Spontaneous Cell Cluster Formation in Human iPSC-Derived Neuronal Spheroid Networks Influences Network Activity.

Three-dimensional neuronal culture systems such as spheroids, organoids, and assembloids constitute a branch of neuronal tissue engineering that has improved our ability to model the human brain in the laboratory. However, the more elaborate the brain model, the more difficult it becomes to study functional properties such as electrical activity at the neuronal level, similar to the challenges of studying neurophysiology in vivo We describe a simple approach to generate self-assembled three-dimensional neuronal spheroid networks with defined human cell composition on microelectrode arrays. Such spheroid networks develop a highly three-dimensional morphology with cell clusters up to 60 µm in thickness and are interconnected by pronounced bundles of neuronal fibers and glial processes. We could reliably record from up to hundreds of neurons simultaneously per culture for ≤90 d. By quantifying the formation of these three-dimensional structures over time, while regularly monitoring electrical activity, we were able to establish a strong link between spheroid morphology and network activity. In particular, the formation of cell clusters accelerates formation and maturation of correlated network activity. Astrocytes both influence electrophysiological network activity as well as accelerate the transition from single cell layers to cluster formation. Higher concentrations of astrocytes also have a strong effect of modulating synchronized network activity. This approach thus represents a practical alternative to often complex and heterogeneous organoids, providing easy access to activity within a brain-like 3D environment.Significance StatementNeuronal "organoid" cultures with multiple cell types grown on elaborate three-dimensional scaffolds have become popular tools to generate brain-like properties in vitro but bring with them similar problems concerning access to physiological function as real brain tissue. Here, we developed a new approach to form simple brain-like spheroid networks from human neurons, but using the normal supporting cells of the brain, astrocytes, as the scaffold. By growing these cultures on conventional microelectrode arrays, we were able to observe development of complex patterns of electrical activity for months. Our results highlight how formation of three-dimensional structures accelerated the formation of synchronized neuronal network activity and provide a promising new simple model system for studying interactions between known human cell types in vitro.

Human neural progenitor cells promote photoreceptor survival in retinal explants.

Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-beta1 and TGF-beta2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of growth factors secreted from the HNPCs.

Analysis of complexes formed by small gold nanoparticles in low concentration in cell culture media.

New nanomaterials are constantly developed with applications in everything from cosmetics to high tech electronics. Assessing their biological impact has been done by analysis of their adsorbed protein corona, in vitro cell assays, and larger scale ecotoxicological studies. This has proved to be a huge challenge due to the wide range of available nanomaterials and their unpredictable behaviour in different environments. Furthermore, the enormous number of experimental variables make comparisons difficult. Concentration is one of these variables and can vary greatly depending on the aim of the study. When analysing the protein corona, concentrations are often higher than in cell assays. Using a combination of complementary techniques, we have characterised 20 nm gold nanoparticles in a concentration level commonly used in cell studies. We compare their behaviour in a commonly used, protein rich medium and one protein poor medium over 24 hours. Under these conditions, the NPs were stable in protein rich environment but underwent gradual aggregation in protein poor medium. We characterise the biomolecular corona in both media. In protein poor medium, we can describe the often overlooked aggregation. The aggregates' morphology is confirmed by cryo-TEM. Finally, in the protein poor medium, by infrared spectroscopy, we have identified the amino acid arginine in the biomolecular corona which drives the aggregation.

Immunocytochemical Profiling of Cultured Mouse Primary Retinal Cells.

Primary retinal cell cultures and immunocytochemistry are important experimental platforms in ophthalmic research. Translation of retinal cells from their native environment to the in vitro milieu leads to cellular stress, jeopardizing their in vivo phenotype features. Moreover, the specificity and stability of many retinal immunochemical markers are poorly evaluated in retinal cell cultures. Hence, we here evaluated the expression profile of 17 retinal markers, that is, recoverin, rhodopsin, arrestin, Chx10, PKC, DCX, CRALBP, GS, vimentin, TPRV4, RBPMS, Brn3a, ß-tubulin III, NeuN, MAP2, GFAP, and synaptophysin. At 7 and 18 days of culture, the marker expression profiles of mouse postnatal retinal cells were compared with their age-matched in vivo retinas. We demonstrate stable in vitro expression of all markers, except for arrestin and CRALBP. Differences in cellular expression and location of some markers were observed, both over time in culture and compared with the age-matched retina. We hypothesize that these differences are likely culture condition dependent. Taken together, we suggest a thorough evaluation of the antibodies in specific culture settings, before extrapolating the in vitro results to an in vivo setting. Moreover, the identification of specific cell types may require a combination of different genes expressed or markers with structural information.

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.

BDNF increases survival and neuronal differentiation of human neural precursor cells cotransplanted with a nanofiber gel to the auditory nerve in a rat model of neuronal damage.

