Ijaz Ahmed

Three-dimensional nanofibrillar surfaces promote self-renewal in mouse embryonic stem cells

The regulation of mouse embryonic stem cell (mESC) fate is controlled by the interplay of signaling networks that either promote self‐renewal or induce differentiation. Leukemia inhibitory factor (LIF) is a cytokine that is required for stem cell renewal in mouse but not in human embryonic stem cells. However, feeder layers of embryonic fibroblasts are capable of inducing stem cell renewal in both cell types, suggesting that the self‐renewal signaling pathways may also be promoted by other triggers, such as alternative cytokines and/or chemical or physical properties of the extracellular matrix (ECM) secreted by feeder fibroblasts. We have recently used a synthetic polyamide matrix (Ultra‐Web) whose three‐dimensional (3D) nanofibrillar organization resembles the ECM/basement membrane. Growth of mESCs on this nanofibrillar surface greatly enhanced proliferation and self‐renewal in comparison with growth on tissue culture surfaces without nanofibers, despite the presence of LIF in both systems. Enhanced proliferation and self‐renewal of the stem cells on nanofibrillar surfaces were correlated with the activation of the small GTPase Rac, the activation of phosphoinositide 3‐kinase (PI3K) pathway, and the enhanced expression of Nanog, a homeoprotein required for maintenance of pluripotency. Inhibitors of PI3K reduced the expression level of Nanog in mESCs cultured on 3D nanofibrillar surfaces. These results provide support for the view that the three‐dimensionality of the culture surface may function as a cue for the activation of Rac and PI3K signaling pathways, resulting in stem cell proliferation and self‐renewal.

Living in three dimensions: 3D nanostructured environments for cell culture and regenerative medicine.

Research focused on deciphering the biochemical mechanisms that regulate cell proliferation and function has largely depended on the use of tissue culture methods in which cells are grown on two-dimensional (2D) plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) and the basement membrane (BM), a structurally compact form of the ECM. Recent work has provided strong evidence that the highly porous nanotopography that results from the 3D associations of ECM and BM nanofibrils is essential for the reproduction of physiological patterns of cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis, and differentiation in cell culture. In vitro approximations of these nanostructured surfaces are therefore desirable for more physiologically mimetic model systems to study both normal and abnormal functions of cells, tissues, and organs. In addition, the development of 3D culture environments is imperative to achieve more accurate cell-based assays of drug sensitivity, high-throughput drug discovery assays, and in vivo and ex vivo growth of tissues for applications in regenerative medicine.

Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces

Growth of cells in tissue culture is generally performed on two-dimensional (2D) surfaces composed of polystyrene or glass. Recent work, however, has shown that such 2D cultures are incomplete and do not adequately represent the physical characteristics of native extracellular matrix (ECM)/basement membrane (BM), namely dimensionality, compliance, fibrillarity, and porosity. In the current study, a three-dimensional (3D) nanofibrillar surface composed of electrospun polyamide nanofibers was utilized to mimic the topology and physical structure of ECM/BM. Additional chemical cues were incorporated into the nanofibrillar matrix by coating the surfaces with fibronectin, collagen I, or laminin-1. Results from the current study show an enhanced response of primary mouse embryonic fibroblasts (MEFs) to culture on nanofibrillar surfaces with more dramatic changes in cell spreading and reorganization of the cytoskeleton than previously observed for established cell lines. In addition, the cells cultured on nanofibrillar and 2D surfaces exhibited differential responses to the specific ECM/BM coatings. The localization and activity of myosin II-B for MEFs cultured on nanofibers was also compared. A dynamic redistribution of myosin II-B was observed within membrane protrusions. This was previously described for cells associated with nanofibers composed of collagen I but not for cells attached to 2D surfaces coated with monomeric collagen. These results provide further evidence that nanofibrillar surfaces offer a significantly different environment for cells than 2D substrates.

