in the body exist in a 3-D microenvironment. Cellular processes like proliferation, differentiation, apoptosis as well as cellular morphology have been shown to be significantly different in 2-D versus 3-D. In part, these differences are due to differential signaling as a consequent of different cytoskeletal assembly and cell-matrix adhesions in 2-D and 3-D. To fully understand the mechanism of gene transfer in cells, L-polyethylenimine (LPEI) mediated non-viral gene transfer mechanism constituting pathways of endocytosis, cytoskeletal dynamics and RhoGTPases, was studied in 3-D using hyaluronic acid hydrogels, in comparison with 2-D using tissue culture plates. Caveolae and clathrin mediated endocytosis were observed to regulate gene transfer in 2-D, while all three pathways of endocytosis namely clathrin and caveolae mediated endocytosis, and macropinocytosis regulated gene transfer in 3-D. Actin polymerization and RhoA mediated contractility highly contributed to efficient transgene expression in 3-D rather than 2-D. Furthermore, Rac and Cdc42 influenced internalization in 2-D but not in 3-D. In conclusion, endocytic pathways, cytoskeletal dynamics and RhoGTPase mediated signaling differentially modulated non-viral gene transfer in cells cultured in 2-D and 3-D. These results provide an understanding of the dimensionality of cell microenvironment to obtain efficient gene transfer for the purpose of tissue regeneration and gene therapy.We believe that the cellular microenvironment can be engineered to enhance the ability of cells to become transfected, and through understanding of the mechanisms by which the ECM affects non-viral gene transfer, better materials and transfection protocols can be achieved
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Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases.
Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer
Effect of RhoGTPase activation using calpeptin (CP) and epidermal growth factor (EGF) on gene transfer.
<p>mMSCs cells were cultured for 8 hours on fibronectin wells, followed by overnight serum starvation and then treated with 0–100 µg/ml CP for 10 minutes or 0–100 ng/ml EGF for 2 minutes in serum free media. (<b>A</b>) After treatment, the changes in cell morphology were visualized through lexa488 conjugated phalloidin (green) and for DNA using Hoechst 33258 dye (blue) staining. Images were taken with a Zeiss AxioObserver Z1 inverted microscope at 100× magnification. (<b>B</b>) Stress fiber quantification. (<b>C</b>) Active RhoGTPase quantification after treatment was performed using GLISA specific for RhoA, Rac1,2,3 and Cdc42. (<b>D</b>) Transfection was performed immediately post treatment in serum free media. 4 hours post transfection media was replaced with complete media. The transgene expression was analyzed 48 hours post transfection using luciferase assay and normalized with total protein analyzed using Peirce BCA assay. (<b>E</b>) Internalization was analyzed 2 hours post transfection using flow cytometry of YOYO-1 labeled polyplexes. A total of 7000 events were analyzed per sample. Statistical analysis for the level of active RhoGTPase, transgene expression and internalization, was done using the unpaired t-test (two tail p value) where treated sample was compared with untreated sample. The symbols ** and *** represents a significant change to the level of p<0.01 and p<0.001, respectively.</p
Effect of expression of constitutively active mutants of RhoGTPases on gene transfer.
<p>mMSCs plated on a 6-well plate were transiently transfected with constitutive active forms of RhoA, Rac1 or Cdc42 conjugated with GFP, using lipofectamine™2000. Cells transfected with pEGFP were used as control. After 24 hours, cells were harvested and replated on fibronectin-coated surfaces. (<b>A</b>) 24 hours post transfection with constitutive active forms of RhoGTPases in cells plated on tissue culture plastic, changes in cell morphology were analyzed through actin and DNA staining using Alexa488 conjugated phalloidin (green), and Hoechst 33258 dye (blue), respectively. (<b>B</b>) The number of stress fibers per cell were counted. (<b>C</b>) Active Rho, Rac and Cdc42-GTPases were assessed using GLISA assays 24 hours post transfection with constitutive active forms of RhoGTPases and overnight serum starvation. (<b>D</b>) The replated cells were cultured for 16 hours on fibronectin before bolus transfection. The transgene expression was analyzed 48 hours post transfection using luciferase assay and normalized with total protein analyzed using Peirce BCA assay. (<b>E</b>) Internalization was analyzed 2 hours post transfection using flow cytometry and YOYO-3 labeled polyplexes. A total of 10,000 events were analyzed per sample and the mean fluorescence of events positive for both GFP and YOYO-3 was analyzed. Statistical analysis for number of stress fibers, ruffles, transgene expression and internalization was done using the Dunnett multiple comparison test.</p
RhoGTPase inhibition using dominat negative gene products decreases transgene expression.
<p>mMSCs were transiently transfected with dominant negative forms of RhoA, Rac1 or Cdc42 with a plasmid also containing EGFP. 24 hours post transfection, cells were replated on fibronectin coated plates and cultured for 16 hours prior to bolus transfection. Cells transfected with pEGFP were used as control. (<b>A</b>) Alexa488 conjugated phalloidin (green), and Hoechst dye (blue) staining. Images were taken with a Zeiss AxioObserver Z1 inverted microscope at 100Ă— magnification. (<b>B</b>) Stress fiber quantification. Statistical analysis was done using a one-way Anova followed by the Dunnett Multiple Comparison test. The Anova p value was 0.0015. The symbol * represents a significant change in stress fibers with respect to untreated cells to the level of p<0.05. (<b>C</b>) Active Rho, Rac and Cdc42, was assessed for cells replated on fibronectin for 16 hours using GLISA assays specific for RhoA, Rac1,2,3 and Cdc42. Statistical analysis was done using the unpaired t-test (two tail p value). The symbol * represents a significant change to the level of p<0.05. (<b>D</b>) The transgene expression was analyzed 48 hours post transfection using luciferase assay and normalized with total protein analyzed using Peirce BCA assay. (<b>E</b>) Internalization was analyzed using YOYO-3 labeled polyplexes 2 hours post transfection using flow cytometry. A total of 10,000 events were analyzed per sample and the mean fluorescence of events positive for both GFP and YOYO-3 was analyzed. Statistical analysis for gene transfer and internalization was done using a one-way Anova followed by the Dunnett Multiple Comparison test. The Anova p values were 0.0219 and <0.0001 for transfection (D) and internalization (E) respectively. The symbols * and ** represents a significant change with respect to cells transfected with control plasmid (pEGFP) to the level of p<0.05 and p<0.01, respectively.</p