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Method Article
The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.
Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology."
Neurite growth is a process fundamental to the formation of the neuronal network and nerve regeneration1,2. Following an injury, neurite outgrowth plays a key role in regeneration of the nervous system. Neurite outgrowth is also an important element of the extracellular signaling in inducing neuronal regenerative activities to enhance the outcomes for neurodegenerative disorders and neuronal injury3,4,5,6.
By maintaining their differentiation potential in producing various neural lineages, human neural progenitor cells (hNPCs) could provide a model system for studies of central nervous system (CNS) function and development7,8,9. High translational potential and physiological relevance of hNPCs as a primary human cell model offer a considerable advantage in neurite outgrowth-related drug discovery screenings. However, the maintenance and scaling of the primary cell models for high-throughput assays could be time-consuming and labor-intensive10,11,12,13.
In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment is another application using the hNPC-derived neurons. There are thousands of commercial chemical compounds that are either not examined or with poorly understood neurotoxicity potential. Therefore, more reliable and effective screening experiments to assess, distinguish, and rank compounds based on their potential to elicit developmental neurotoxicity is in high demand14. The increase in prevalence and incidence of neurological disorders along with the abundance of untested compounds in the environment necessitates the development of more trustworthy and efficient experiments to identify hazardous environmental compounds that may pose neurotoxicity15.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.
Ethics Statement: Fetal specimens were received from the Birth Defects Research Laboratory at the University of Washington in Seattle through a tissue distribution program supported by the National Institute of Health (NIH). The Birth Defects Research Laboratory obtained appropriate written informed consent from the parents and the procurement of tissues was monitored by the Institutional Review Board of the University of Washington. All the work was performed with approval by the Human Subject Research Office at the University of Miami8.
1. Isolation and culture of human neural progenitor cells (hNPCs)
Amount | Component |
100 µL | EGF (20 ng/mL) |
100 µL | FGF (10 ng/mL) |
2 mL | B-27, Minus vitamin A (50X) |
1 mL | L-alanyl-L-glutamine (100X) (see table of materials) |
4 µL | Heparin (2 μg/mL) |
96.8 mL | Neuronal cell culture medium (see table of materials) |
Table 1. Components required for making 100 mL of culture media
2. Passaging the hNPCs
3. Freezing the hNPCs
4. Differentiation and treatment of hNPCs
NOTE: To induce differentiation, neurospheres are disaggregated into single cells, counted and then seeded on coated plates for 5 days. Then differentiated cells are treated for 24 h with test compounds before immunostaining and fluorescence quantification.
Amount | Component |
49 mL | DMEM/F-12 |
0.5 mL | N2 supplement (100X) |
0.5 mL | MEM non-essential amino acids (100X) |
2 µL | Heparin (2 µg/mL) (Stock Conc. is 50 mg/mL) |
Table 2. Components required for making 50 mL of NIM
Amount | Component |
1 mL | B-27 (50X) |
500 µL | Antibiotic-Antimycotic (100X) |
5 µL | Retinoic acid (0.1 µM) |
50 µL | GDNF (10 µg/mL) |
50 µL | BDNF (10 µg/mL) |
5 µL | Ascorbic acid (0.2 µg/mL) (Stock Conc. is 2 mg/mL) NOTE: Recommended to be made fresh. |
48.5 mL | NIM |
Table 3. Components required for making 50 mL of differentiation media
5. Immunocytochemistry (ICC)
NOTE: Cells are fixed with 4% formaldehyde. Permeabilization and blocking is then performed to improve penetration and prevent nonspecific binding of antibodies. Cells are then incubated with primary antibodies overnight. Subsequently, cells are incubated with fluorescently labeled secondary antibodies. Finally, after using DAPI to stain the nucleus, chamber slides are mounted.
Amount | Component |
1.75 g | NaCl (150 mM) |
1.2 g | TrisBase (50 mM) |
2 g | BSA 1% |
3.6 g | L-lysine (100 mM) |
8 g | Sodium Azide (4%) |
200 mL | Distilled water. NOTE: Initially dissolve the required components in 150 mL of water, then adjust to 200 mL. |
Table 4. Components required for making 200 mL of Antibody buffer
Amount | Component |
600 µL | 20% Goat serum |
6 µL | 0.2% Triton-X100 |
2394 µL | Antibody buffer. NOTE: Initially dissolve the required components in 2 mL of Ab buffer, and then adjust to pH 7.4. Then add more Ab buffer to adjust to final volume of 3 mL and filter sterilize. |
Table 5. Components required for making 3 mL of cell permeabilization and blocking solution
6. Image acquisition, neurite outgrowth and fluorescence intensity quantification
NOTE: Following staining, use a confocal microscope with a 20x objective and an image size of 1024 x 1024 pixels to acquire the images of the treated cells. Take image at least from two fields per biological replicate per condition. Then use Fiji image analysis software (ImageJ 1.51u) for quantification of the neurite length. Briefly, measure the length of the longest neurite for each neuron and after averaging the values per treatment, use student’s t test for independent groups to compare the means between experimental groups and control group.