Covalently attached FGF-2 to three-dimensional polyamide nanofibrillar surfaces demonstrates enhanced biological stability and activity

Activation of fibroblast growth factor receptors (FGFRs) requires the formation of a ternary complex between fibroblast growth factors (FGFs), FGFRs, and heparan sulfate proteoglycans, which are all located on the cell surface and the basement membrane (BM)/extracellular matrix (ECM). Heparan sulfate proteoglycans appear to stabilize FGFs by inhibiting the rapid degradation of FGFs normally observed in solution. Because of the pivotal role of FGFs in proliferative and developmental pathways, a number of recent studies have attempted to engineer microenvironments to stabilize growth factors for use in applications in tissue culture and regenerative medicine. In this communication, we demonstrate that covalent linkage of FGF-2 to nanofibrillar surfaces (defined as covalently bound FGF-2) composed of a network of polyamide nanofibers resulted in the maintenance of the biological efficacy of FGF-2 when stored dry for at least 6xa0months at 25°C or 4°C. Moreover, covalently bound FGF-2 was more potent than FGF-2 in solution when measured in cellular assays of proliferation and viability using a variety of cell types. Covalently bound FGF-2 also strongly activated FGFR, extracellular signal-regulated kinase (ERK1/2), and c-fos. Hence cell-signaling molecules can be incorporated into a synthetic nanofibrillar surface, providing a novel means to enhance their stability and biological activity.

Increased FGF-2 secretion and ability to support neurite outgrowth by astrocytes cultured on polyamide nanofibrillar matrices

An electrospun nonwoven matrix of polyamide nanofibers was employed as a new model for the capillary basement membrane at the blood-brain barrier (BBB). The basement membrane separates astrocytes from endothelial cells and is associated with growth factors, such as fibroblast growth factor-2 (FGF-2). FGF-2 is produced by astrocytes and induces specialized functions in endothelial cells, but also has actions on astrocytes. To investigate potential autocrine actions of FGF-2 at the BBB, astrocytes were cultured on unmodified nanofibers or nanofibers covalently modified with FGF-2. The former assumed an in vivo-like stellate morphology that was enhanced in the presence of cross-linked FGF-2. Furthermore, astrocyte monolayers established on unmodified nanofibers were more permissive for neurite outgrowth when cultured with an overlay of neurons than similar monolayers established on standard tissue culture surfaces, while astrocytes cultured on FGF-2-modifed nanofibers were yet more permissive. The observed differences were due in part to progressively increasing amounts of FGF-2 secreted by the astrocytes into the medium; hence FGF-2 increases its own expression in astrocytes to modulate astrocyte-neuron interactions. Soluble FGF-2 was unable to replicate the effects of cross-linked FGF-2. Nanofibers alone up-regulated FGF-2, albeit to a lesser extent than nanofibers covalently modified with FGF-2. These results underscore the importance of both surface topography and growth factor presentation on cellular function. Moreover, these results indicate that FGF-2-modified nanofibrillar scaffolds may demonstrate utility in tissue engineering applications for replacement and regeneration of lost tissue following central nervous system (CNS) injury or disease.

Role of DNA topoisomerase IIβ in neurite outgrowth

Abstract Failure to establish neuromuscular junctions is a major phenotype of top2β knockout mice. However, the precise mechanism for this defect is not known. In the current study, we have investigated the role of TopIIβ in cultured neurons. We showed that the TopII inhibitor ICRF-193 significantly blocked neurite outgrowth and growth cone formation in cultured cerebellar granule neurons (CGNs), dorsal root ganglions (DRGs) and cortical neurons (CNs). In addition, ICRF-193 also blocked neurite outgrowth and growth cone formation of PC12 cells undergoing NGF-induced differentiation. Isolated cortical neurons from top2β knockout embryos elaborated shorter neurites than did those from their wild type counterparts, confirming the role of TopIIβ in neurite outgrowth. Together, these results demonstrate a critical role of TopIIβ in neurite outgrowth in cultured neurons. Furthermore, we demonstrated that neurons derived from top2β knockout mice failed to form contacts with muscle cells in co-cultures. These results suggest that the defect in establishing neuromuscular junctions in top2β knockout mice could be due to the lack of TopIIβ-mediated neurite outgrowth.

Neuroprotection by genipin against reactive oxygen and reactive nitrogen species-mediated injury in organotypic hippocampal slice cultures

