Julian George

Electrophysiological properties and synaptic function of mesenchymal stem cells during neurogenic differentiation - a mini-review.

Introduction: Mesenchymal stem cells (MSCs) have gained considerable interest due to their potential use in cell therapies and tissue engineering. They have been reported to differentiate into various anchorage-dependent cell types, including bone, cartilage, and tendon. Our focus is on the differentiation of MSCs into neuron-like cells through the use of soluble chemical stimuli or specific growth factor supplements. The resulting cells appear to adopt neural phenotypes and express some typical neuronal markers, however, their electrophysiological properties and synaptic function remains unclear. Results: This mini-review illustrates how particular characteristics, electrophysiological properties, and synaptic functions of MSCs change during their neuronal differentiation. In particular we focus on changes in ion currents, ion channels, synaptic communication, and neurotransmitter release. We also highlight conflicting results, caused by inconsistencies in the experimental conditions used and in the methodologies adopted. Conclusions: We conclude that there is insufficient data and that further, carefully controlled investigations are required in order to ascertain whether MSC-derived neuron-like cells can exhibit the necessary neuronal functions to become clinically relevant for use in neural repairs.

Electrical Property Characterization of Neural Stem Cells in Differentiation.

Electrical property characterization of stem cells could be utilized as a potential label-free biophysical approach to evaluate the differentiation process. However, there has been a lack of technology or tools that can quantify the intrinsic cellular electrical markers (e.g., specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm)) for a large amount of stem cells or differentiated cells. In this paper, a microfluidic platform enabling the high-throughput quantification of Cspecific membrane and σcytoplasm from hundreds of single neural stem cells undergoing differentiation was developed to explore the feasibility to characterize the neural stem cell differentiation process without biochemical staining. Experimental quantification using biochemical markers (e.g., Nestin, Tubulin and GFAP) of neural stem cells confirmed the initiation of the differentiation process featured with gradual loss in cellular stemness and increased cell markers for neurons and glial cells. The recorded electrical properties of neural stem cells undergoing differentiation showed distinctive and unique patterns: 1) in the suspension culture before inducing differentiation, a large distribution and difference in σcytoplasm among individual neural stem cells was noticed, which indicated heterogeneity that may result from the nature of suspension culture of neurospheres; and 2) during the differentiation in adhering monolayer culture, significant changes and a large difference in Cspecific membrane were located indicating different expressions of membrane proteins during the differentiation process, and a small distribution difference in σcytoplasm was less significant that indicated the relatively consistent properties of cytoplasm during the culture. In summary, significant differences in Cspecific membrane and σcytoplasm were observed during the neural stem cell differentiation process, which may potentially be used as label-free biophysical markers to monitor this process.

Study of neuroprotective function of Ginkgo biloba extract (EGb761) derived-flavonoid monomers using a three-dimensional stem cell-derived neural model

An in vitro three‐dimensional (3D) cell culture system that can mimic organ and tissue structure and function in vivo will be of great benefit for drug discovery and toxicity testing. In this study, the neuroprotective properties of the three most prevalent flavonoid monomers extracted from EGb 761 (isorharmnetin, kaempferol, and quercetin) were investigated using the developed 3D stem cell‐derived neural co‐culture model. Rat neural stem cells were differentiated into co‐culture of both neurons and astrocytes at an equal ratio in the developed 3D model and standard two‐dimensional (2D) model using a two‐step differentiation protocol for 14 days. The level of neuroprotective effect offered by each flavonoid was found to be aligned with its effect as an antioxidant and its ability to inhibit Caspase‐3 activity in a dose‐dependent manner. Cell exposure to quercetin (100 µM) following oxidative insult provided the highest levels of neuroprotection in both 2D and 3D models, comparable with exposure to 100 µM of Vitamin E, whilst exposure to isorhamnetin and kaempferol provided a reduced level of neuroprotection in both 2D and 3D models. At lower dosages (10 µM flavonoid concentration), the 3D model was more representative of results previously reported in vivo. The co‐cultures of stem cell derived neurons and astrocytes in 3D hydrogel scaffolds as an in vitro neural model closely replicates in vivo results for routine neural drug toxicity and efficacy testing.

4.13 Use and Manipulation of Microporous Membranes in Mammalian Cell Cultures

Porous filter membranes have been used in mammalian cell culture since the early 1950s, and their use in cell culture continues to plays a significant role. Thin tissue scaffolds, created using tissue engineering techniques, have also been widely investigated, and microfabrication techniques have been used to create porous culture substrates with submicron control over membrane thickness, porosity, arrangement and diameter of pores and channel shape. This work reviews common and emerging uses of the many different types of microporous membrane in mammalian cell culture.

