The overall goal of the following experiment is to develop a human neuronal culture model, identify neuronal markers and isolate a pure population of neurons for gene expression analysis. This is achieved by first expanding human neuro progenitor cells to create a continuous supply of progenitor cells. As a second step, the neuro progenitor cells are cultured in the presence of differentiation supplements and neurotrophic growth factors in dishes coated with attachment factors.
Next, immunofluorescent staining for neuronal markers is performed to determine the fate of the differentiated neuro progenitor cells. Finally, laser capture microdissection of neurons cultured on PEN membrane Slides is performed to facilitate neuron specific gene expression analysis. Results are obtained that show differentiated neuro progenitor cells to be predominantly neuronal in nature.
Based on immunofluorescent staining and capture of neurons. By laser capture microdissection, The human neuro progenitor cells can be expanded and differentiated into neuron rich cultures. For in vitro studies, as these cultures have some gal cells, we can use laser capture micro dissection to isolate a pure population of neurons.
Demonstrating these procedures will be Ron Bouchard, a senior research associate in our microscope core facility. To begin this procedure, revive a frozen stock of human neuro progenitor cells isolated from fetal brain by quickly warming them to 37 degrees Celsius, and then adding them to a T 75 containing 15 milliliters of prewarm neuro basal medium, supplemented with 10 nanograms per milliliter, EG, F, and FGF. After culturing the cells for three days, transfer the neurospheres into a 15 milliliter tube and pellet them by centrifugation at 1000 RPM for five minutes following centrifugation, discard the supernatant, leaving behind about 100 microliters medium, and transfer the cell pellet into an einor tube.
A 100 microliter cell pellet is enough to split into two T 75 flasks. If less, reduce the number of flasks as neuro progenitor cells fail to proliferate. If split thin, then thoroughly break up the neurospheres into a single cell suspension by pipetting up and down 50 times while keeping the 200 microliter tip against the bottom of the tube.
Visually check the progress using a microscope to ensure disassociation of the neurospheres. Next, divide the cell suspension equally and add it to two T 75 flasks. One for continued expansion and another for differentiation.
Then incubate the flasks. Change the medium of the neuro progenitor cells in the flask marked for expansion by centrifugation. Then discarding the old medium and adding new medium.
Change the medium every four to five days until the neurospheres reach their original size of 300 to 500 microns. To begin neuro progenitors cell differentiation into a neuron rich culture, place a single cover slip into each well of a 24 well plate and coat by adding 500 microliters of 100 micrograms per milliliter poly L lysine to each. Well incubate the dishes for 30 minutes at room temperature, then aspirate the polyol lysine solution.
Rinse the plate with deionized sterile water and aspirate again. Next, coat the wells of the plate with 500 microliters of five micrograms per milliliter, mouse laminin and incubate for 30 minutes following incubation, wash the wells with PBS aspirate again and set them to dry in the tissue culture hood. Four days after splitting the cells, transfer the small neurospheres from the flask labeled for differentiation into the coated 24 well plate.
Add a density of about 500 neurospheres per well after incubating for six hours when the neurospheres have attached to the dish, remove the proliferation medium and add differentiation. Medium consisting of neuro basal medium B 27 supplement N-G-F-B-D-N-F-D-B-C and retinoic acid. Return the plate to the incubator following culture of neuro progenitor cells in neuronal differentiation medium for two weeks, a neuron rich culture is obtained.
Rinse the neurons once in PBS and then fix them in 4%paraform aldehyde for 30 minutes. Once fixed, rinse the cells three times with PBS. Then prepare the cells for immuno staining by incubating them with permeable buffer at room temperature for 60 minutes.
Next, add polyclonal and monoclonal antibodies in 3%BSA in PBS to the wells. Adding a different antibody pair into each, well incubate them overnight at four degrees Celsius in a shaker the next morning. Wash the cover slips three times with PBS and then incubate them with anti rabbit P SI three and anti-US fitzy secondary antibodies at room temperature in the dark for 90 minutes following incubation.
Wash the cover slips three times with PBS. Then place 10 microliters of mounting medium onto a glass slide. Take out the cover slip from the culture dish and place it upside down on the mounting medium.
Gently wipe away any excess mounting medium and seal the edges with nail polish. To prepare the cells for microdissection first, differentiate neuro progenitor cells into a neuron rich culture on PEN membrane. Slides coated with poly L lysine and laminin.
Perform all the subsequent steps under RNA free conditions. Next, stain the cultures using histo gene stain to visualize the cells histo gene stains the nuclei purple and the cytoplasm, a light pink, making it easy to differentiate neurons and glial cells by their distinct morphologies. Place the slide in 75%ethanol and deionized sterile water for 30 seconds.
Then place the slide on a Kim wipe. Add 200 microliters of histo gene and wait for 20 seconds. Next, place the slide sequentially for 30 seconds each in deionized sterile water, 75%ethanol, 95%ethanol, and 100%ethanol.
Then place in xylene for five minutes and dry on a Kim wipe for five minutes. Then place a PEN membrane Slide over the regular slide and place them into the Veritas laser capture micro dissection system or LCM using the software. Take a roadmap image to find the areas of pure neuronal populations devoid of astrocytes.
Mark these neurons using the drawing tools. Next, perform laser capture of these areas using computer controlled precision and automation in a two-step process. First, IR is fired at multiple spots with a laser power setting of 70 milliwatts and a pulse of 2, 500 microseconds to attach the membrane to the cap.
Next, the marked area is excised using a UV cutting tool at a low level setting of 10 millivolts. The combination of these processes selectively captures the marked areas onto capture LCM macro caps. When a minimum of 100 neurons have been captured, use a microscope to remove the membrane from the cap and place it on a tube.
Isolate total RNA from LCM samples using a P OPU RNA isolation kit and treat with DNAs. Then amplify the isolated RNA using ribo amp RNA amplification kit by following the instructions from the kit. Finally, perform real-time R-T-P-C-R analysis using TAC MAN probes to detect human neurofilament heavy chain.
A neuron rich culture is obtained by the differentiation of four day old neurospheres using poly L lysine and laminate coated surfaces in combination with differentiation specific growth factors. After two weeks, a neuron rich culture shown here is obtained in areas of low density. The cells may differentiate into astrocytes like the ones seen here.
Astrocyte differentiation can also be directed by using the growth factor CNTF instead of those used for neuron differentiation. Neuron differentiation can be identified using antibodies towards the specific neuronal markers, new N and synapsin, acetylcholinesterase and synaptophysin, and BDNF and GAAP 43 Positive staining is evidence that the differentiated cells have characteristics of primary neurons. Additionally, astrocytes can be identified through positive stat three and GAP immuno staining.
Laser capture microdissection of neurons is performed on cells that have been differentiated on poly L lysine and laminin coated PEN membrane Slides shown here are neurons stained with histo stain. To visualize the cells, the cells of interest can then be outlined using a laser, which melts the PEN membrane, causing it to stick to the capture cap placed above it. In the end, numerous cells can be outlined and captured onto the same cap for DNA or RNA analysis.
After watching this video, you should have a good understanding of how to isolate neurons using laser capture microdissection.