Genipin, the multipotent ingredient in Gardenia jasmenoides fruit extract (GFE), may be an effective candidate for treatment following stroke or traumatic brain injury (TBI). Secondary injury includes damage mediated by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can alter the biological function of key cellular structures and eventually lead to cell death. In this work, we studied the neuroprotective potential of genipin against damage stemming from ROS and RNS production in organotypic hippocampal slice cultures (OHSC), as well as its potential as a direct free radical scavenger. A 50 µM dose of genipin provided significant protection against tert-butyl hydroperoxide (tBHP), a damaging organic peroxide. This dosage of genipin significantly reduced cell death at 48 h compared to vehicle control (0.1% DMSO) when administered 0, 1, 6, and 24 h after addition of tBHP. Similarly, genipin significantly reduced cell death at 48 h when administered 0, 1, 2, and 6h after addition of rotenone, which generates reactive oxygen species via a more physiologically relevant mechanism. Furthermore, genipin significantly reduced both cell death and nitrite levels at 24 h caused by S-nitroso-N-acetylpenicillamine (SNAP), a direct nitric oxide (NO) donor, and successfully quenched 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, suggesting that genipin may act as a direct free radical scavenger. Our encouraging findings suggest that genipin should be tested in animal models of CNS injury with a significant component of ROS- and RNS-mediated damage, such as TBI and stroke, to assess its in vivo efficacy.

Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

Cerebral cortical astrocyte responses to polyamide nanofibrillar scaffolds versus poly-L-lysine (PLL)-functionalized planar glass, unfunctionalized planar Aclar coverslips, and PLL-functionalized planar Aclar surfaces were investigated by atomic force microscopy and immunocytochemistry. The physical properties of the cell culture environments were evaluated using contact angle and surface roughness measurements and compared. Astrocyte morphological responses, including filopodia, lamellipodia, and stress fiber formation, and stellation were imaged using atomic force microscopy and phalloidin staining for F-actin. Activation of the corresponding Rho GTPase regulators was investigated using immunolabeling with Cdc42, Rac1, and RhoA. Astrocytes cultured on the nanofibrillar scaffolds showed a unique response that included stellation, cell–cell interactions by stellate processes, and evidence of depression of RhoA. The results support the hypothesis that the extracellular environment can trigger preferential activation of members of the Rho GTPase family, with demonstrable morphological consequences for cerebral cortical astrocytes.

Role of Cdc42 in neurite outgrowth of PC12 cells and cerebellar granule neurons.

Inactivation of Rho GTPases inhibited the neurite outgrowth of PC12 cells. The role of Cdc42 in neurite outgrowth was then studied by selective inhibition of Cdc42 signals. Overexpression of ACK42, Cdc42 binding domain of ACK-1, inhibited NGF-induced neurite outgrowth in PC12 cells. ACK42 also inhibited the neurite outgrowth of PC12 cells induced by constitutively activated mutant of Cdc42, but not Rac. These results suggest that Cdc42 plays an important role in mediating NGF-induced neurite outgrowth of PC12 cells. Inhibition of neurite outgrowth was also demonstrated using a cell permeable chimeric protein, penetratin-ACK42. A dominant negative mutant of Rac, RacN17 inhibited Cdc42-induced neurite outgrowth of PC12 cells suggesting that Rac acts downstream of Cdc42. Further studies, using primary-cultures of rat cerebellar granule neurons, showed that Cdc42 is also involved in the neurite outgrowth of cerebellar granule neurons. Both penetratin-ACK42 and Clostridium difficile toxin B, which inactivates all members of Rho GTPases strongly inhibited the neurite outgrowth of cerebellar granule neurons. These results show that Cdc42 plays a similar and essential role in the development of neurite outgrowth of PC12 cells and cerebellar granule neurons. These results provide evidence that Cdc42 produces signals that are essential for the neurite outgrowth of PC12 cells and cerebellar granule neurons.

Differentiation of reactive-like astrocytes cultured on nanofibrillar and comparative culture surfaces

AIMnTo investigate the directive importance of nanophysical properties on the morphological and protein expression responses of dibutyryladenosine cyclic monophosphate (dBcAMP)-treated cerebral cortical astrocytes in vitro.nnnMATERIALS & METHODSnElasticity and work of adhesion characterizations of culture surfaces were performed using atomic force microscopy and combined with previous surface roughness and polarity results. The morphological and biochemical differentiation of dBcAMP-treated astrocytes cultured on promising nanofibrillar scaffolds and comparative culture surfaces were investigated by immunocytochemistry, colocalization, super resolution microscopy and atomic force microscopy. The dBcAMP-treated astrocyte responses were further compared with untreated astrocyte responses.nnnRESULTS & CONCLUSIONnNanofibrillar scaffold properties were shown to reduce immunoreactivity responses while poly-L-lysine-functionalized Aclar® (Ted Pella Inc., CA, USA) properties were shown to induce responses reminiscent of glial scar formation. The comparison study indicated that directive cues may differ in wound-healing versus quiescent situations.