Rapid and efficient differentiation of functional motor neurons from human iPSC for neural injury modelling

Primary rodent neurons and immortalised cell lines have overwhelmingly been used for in vitro studies of traumatic injury to peripheral and central neurons, but have some limitations of physiological accuracy. Motor neurons (MN) derived from human induced pluripotent stem cells (iPSCs) enable the generation of cell models with features relevant to human physiology. To facilitate this, it is desirable that MN protocols both rapidly and efficiently differentiate human iPSCs into electrophysiologically active MNs. In this study, we present a simple, rapid protocol for differentiation of human iPSCs into functional spinal (lower) MNs, involving only adherent culture and use of small molecules for directed differentiation, with the ultimate aim of rapid production of electrophysiologically functional cells for short-term neural injury experiments. We show successful differentiation in two unrelated iPSC lines, by quantifying neural-specific marker expression, and by evaluating cell functionality at different maturation stages by calcium imaging and patch clamping. Differentiated neurons were shown to be electrophysiologically altered by uniaxial mechanical deformation. Spontaneous network activity decreased with applied stretch, indicating aberrant network connectivity. These results demonstrate the feasibility of this rapid, simple protocol for differentiating iPSC-derived MNs, suitable for in vitro neural injury studies focussing on electrophysiological alterations caused by mechanical deformation or trauma.

A closer look at neuron interaction with track-etched microporous membranes

Microporous membranes support the growth of neurites into and through micro-channels, providing a different type of neural growth platform to conventional dish cultures. Microporous membranes are used to support various types of culture, however, the role of pore diameter in relation to neurite growth through the membrane has not been well characterised. In this study, the human cell line (SH-SY5Y) was differentiated into neuron-like cells and cultured on track-etched microporous membranes with pore and channel diameters selected to accommodate neurite width (0.8 µm to 5 µm). Whilst neurites extended through all pore diameters, the extent of neurite coverage on the non-seeded side of the membranes after 5 days in culture was found to be directly proportional to channel diameter. Neurite growth through membrane pores reduced significantly when neural cultures were non-confluent. Scanning electron microscopy revealed that neurites bridged pores and circumnavigated pore edges – such that the overall likelihood of a neurite entering a pore channel was decreased. These findings highlight the role of pore diameter, cell sheet confluence and contact guidance in directing neurite growth through pores and may be useful in applications that seek to use physical substrates to maintain separate neural populations whilst permitting neurite contact between cultures.

Sacrificial Core-Based Electrospinning: A Facile and Versatile Approach to Fabricate Devices for Potential Cell and Tissue Encapsulation Applications

Electrospinning uses an electric field to produce fine fibers of nano and micron scale diameters from polymer solutions. Despite innovation in jet initiation, jet path control and fiber collection, it is common to only fabricate planar and tubular-shaped electrospun products. For applications that encapsulate cells and tissues inside a porous container, it is useful to develop biocompatible hollow core-containing devices. To this end, by introducing a 3D-printed framework containing a sodium chloride pellet (sacrificial core) as the collector and through post-electrospinning dissolution of the sacrificial core, we demonstrate that hollow core containing polyamide 66 (nylon 66) devices can be easily fabricated for use as cell encapsulation systems. ATR-FTIR and TG/DTA studies were used to verify that the bulk properties of the electrospun device were not altered by contact with the salt pellet during fiber collection. Protein diffusion investigations demonstrated that the capsule allowed free diffusion of model biomolecules (insulin, albumin and Ig G). Cell encapsulation studies with model cell types (fibroblasts and lymphocytes) revealed that the capsule supports the viability of encapsulated cells inside the capsule whilst compartmentalizing immune cells outside of the capsule. Taken together, the use of a salt pellet as a sacrificial core within a 3D printed framework to support fiber collection, as well as the ability to easily remove this core using aqueous dissolution, results in a biocompatible device that can be tailored for use in cell and tissue encapsulation applications.

Engineered method for directional growth of muscle sheets on electrospun fibers: Engineered method for directional growth

Research on the neuromuscular junction (NMJ) and its function and development spans over a century. However, researchers are limited in their ability to conduct experimentation on this highly specialized synapse between motor neurons and muscle fibers, as NMJs are not easily accessible outside the body. The aim of this work is to provide a reliable and reproducible muscle sheet model for in vitro NMJ study. A novel culture system was designed by engineering a method for the directional growth of myofiber sheets, using muscle progenitor cells cultured on electrospun fiber networks. Myoblastic C2C12 cells cultured on suspended aligned fibers were found to maintain directionality, with alignment angle standard deviations approximately two-thirds lower on fibers than on regular culture surfaces. Morphological studies found nuclei and cytoskeleton aspect ratios to be elongated by 20 and 150%, respectively. Furthermore, neurons were shown to form innervation patterns parallel to suspended fibers when co-cultured on developed muscle sheets, with alignment angle standard deviations three times lower compared with those on typical surfaces. The effect of agrin on samples was quantified through the slow release of agrin medium, encapsulated in alginate pellets and imbedded within culture chambers. Samples exposed to agrin showed significantly increased percentage of AChR-covered area. The developed model has potential to serve as the basis for synaptogenesis and NMJ studies, providing a novel approach to bio-artificial muscle alignment and setting the groundwork for further investigations in innervation.