NOTE: Several commercial (Imaris, Volocity, Amira) and open source (ImajeJ, CellProfiler, Vaa3D, BioImageXD, Icy, KNIME) image processing programs are available. Among these programs, ImageJ has become the tool of choice for biological image analysis20,21. The ImageJ portal at https://imagej.net/Introduction is a useful source of information providing a thorough description of ImageJ’s basic, and built-in functions including image processing, colocalization, deconvolution, registration, segmentation, tracking, and visualization.
7. Neurotoxicity assessment
NOTE: Cytotoxicity of test compounds are evaluated in 384-well plates (see Table of Materials) using a luminescent cell viability assay (see Table of Materials). The hNPCs are prepared following the same method, except slight modifications, described in the “Differentiation and Treatment of hNPCs” section. Subsequently, a luminescent signal generated by luminescent cell viability assay is measured utilizing a microplate reader. The luminescent signal is proportional to the cellular ATP concentration which itself is directly proportional to the number of viable cells present in each well.
The protocol presented in the manuscript has successfully been used in two recently published papers22,23. Figure 3 demonstrates the use of hNPCs-derived neurons in examining the effect of HDAC inhibitors as epigenetic compounds on the extension of neurites as a marker for neurite outgrowth and subsequent neurogenic ability of small molecule compounds.
Furthermore, in Figure 4...
This protocol is one of the few published papers describing the test for neurite length upon treatment with test compounds. Furthermore, we describe how to use hNPCs for a neurite outgrowth assay and neurotoxicity assessment. By utilizing this neurite outgrowth assay and neurotoxicity assessment on hNPCs-derived neurons, the neurogenic potential of a category of epigenetic small-molecule compounds, HDAC inhibitors, in inducing neurite outgrowth is demonstrated22. Furthermore, in another paper pres...
All authors indicate no potential conflicts of interest.
This research was funded by NIMAD research grant (940714) awarded to MAF.
Name | Company | Catalog Number | Comments |
4-well Glass Chamber Slides | Sigma | PEZGS0816 | |
Alexa Fluor 488 | Invitrogen | A-11001 | |
Alexa Fluor 594 | Invitrogen | R37117 | |
Antibiotic-Antimycotic | Gibco | 15240062 | |
Anti-β-Tubulin III | Thermo | MA1-118X | |
B27 | Thermo | 17504001 | |
B27 - minus vitamin A | Thermo | 12587010 | |
BDNF | PeproTech | 450-02 | |
BSA | Sigma | A8531 | |
CellTiter-Glo | Promega | G7572 | |
CoolCell | Corning | 432000 | Cell freezing containers ensuring standardized controlled-rate -1℃/minute cell freezing in a -80℃ freezer |
CryoStor CS10 | StemCell Technologies | 7930 | Cryopreservation medium containing 10% DMSO |
DAPI | Thermo | D1306 | |
DMEM/F12 | Gibco | 11320033 | |
DMSO | Sigma | 34869-100ML | |
EGF | Gibco | PHG0311 | |
FGF | Gibco | PHG6015 | |
Formaldehyde | Thermo | FB002 | |
GDNF | PeproTech | 450-10 | |
Glutamax | Gibco | 35050061 | L-alanyl-L-glutamine supplement |
Goat Serum | Thermo | 50062Z | |
Heparin | Calbiochem | 375095 | |
Laminin | Sigma | L2020-1MG | |
L-Ascorbic Acid | Sigma | A92902-25G | |
L-lysine | Sigma | L5501 | |
MEM non-essential amino acids | Gibco | 11140050 | |
mFreSR | StemCell Technologies | 5854 | Serum-free cryopreservation medium designed for the cryopreservation of human embryonic and induced pluripotent stem cells |
N2 | Gibco | 17502048 | |
NaCl | Sigma | 71376 | |
Neurobasal Medium | Gibco | 21103049 | |
Nunc 384-Well Polystyrene White Microplates | Thermo | 164610 | |
PBS | Thermo | 10010-049 | |
Poly‐L‐lysine | Sigma | P5899-5MG | |
ProLong Gold Antifade Mountant | Thermo | P10144 | |
Retinoic Acid | Sigma | R2625 | |
Sodium Azide | Sigma | S2002 | |
StemPro Accutase | Gibco | A1110501 | Cell dissociation reagent containing proteolytic and collagenolytic enzymes |
Synaptophysin | Thermo | MA5-14532 | |
Tris Base | Sigma | 10708976001 | |
Triton X-100 | Sigma | X100-100ML |
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