OBJECTIVES: To study possible nerve regeneration of a damaged auditory nerve by the use of stem cell transplantation. METHODS: We transplanted HNPCs to the rat AN trunk by the internal auditory meatus (IAM). Furthermore, we studied if addition of BDNF affects survival and phenotypic differentiation of the grafted HNPCs. A bioactive nanofiber gel (PA gel), in selected groups mixed with BDNF, was applied close to the implanted cells. Before transplantation, all rats had been deafened by a round window niche application of ß-bungarotoxin. This neurotoxin causes a selective toxic destruction of the AN while keeping the hair cells intact. RESULTS: Overall, HNPCs survived well for up to six weeks in all groups. However, transplants receiving the BDNF-containing PA gel demonstrated significantly higher numbers of HNPCs and neuronal differentiation. At six weeks, a majority of the HNPCs had migrated into the brain stem and differentiated. Differentiated human cells as well as neurites were observed in the vicinity of the cochlear nucleus. CONCLUSION: Our results indicate that human neural precursor cells (HNPC) integration with host tissue benefits from additional brain derived neurotrophic factor (BDNF) treatment and that these cells appear to be good candidates for further regenerative studies on the auditory nerve (AN).

Neuronal differentiation and extensive migration of human neural precursor cells following co-culture with rat auditory brainstem slices.

Congenital or acquired hearing loss is often associated with a progressive degeneration of the auditory nerve (AN) in the inner ear. The AN is composed of processes and axons of the bipolar spiral ganglion neurons (SGN), forming the connection between the hair cells in the inner ear cochlea and the cochlear nuclei (CN) in the brainstem (BS). Therefore, replacement of SGNs for restoring the AN to improve hearing function in patients who receive a cochlear implantation or have severe AN malfunctions is an attractive idea. A human neural precursor cell (HNPC) is an appropriate donor cell to investigate, as it can be isolated and expanded in vitro with maintained potential to form neurons and glia. We recently developed a post-natal rodent in vitro auditory BS slice culture model including the CN and the central part of the AN for initial studies of candidate cells. Here we characterized the survival, distribution, phenotypic differentiation, and integration capacity of HNPCs into the auditory circuitry in vitro. HNPC aggregates (spheres) were deposited adjacent to or on top of the BS slices or as a monoculture (control). The results demonstrate that co-cultured HNPCs compared to monocultures (1) survive better, (2) distribute over a larger area, (3) to a larger extent and in a shorter time-frame form mature neuronal and glial phenotypes. HNPC showed the ability to extend neurites into host tissue. Our findings suggest that the HNPC-BS slice co-culture is appropriate for further investigations on the integration capacity of HNPCs into the auditory circuitry.

Gold- and silver nanoparticles affect the growth characteristics of human embryonic neural precursor cells.

Rapid development of nanotechnologies and their applications in clinical research have raised concerns about the adverse effects of nanoparticles (NPs) on human health and environment. NPs can be directly taken up by organs exposed, but also translocated to secondary organs, such as the central nervous system (CNS) after systemic- or subcutaneous administration, or via the olfactory system. The CNS is particularly vulnerable during development and recent reports describe transport of NPs across the placenta and even into brain tissue using in vitro and in vivo experimental systems. Here, we investigated whether well-characterized commercial 20 and 80 nm Au- and AgNPs have an effect on human embryonic neural precursor cell (HNPC) growth. After two weeks of NP exposure, uptake of NPs, morphological features and the amount of viable and dead cells, proliferative cells (Ki67 immunostaining) and apoptotic cells (TUNEL assay), respectively, were studied. We demonstrate uptake of both 20 and 80 nm Au- and AgNPs respectively, by HNPCs during proliferation. A significant effect on the sphere size- and morphology was found for all cultures exposed to Au- and AgNPs. AgNPs of both sizes caused a significant increase in numbers of proliferating and apoptotic HNPCs. In contrast, only the highest dose of 20 nm AuNPs significantly affected proliferation, whereas no effect was seen on apoptotic cell death. Our data demonstrates that both Au- and AgNPs interfere with the growth profile of HNPCs, indicating the need of further detailed studies on the adverse effects of NPs on the developing CNS.

Hypoxia-inducible factor and vascular endothelial growth factor in the neuroretina and retinal blood vessels after retinal ischemia.

Retinal ischemia arises from circulatory failure. As the retinal blood vessels are key organs in circulatory failure, our aim was to study the retinal vasculature separately from the neuroretina to elucidate the role of hypoxia-inducible factor (HIF) 1α and 1ß and vascular endothelial growth factor (VEGF) in retinal ischemia. Retinal ischemia was induced in porcine eyes by applying an intraocular pressure, followed by 12 h of reperfusion. HIF-1α mRNA expression was not affected by ischemia, while immunofluorescence staining was higher after ischemia in the neuroretina. HIF-1ß immunoreactivity and mRNA expression were unaffected. VEGF protein levels in the vitreous humor and VEGF staining in the neuroretina were more pronounced in eyes subjected to ischemia than in the sham eyes. VEGF may be activated downstream of HIF-1 and is known to stimulate retinal neovascularization, which causes sight-threatening complications. These results emphasize the need for pharmacological treatment to block the HIF and VEGF signaling pathways in retinal ischemia.