Human Neurotoxicity Screening
Toxicological HTS screening for neurobiological targets using hNP1™ Human Neural Progenitor Cells or hN2™ Neuronal Cells has now become easier, faster, and standardized thanks to HemoGenix® Bioluminomics technology.
Fig. 1. Differences in cellular ATP levels between hNP1 & hN2 cells indicate differences in cell proliferation.
Cell Migration Assay
Using hNP1™ neural progenitors, the Oris™ Cell Migration Assay, developed by Platypus Technologies, can quantitatively detect both stimulators and inhibitors of cell migration and has the potential for adaptation as a homogeneous HTS-amenable cell-based assay. For data generated using this assay, please click here (PDF).
Fig. 1. Schematic of the Oris Cell Migration Assay
Fig. 2. Measurement of hNP1 cell migration using fluorescence microplate readers and image analysis.
hNP1™ neural progenitors were plated at 60,000 cells per well onto Matrigel-coated Oris™ Assay plates then incubated at 37C for 72 hrs to allow for migration. Plates were read using a Molecular Devices' Flexstation 3 microplate reader (ex 494 nm / em517 nm) and then imaged by epifluorescence microscopy and analyzed fro area closure using imageJ. Over a wide range of conditions, calcein fluroescence correlated well with area closure measurements, allowing fo ruse of the assay in fluorescent plate readers as well as imaging platforms.
Fig. 3. Inhibition and stimulation of hNP1™ cell migration.
Left, Cytocalcein D inhibited hNP1™ migration in a concentration dependent manner with an IC50 of ~15 nM, indicating that inhibitors of migration can be readily detected.
Right, Preliminary results indicate that bFGF alone stimulates hNP1™ migration (*, P < 0.05 when compared to no growth factor control). In contrast, the combination of LIF and GDNF has a synergistic chemokinetic effect (*, P < 0.05).
Neural Progenitor Proliferation
Using hNP1™ neural progenitors, Molecular Devices' ImageXpress platform can be used to quantitatively measure stimulators and inhibitors of neural progenitor proliferation through high content imaging (HCI). For data generated using this assay, please click here (PDF).
Fig. 1. Overlayed images from wells containing hNP1™ neural progenitors and hN2™ differentiated neuronal cells. Cells were stained with Hoeschst (nuclei; blue) and beta-III-tubulin (cytoskeleton; green). Images were acquired with ImageXpress Mircro system using a 20X objective.
Fig. 2. Image analysis results using Molecular Devices' MexaXpress Software (Cell Scoring Module) to measure the effects of growth factors on hNP1™ neural progenitor proliferation (number of cells per field).
Neural Progenitor Differentiation
Using hNP1™ neural progenitors, Molecular Devices' ImageXpress platform can be used to quantitatively measure stimulators and inhibitors of neural progenitor differentiation through high content imaging (HCI). For data generated using this assay, please click here (PDF).
Fig. 1. Effects of different growth factors on the neuronal differentiation of hNP1™ neural progenitors were evaluated in a 14-day assay in 96-well formate. Cells were cultured in teh presence of different growth factors and analyzed with the Cell Scoring and Neurite Outgrowth modules of MetaXpress Software. The presence of LIF or EPO increases neuronal differentiation. Shh and BDNF had marginal effects.
In conjunction with Molecular Devices' ImageeXpress HCI platform, hN2™ differentiated neuronal cells can be used to evaluate potential toxic effects of test compounds on nuerite outgrowth through visualizing cells stained with β-lll tubulin.
Fig. 1. Dose response curves for the effect of neurotoxic agents on neurite outgrowth. hN2™ cells were cultured in the presence of cytotoxic compounds for 72h, stained for β-lll tubulin, imaged with the ImageeXpress and analyzed for neurite outgrowth.
hNP1™ neural progenitors can be deployed for cytotoxicity testing using the widely used Alamar Blue assay.
Fig. 1. Mitochondrial reductase activity of hNP1™ cells as a function of number of cell plated and Alamar Blue assay time. Cells were plated at the indicated density in 96-well formate, incubated for 48 hours in normal medium, then assayed for reductase activity with the edition of Alamar Blue reagent (available via Life Technologies) and subsequent incubation for 1-8 hours.
Effective genetic modification of hNP1™ Human Neural Progenitor Cells demonstrates the extensive versatility of this product and broadens their potential further – enabling selection, directed differentiation and live cell tracking and monitoring, etc.
The following application note describes the successful use of a non-viral vector to genetically modify hNP1 cells and demonstrates thier utility for use in High Content Screeing.
High Content Screening assays often utilize primary or stem cell sources which are not amenable to large scale screening and can require extensive cell culture, fixation, permeation and immunocytochemistry prior to imaging. ArunA Biomedical has demonstrated the feasibility of utilizing a non-viral vector encoding a Green Flourescent Protein (GFP) reporter gene driven by a ubiquitous promoter. GFP+ cells were Puromycin resistent and Puromycin was used as a selection agent enabling yields of >99% GFP positive populations. The resulting population of Neural Progenitor cells were further differentiated and used in a neurite outgrowth assay using bisindolylmaleimide 1 (BIS1). Read more.
Figure 2: Clonal GFP+ hNP1™ cells. hNP1 Human Neural Progenitor Cells were cultured in ArunA's Expansion Medium and passaged upon reaching 100% confluence. These images were taken at 24 hours post-passage and at 20X magnification. Panel A shows GFP image of a clonal culture. Panel B shows the same image in phase contrast.
Figure 2: Puromycin selected GFP+ cells were further differentiated to Neurons using ArunA's proprietary Differentiation Medium then cryopreserved.
The following application note describes both lipid‐based transfection with non‐integrating plasmid DNA and transduction with integrating recombinant lentivirus of ArunA Biomedical’s hNP1™ Human Neural Progenitor Cells (hNP1™ cells).