Exploration of physical and chemical cues on retinal cell fate

Identification of the key components in the physical and chemical milieu directing donor cells into a desired phenotype is a requirement in the investigation of bioscaffolds for the advancement of cell-based therapies for retinal neurodegeneration. We explore the effect of electrospun poly-ε-caprolactone (PCL) fiber scaffold topography and functionalization and culture medium, on the behavior of mouse retinal cells. Dissociated mouse retinal post-natal cells were seeded on random or aligned oriented fibers, with or without laminin coating and cultured with either basic or neurotrophins enriched medium for 7 days. Addition of laminin in combination with neurotrophins clearly promoted cell– morphology, fate, and neurite extension. Nanotopography per se significantly affected cell morphology, with mainly bipolar profiles on aligned fibers and more multipolar profiles on random fibers. Laminin induced a remarkable 90° switch of neurite orientation. Herewith, we demonstrate that the chemical cue is stronger than the physical cue for the orientation of retinal neurites and describe the requirement of both neurotrophins and extracellular matrix proteins for extended neurite outgrowth and formation of complex retinal neuronal networks. Therefore, tailor-made PCL fiber mats, which can be physically and chemically modified, indeed influence cell behavior and hence motivate further retinal restorative studies using this system

Inflamed In Vitro Retina: Cytotoxic Neuroinflammation and Galectin-3 Expression.

BACKGROUND:Disease progression in retinal neurodegeneration is strongly correlated to immune cell activation, which may have either a neuroprotective or neurotoxic effect. Increased knowledge about the immune response profile and retinal neurodegeneration may lead to candidate targets for treatments. Therefore, we have used the explanted retina as a model to explore the immune response and expression of the immune modulator galectin-3 (Gal-3), induced by the cultivation per se and after additional immune stimulation with lipopolysaccharide (LPS), and how this correlates with retinal neurotoxicity. METHODS:Post-natal mouse retinas were cultured in a defined medium. One group was stimulated with LPS (100 ng/ml, 24 h). Retinal architecture, apoptotic cell death, and micro- and macroglial activity were studied at the time of cultivation (0 days in vitro (DIV)) and at 3, 4 and 7 DIV using morphological staining, biochemical- and immunohistochemical techniques. RESULTS:Our results show that sustained activation of macro- and microglia, characterized by no detectable cytokine release and limited expression of Gal-3, is not further inducing apoptosis additional to the axotomy-induced apoptosis in innermost nuclear layer. An elevated immune response was detected after LPS stimulation, as demonstrated primarily by release of immune mediators (i.e. interleukin 2 (IL-2), IL-6, KC/GRO (also known as CLCX1) and tumour necrosis factor-α (TNF-α)), increased numbers of microglia displaying morphologies of late activation stages as well as Gal-3 expression. This was accompanied with increased apoptosis in the two additional nuclear layers, and damage to retinal gross architecture. CONCLUSION:We demonstrate that an immune response characterized by sustained and increased release of cytokines, along with an increase in Gal-3 expression, is accompanied by significant increased neurotoxicity in the explanted retina. Further investigations using the current setting may lead to increased understanding on the mechanisms involved in neuronal loss in retinal neurodegenerations

Three-dimensional functional human neuronal networks in uncompressed low-density electrospun fiber scaffolds

We demonstrate an artificial three-dimensional (3D) electrical active human neuronal network system, by the growth of brain neural progenitors in highly porous low density electrospun poly-ε-caprolactone (PCL) fiber scaffolds. In neuroscience research cell-based assays are important experimental instruments for studying neuronal function in health and disease. Traditional cell culture at 2D-surfaces induces abnormal cell-cell contacts and network formation. Hence, there is a tremendous need to explore in vivo-resembling 3D neural cell culture approaches. We present an improved electrospinning method for fabrication of scaffolds that promote neuronal differentiation into highly 3D integrated networks, formation of inhibitory and excitatory synapses and extensive neurite growth. Notably, in 3D scaffolds in vivo-resembling intermixed neuronal and glial cell network were formed, whereas in parallel 2D cultures a neuronal cell layer grew separated from an underlying glial cell layer. Hence, the use of the 3D cell assay presented will most likely provide more physiological relevant results

Label-free concentration of viable neurons, hESCs and cancer cells by means of acoustophoresis.

