Extremely low frequency magnetic field induces human neuronal differentiation through NMDA receptor activation.

Magnetic fields with different frequency and intensity parameters exhibit a wide range of effects on different biological models. Extremely low frequency magnetic field (ELF MF) exposure is known to augment or even initiate neuronal differentiation in several in vitro and in vivo models. This effect holds potential for clinical translation into treatment of neurodegenerative conditions such as autism, Parkinson's disease and dementia by promoting neurogenesis, non-invasively. However, the lack of information on underlying mechanisms hinders further investigation into this phenomenon. Here, we examine involvement of glutamatergic Ca2+ channel, N-methyl-D-aspartate (NMDA) receptors in the process of human neuronal differentiation under ELF MF exposure. We show that human neural progenitor cells (hNPCs) differentiate more efficiently under ELF MF exposure in vitro, as demonstrated by the abundance of neuronal markers. Furthermore, they exhibit higher intracellular Ca2+ levels as evidenced by c-fos expression and more elongated mature neurites. We were able to neutralize these effects by blocking NMDA receptors with memantine. As a result, we hypothesize that the effects of ELF MF exposure on neuronal differentiation originate from the effects on NMDA receptors, which sequentially triggers Ca2+-dependent cascades that lead to differentiation. Our findings identify NMDA receptors as a new key player in this field that will aid further research in the pursuit of effect mechanisms of ELF MFs.

Cell adhesion molecule immobilized gold surfaces for enhanced neuron-electrode interfaces.

To provide a long-term solution for increasing the biocompatibility of neuroprosthetics, approaches to reduce the side effects of invasive neuro-implantable devices are still in need of improvement. Physical, chemical, and bioactive design aspects of the biomaterials are proven to be important for providing proper cell-to-cell, cell-to-material interactions. Particularly, modification of implant surfaces with bioactive cues, especially cell adhesion molecules (CAMs) that capitalize on native neural adhesion mechanisms, are promising candidates in favor of providing efficient interfaces. Within this concept, this study utilized specific CAMs, namely N-Cadherin (Neural cadherin, N-Cad) and neural cell adhesion molecule (NCAM), to enhance neuron-electrode contact by mimicking the cell-to-ECM interactions for improving the survival of cells and promoting neurite outgrowth. For this purpose, representative gold electrode surfaces were modified with N-Cadherin, NCAM, and the mixture (1:1) of these molecules. Modifications were characterized, and the effect of surface modification on both differentiated and undifferentiated neuroblastoma SH-SY5Y cell lines were compared. The findings demonstrated the successful modification of these molecules which subsequently exhibited biocompatible properties as evidenced by the cell viability results. In cell culture experiments, the CAMs displayed promising results in promoting neurite outgrowth compared to conventional poly-l-lysine coated surfaces, especially NCAM and N-Cad/NCAM modified surfaces clearly showed significant improvement. Overall, this optimized approach is expected to provide an insight into the action mechanisms of cells against the local environment and advance processes for the fabrication of alternative neural interfaces.

Electroconductive Collagen-Carbon Nanodots Nanocomposite Elicits Neurite Outgrowth, Supports Neurogenic Differentiation and Accelerates Electrophysiological Maturation of Neural Progenitor Spheroids.

Neuronal disorders are characterized by the loss of functional neurons and disrupted neuroanatomical connectivity, severely impacting the quality of life of patients. This study investigates a novel electroconductive nanocomposite consisting of glycine-derived carbon nanodots (GlyCNDs) incorporated into a collagen matrix and validates its beneficial physicochemical and electro-active cueing to relevant cells. To this end, this work employs mouse induced pluripotent stem cell (iPSC)-derived neural progenitor (NP) spheroids. The findings reveal that the nanocomposite markedly augmented neuronal differentiation in NP spheroids and stimulate neuritogenesis. In addition, this work demonstrates that the biomaterial-driven enhancements of the cellular response ultimately contribute to the development of highly integrated and functional neural networks. Lastly, acute dizocilpine (MK-801) treatment provides new evidence for a direct interaction between collagen-bound GlyCNDs and postsynaptic N-methyl-D-aspartate (NMDA) receptors, thereby suggesting a potential mechanism underlying the observed cellular events. In summary, the findings establish a foundation for the development of a new nanocomposite resulting from the integration of carbon nanomaterials within a clinically approved hydrogel, toward an effective biomaterial-based strategy for addressing neuronal disorders by restoring damaged/lost neurons and supporting the reestablishment of neuroanatomical connectivity.

Unraveling the assembloid: Real-time monitoring of dopaminergic neurites in an inter-organoid pathway connecting midbrain and striatal regions.

