A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures

Abstract

BackgroundPluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca2+-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate.Methodology/Principal FindingsIn this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters.Conclusions/SignificanceOur method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture.

Impedimetric real-time monitoring of neural pluripotent stem cell differentiation process on microelectrode arrays

“…Most studies on neuronal differentiation processes still rely on classical qualitative or quantitative endpoint analysis of neuronal marker genes/proteins, which depend on time-and costconsuming cell labeling and destruction of the analyzed sample (Mertens et al 2015;Pasca et al 2015), while electrophysiological analysis of neuronal activity is only suitable for matured cultures after several weeks/months (Heikkila et al 2009;Mertens et al 2015;Ullah et al 2016). Although, there is ongoing research to overcome these limitations, existent studies as well require artificial handling such as cell isolation or genetic manipulation and are thus not able to assure and predict the progress of neuronal maturation of a complex iPS-derived neural network (Ilieva et al 2013;Leao et al 2010;Mir and Shinohara 2013).…”

“…Besides time-consuming conventional molecular biological methods mainly restricted to end-point analysis, some techniques have emerged that allow the fast and real-time study of differentiating cell lines. However, these technologies are mostly dependent on labels or single cell applications that do not comprise the analysis of a complex developing neuronal network (Leao et al 2010;Mir and Shinohara 2013).…”

Delivery of Differentiation Factors by Mesoporous Silica Particles Assists Advanced Differentiation of Transplanted Murine Embryonic Stem Cells

“…For imaging, the transplants were removed from live animals and cut on a vibratome with 200‐μm slices, which were transferred to the stage of an upright microscope and continuously perfused with aCSF with no HEPES equilibrated with 95% O 2 + 5% CO 2 . Excitation was provided by a light‐emitting diode array [11]. For VSFP2.42 imaging, excitation was centered at 520 nm, and a 605 nm low‐pass filter was used for emission, thus avoiding cross‐talk from EGFP fluorescence emission.…”

Recombinant spider silk protein matrices facilitate multi-analysis of calcium-signaling in neural stem cell-derived AMPA-responsive neurons

“…To investigate if NSCs grown on 4RepCT foam shaped matrices in 3D can differentiate into neurons we exposed the cells to treatment with bone morphogenetic protein (BMP) 4 or co-treatment with BMP4 and the signaling factor Wnt3a for 14 days. We have previously shown that BMP4 and BMP4+Wnt3a stimulation induces differentiation of mature, functional glutamate-receptor (GluR) -agonist responsive neuronal cells when NSCs are seeded at relatively high densities [16,25]. NSCs grown in 3D on 4RepCT foam matrices with pore sizes of around 5-30 µm and exposed to 10 ng/ml BMP4 or BMP4 and Wnt3a every 24 hours for 14 days differentiated into neuronal cells positive for the dendritic marker of mature, post-mitotic neurons, microtubule associated protein 2 (MAP2; in average 30 cells/5 micrographs/well (BMP4) and 41 cells/5 micrographs/well (BMP4+Wnt3a) in control conditions, and 71 cells/5 micrographs/well (BMP4) and 94 cells/5 micrographs/well (BMP4+Wnt3a) when grown on 4RepCT foam) (Figure 1,2).…”