A quantificação tridimensional das espinhas dendríticas de Piramidal neurônios derivados pluripotentes induzidas pelo homem Células-Tronco

The overall goal of this procedure is to image dendritic spines from peral neurons derived from human-induced pluripotent stem cells and quantify them in three dimensions. This is accomplished by first treating cover slips in six well plates with polyurethane and laminin for culturing neural stem cells. Next neural stem cells diluted in culture.

Medium are plated, using a gentle rotating pipette movement and are allowed to differentiate up to 70 days in culture. By renewing the medium regularly, then the cells are transduced with GFP lentivirus stained with anti GFP antibodies and visualized. Finally, the cultures are fixed and the dendritic spines are in imaged using confocal microscopy.

Ultimately, the image data of human dendritic spines are reconstructed in 3D for their classification and quantification. This protocol provides a stepwise procedure to obtain glutamatergic neurons of layer two to four of human brain cortex. It involves the use of neural stem cells derived from human induced pre potent stem cells that originate from human fibroblast based on the previously published method.

The main advantage of this technique of our existing methods is that allow for the automatic segmentation of DON right and PY in 3D. This with an appreciable gain of time over existing softwares, which typically prepare from 2D analysis only. The donr and spy in classifications is very handy and easy to use as well.

To Prepare neuronal cultures, place glass cover slips into six well culture dishes and add poly ornithine incubate overnight under a flow hood the following day. Use DPBS to wash the cover slips three times and add laminin incubate under a flow hood for at least 10 hours. Prepare culture medium containing the following components.

Then use the medium to plate and dispatch neural stem cells or NSCS at a density of 50, 000 cells per square centimeter Pipetting with slow rotating movements in order to reduce cell clustering to transduce human IPSC derived neurons. At any step of maturation, add one microliter of a stock solution containing 40 nanograms of GFP lentiviral vector per culture well containing fresh culture medium, and incubate at 37 degrees Celsius for 48 hours. To prepare cells for immunofluorescence, remove the culture medium and use 4%formaldehyde to fix the transduced cells at room temperature for 10 minutes.

Then use one XPBS to wash the cells three times for 10 minutes each. Next, submerge the cover slips in PBS supplemented with 0.05%Triton X 110%horse serum and incubate at room temperature for one hour. Then use PBS to wash the cover slips three times.

Add one 1000th GFP primary antibody in 100 microliters of PBS 4%horse serum to each cover slip. Incubate in a dark box at four degrees celsius overnight. Now dilute Alexa Fluor 4 88 conjugated antibody one to 200 in PBS 0.5%tween 20, and incubate the cells with the antibody solution at room temperature for one hour after using PBS to wash the slides three times.

Use mounting medium to mount the slides for fluorescence microscopy. Under a confocal microscope, select at least 10 healthy neurons per condition with a parametal morphology and a fold dendritic arborization to quantify 60 to 100 micrometers per dendrite. Acquire images with a 40 x na, 1.3 oil objective and a 488 nanometer laser with a typical peak power at sample level around 20 micro watts, set the pixel size around 80 nanometers to properly sampled dendritic spines.

To sample the whole neuron volume, acquire a Z stack with a Z spacing from 150 to 300 nanometers, yielding 20 to 30 Z slices to carry out 3D quantification of dendritic spines. Use the following settings for the analysis software for pre-processing. Use Gaussian filtering by choosing image processing smoothing Gaussian filter.

Set the filter width equal to the pixel size in the XY plane to perform a semi-automatic tracing of dendrites from the filament tracer module. In the slice tab, use the distance tool to estimate the dendrite diameter. Next in the surpassed tab, click on the filaments tool and choose skip automatic creation for better robustness In the draw tab.

Now presented select auto path as a method, dendrite as a type, and input the estimated dendrite diameter. Using the select mode of the pointer, turn the cursor into a box and choose shift and right click on the dendrite starting point. The software will perform initial calculations.

Now move the pointer along the dendrite from the starting point. A yellow line representing the most likely dendrite path is shown. Press shift and left.

Click on the dendrite endpoint to carry out automated spine segmentation in the module interface. Click on the creation tab in the rebuild drop list. Choose rebuild dendrite diameter and check the keep data box.

Then click rebuild. Set the threshold so that the segmented volume corresponds to the actual dendrite volume. As an algorithm, select shortest distance from distance map.

Then click the next button under the slice tab, determine the smallest spine, head diameter and the maximal length. Then under the surpass tab, enter the parameters values around 200 to 300 nanometers for minimal diameter and four micrometers for maximal length are good Starting points without checking the allow spines box, click the next button. Next, adjust the seed points threshold so that the blue points representing spines localized to the actual spine heads.

Then click next. The core calculation will now be performed and can take time to classify spines under the tools tab of the module interface, click on classify spine. After verifying that there are four classes as outlined in the text protocol, the module interface will generate four new filament objects with the results of the classification.

Finally, export the statistical data under the statistics tab by clicking on export all statistics to file. This figure illustrates the culture of human neurons labeled an anti beta three tubulin antibody. The tendency for cell clustering is also evident here.

Shown here is A GFP labeled parametal neuron 40 days post differentiation from a late cortical progenitor. From the superficial cortical layers. The inset shows a lower magnification with the apparent cell body.

These panels represent the 3D reconstruction of segments of dendritic spines at different stages of maturation. The quantitative analysis of spine density and spine head volume is shown here. As expected, these two parameters increase over the culture period.

While item attempting the culture procedure, it is important to plate and dispatch very carefully the neuro stem cells by adding cells slowly with a gentle rotating movement in order to reduce cell choline. Indeed, this method requires the identification of individual neurons.