Modern in vitro technologies for preclinical research, including organ-on-a-chip, organoids- and assembloid-based systems, have rapidly emerged as pivotal tools for elucidating disease mechanisms and assessing the efficacy of putative therapeutics. In this context, advanced in vitro models of Parkinson's Disease (PD) offer the potential to accelerate drug discovery by enabling effective platforms that recapitulate both physiological and pathological attributes of the in vivo environment. Although these systems often aim at replicating the PD-associated loss of dopaminergic (DA) neurons, only a few have modelled the degradation of dopaminergic pathways as a way to mimic the disruption of downstream regulation mechanisms that define the characteristic motor symptoms of the disease. To this end, assembloids have been successfully employed to recapitulate neuronal pathways between distinct brain regions. However, the investigation and characterization of these connections through neural tracing and electrophysiological analysis remain a technically challenging and time-consuming process. Here, we present a novel bioengineered platform consisting of surface-grown midbrain and striatal organoids at opposite sides of a self-assembled DA pathway. In particular, dopaminergic neurons and striatal GABAergic neurons spontaneously form DA connections across a microelectrode array (MEA), specifically integrated for the real-time monitoring of electrophysiological development and stimuli response. Calcium imaging data showed spiking synchronicity of the two organoids forming the inter-organoid pathways (IOPs) demonstrating that they are functionally connected. MEA recordings confirm a more robust response to the DA neurotoxin 6-OHDA compared to midbrain organoids alone, thereby validating the potential of this technology to generate highly tractable, easily extractable real-time functional readouts to investigate the dysfunctional dopaminergic network of PD patients.

Mesenchymal stem cells derived-exosomes enhanced amniotic membrane extract promotes corneal keratocyte proliferation.

Amniotic membrane extract (AME) and Wharton's jelly mesenchymal stem cells derived-exosomes (WJ-MSC-Exos) are promising therapeutic solutions explored for their potential in tissue engineering and regenerative medicine, particularly in skin and corneal wound healing applications. AME is an extract form of human amniotic membrane and known to contain a plethora of cytokines and growth factors, making it a highly attractive option for topical applications. Similarly, WJ-MSC-Exos have garnered significant interest for their wound healing properties. Although WJ-MSC-Exos and AME have been used separately for wound healing research, their combined synergistic effects have not been studied extensively. In this study, we evaluated the effects of both AME and WJ-MSC-Exos, individually and together, on the proliferation of corneal keratocytes as well as their ability to promote in vitro cell migration, wound healing, and their impact on cellular morphology. Our findings indicated that the presence of both exosomes (3 × 105 Exo/mL) and AME (50 µg/mL) synergistically enhance the proliferation of corneal keratocytes. Combined use of these solutions (3 × 105 Exo/mL + 50 µg/mL) increased cell proliferation compared to only 50 µg/mL AME treatment on day 3 (**** p < 0.0001). This mixture treatment (3 × 105 Exo/mL + 50 µg/mL) increased wound closure rate compared to isolated WJ-MSC-Exo treatment (3 × 105 Exo/mL) (*p < 0.05). Overall, corneal keratocytes treated with AME and WJ-MSC-Exo (3 × 105 Exo/mL + 50 µg/mL) mixture resulted in enhanced proliferation and wound healing tendency. Utilization of combined use of AME and WJ-MSC-Exo can pave the way for a promising foundation for corneal repair research.

Biomaterials-based strategies for in vitro neural models.

In vitro models have been used as a complementary tool to animal studies in understanding the nervous system's physiological mechanisms and pathological disorders, while also serving as platforms to evaluate the safety and efficiency of therapeutic candidates. Following recent advances in materials science, micro- and nanofabrication techniques and cell culture systems, in vitro technologies have been rapidly gaining the potential to bridge the gap between animal and clinical studies by providing more sophisticated models that recapitulate key aspects of the structure, biochemistry, biomechanics, and functions of human tissues. This was made possible, in large part, by the development of biomaterials that provide cells with physicochemical features that closely mimic the cellular microenvironment of native tissues. Due to the well-known material-driven cellular response and the importance of mimicking the environment of the target tissue, the selection of optimal biomaterials represents an important early step in the design of biomimetic systems to investigate brain structures and functions. This review provides a comprehensive compendium of commonly used biomaterials as well as the different fabrication techniques employed for the design of neural tissue models. Furthermore, the authors discuss the main parameters that need to be considered to develop functional platforms not only for the study of brain physiological functions and pathological processes but also for drug discovery/development and the optimization of biomaterials for neural tissue engineering.

Magnetic field-induced Ca2+ intake by mesenchymal stem cells is mediated by intracellular Zn2+ and accompanied by a Zn2+ influx.

Chronic exposure to magnetic fields (MFs) has a diverse range of effects on biological systems but definitive molecular mechanisms of the interaction remain largely unknown. One of the most frequently reported effects of MF exposure is an elevated concentration of intracellular Ca2+ through disputed pathways. Other prominent effects include increased oxidative stress and upregulation of neural markers through EGFR activation in stem cells. Further characterization of cascades triggered by MF exposure is hindered by the phenotype diversity of biological models used in the literature. In an attempt to reveal more mechanistic data in this field, we combined the most commonly used biological model and MF parameters with the most commonly reported effects of MFs. Based on clues from the pathways previously defined as sensitive to MFs (EGFR and Zn2+-binding enzymes), the roles of different types of channels (voltage gated Ca2+ channels, NMDA receptors, TRP channels) were inquired in the effects of 50 Hz MFs on bone marrow-derived mesenchymal stem cells. We report that, an influx of Zn2+ accompanies MF-induced Ca2+ intake, which is only attenuated by the broad-range inhibitor of TRP channels and store-operated Ca2+ entry (SOCE), 2-Aminoethoxydiphenyl borate (2-APB) among other blockers (memantine, nifedipine, ethosuximide and gabapentin). Interestingly, cation influx completely disappears when intracellular Zn2+ is chelated. Our results rule out voltage gated Ca2+ channels as a gateway to MF-induced Ca2+ intake and suggest Zn2+-related channels as a new focus in the field.