Concentration of viable cell populations in suspension is of interest for several clinical and pre-clinical applications. Here, we report that microfluidic acoustophoresis is an effective method to efficiently concentrate live and viable cells with high target purity without any need for protein fluorescent labeling using antibodies or over-expression. We explored the effect of the acoustic field acoustic energy density and systematically used different protocols to induce apoptosis or cell death and then determined the efficiency of live and dead cell separation. We used the breast cancer cell line MCF-7, the mouse neuroblastoma N2a as well as human embryonic stem cells (hESCs) to demonstrate that this method is gentle and can be applied to different cell populations. First, we induced cell death by means of high osmotic shock using a high concentration of PBS (10×), the protein kinase inhibitor staurosporine, high concentrations of dimethyl sulfoxide (DMSO, 10%), and finally, cell starvation. In all the methods employed, we successfully induced cell death and were able to purify and concentrate the remaining live cells using acoustophoresis. Importantly, the concentration of viable cells was not dependent on a specific cell type. Further, we demonstrate that different death inducing stimuli have different effects on the intrinsic cell properties and therefore affect the efficiency of the acoustophoretic separation

Quantification of ED1 and Gal-3-positive microglial cells.

<p>Quantification of fractions of cells expressing ED1 and Gal-3, respectively, of the total number of Iba1-stained cells. ANOVA analysis was performed for comparison between the LPS-treated group and the control groups. Data are expressed as mean±StDev (n = 3/group) *p<0.05, ***p<0.001.</p

Cytokine release profiles.

<p>In collected conditioned media at 3, 4 and 7 DIV, the immune mediator release profile was analyzed using a biochemical assay. Ten well-described cytokines were included; interferon gamma (IFN-γ), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), KC/GRO and tumour necrosis factor alpha (TNF-α). No detectable levels of release of any of the mediators analyzed were found in controls at any of the time-points included. However, after LPS stimulation at 3 DIV, a significant increase of four of the mediators was found, <i>i</i>.<i>e</i>. IL-2, IL-6, KC/GRO and TNF-α. Note that the content of these factors further increased over time in culture to higher levels compared to at 3 DIV. Filled bar = control group, Striped bar = LPS group. ANOVA analysis was performed for comparison between the LPS treated group and the control groups. Data are expressed as mean±StDev (n = 3/group), *p<0.05, **p<0.01.</p

Iba1/ED1- fluorescent immunohistochemistry.

<p>Microglial cells were detected using the Iba1 (green) marker, expressed in all microglial stages and ED1 (red) for detecting activated cells. Cells were counterstained with the nuclei marker DAPI (blue). A-C. At 0 DIV, microglial cells were mainly found in the GCL, IPL and OPL, with a small fraction located in the GCL expressing ED1. At 3 and 4 DIV microglial cells were also found in the INL in both LPS-treated retinas and controls (D-F, G-I). At 3, 4 and 7 DIV the majority of the microglia expressed ED1 in both groups. At 7 DIV microglial cells were only found in the INL in the LPS-treated retinas (S-U cf. to P-R). Primarily three morphologies were found and used in the classification of activation stage, <i>i</i>.<i>e</i>. ramified (see cell #1 in E), round (see cell #2 in E) and amoeboid (see cell #3 in E). GCL = ganglion cell layer, INL = inner nuclear layer, IPL = inner plexiform layer, ONL = outer nuclear layer, NFL = nerve fibre layer, OLM = outer limiting membrane. Scale bar: 200 μm.</p

Gal-3 expression in microglia cells.

<p>Immunohistochemical staining of Gal-3 (red) expressing microglia. All microglia were detected using the microglia marker Iba1 (green). At 0 DIV no Gal-3-expressing cells were found (A, B). In controls, at 3, 4 and 7 DIV Iba1/Gal-3 co-expressing cells were found and only in the GCL (C, D, G, H, K, and L). LPS-treated retinas displayed larger numbers of Iba1/Gal-3 co-expressing cells that were located in the GCL, INL and OPL at 3, 4 and 7 DIV. Scale bar: 200 μm.</p

Macroglial activity detected by GFAP- staining.

<p>GFAP-immunohistochemistry was used to label the macroglial cells of the retina, <i>i</i>.<i>e</i>. astrocytes and Müller cells. At 0 DIV, GFAP-staining was primarily detected in the NFL (A). An increase in GFAP-staining was detected at 3, 4, and 7 DIV (B, D and F); with Müller cell up-regulation of GFAP, seen by labeled process. The LPS-treated retinas showed a similar GFAP-staining pattern with thin and thick GFAP-positive Müller cell processes spanning the retina from the inner to the outer part (C, arrows). GCL = ganglion cell layer, INL = inner nuclear layer, IPL = inner plexiform layer, ONL = outer nuclear layer, NFL = nerve fibre layer, OLM = outer limiting membrane. Scale bar: 200 μm.</p