Substrate stiffness effects on SH-SY5Y: The dichotomy of morphology and neuronal behavior.

Like many other cell types, neuroblastoma cells are also known to respond to mechanical cues in their microenvironment in vitro. They were shown to have mechanotransduction pathways, which result in enhanced neuronal morphology on stiff substrates. However, in previous studies, the differentiation process was monitored only by morphological parameters. Motivated by the lack of comprehensive studies that investigate the effects of mechanical cues on neuroblastoma differentiation, we used SH-SY5Y cells differentiated on polyacrylamide (PA) gels as a model. Cells differentiated on the surface of PA hydrogels with three different elastic moduli (0.1, 1, and 50 kPa) were morphologically evaluated and their electrophysiological responsiveness was probed using calcium imaging. Immunodetection of neural marker TUJ1 and p-FAK was used for biochemical characterization. Groups with defined stiffness that are matching and nonmatching to neural tissue extracellular matrix were used to distinguish biomimetic results from other effects. Results show that while cells display morphologies that do not resemble neurons on soft substrates, they are in fact electrophysiologically more responsive and abundant in neuronal marker TUJ1. Our findings suggest that while neuronal differentiation occurs more efficiently in microenvironments mechanically mimicking neural tissue, the SH-SY5Y model demonstrates morphologies that conflict with neuronal behavior under these conditions. These results are expected to contribute considerable input to researchers that use SH-SY5Y as a neuron model.

Bioinspired hydrogel surfaces to augment corneal endothelial cell monolayer formation.

Corneal endothelial cells (CECs) have limited proliferation ability leading to corneal endothelium (CE) dysfunction and eventually vision loss when cell number decreases below a critical level. Although transplantation is the main treatment method, donor shortage problem is a major bottleneck. The transplantation of in vitro developed endothelial cells with desirable density is a promising idea. Designing cell substrates that mimic the native CE microenvironment is a substantial step to achieve this goal. In the presented study, we prepared polyacrylamide (PA) cell substrates that have a microfabricated topography inspired by the dimensions of CECs. Hydrogel surfaces were prepared via two different designs with small and large patterns. Small patterned hydrogels have physiologically relevant hexagon densities (∼2000 hexagons/mm2 ), whereas large patterned hydrogels have sparsely populated hexagons (∼400 hexagons/mm2 ). These substrates have similar elastic modulus of native Descemet's membrane (DM; ∼50 kPa) and were modified with Collagen IV (Col IV) to have biochemical content similar to native DM. The behavior of bovine corneal endothelial cells on these substrates was investigated and results show that cell proliferation on small patterned substrates was significantly (p = 0.0004) higher than the large patterned substrates. Small patterned substrates enabled a more densely populated cell monolayer compared to other groups (p = 0.001 vs. flat and p < 0.0001 vs. large patterned substrates). These results suggest that generating bioinspired surface topographies augments the formation of CE monolayers with the desired cell density, addressing the in vitro development of CE layers.

A Transient Survival Model of Alteration of Electrophysiological Properties Due to Amyloid Beta Toxicity Based on SH-SY5Y Cell Line.

BACKGROUND: Accumulation of toxic strands of amyloid beta (AB), which cause neurofibrillary tangles and, ultimately, cell death, is suspected to be the main culprit behind clinical symptoms of Alzheimer's disease. Although the mechanism of cell death due to AB accumulation is well known, the intermediate phase between the start of accumulation and cell death is less known and investigated, partially due to technical challenges in identifying partially affected cells. OBJECTIVE: First, we aimed to establish an in vitro model that would show resilience against AB toxicity. Then we used morphological, molecular and electrophysiological assays to investigate how the characteristics of the surviving cells changed after AB toxicity. METHODS: To investigate this phase, we used differentiation of SH-SY5Y neuroblastoma stem cells by Retinoic Acid (RA) and Brain Derived Neurotrophic Factor (BDNF) to establish an in vitro model which would be able to demonstrate various levels of resistance to AB toxicity. We utilized fluorescent microscopy and whole cell patch clamp recordings to investigate behavior of the model. RESULTS: We observed significantly higher morphological resilience against AB toxicity in cells which were differentiated by both Retinoic Acid and Brain Derived Neurotrophic Factor compared to Retinoic Acid only. However, the electrophysiological properties of the Retinoic Acid + Brain-Derived Neurotrophic Factor differentiated cells were significantly altered after AB treatment. CONCLUSION: We established a transient survival model for AB toxicity and observed the effects of AB on transmembrane currents of differentiated neurons.