The overall goal of this procedure is to differentiate immortalized multipotent otic progenitor cells into inner ear cell types. So this method can help us address key questions in auditory regeneration, such as how otic progenitors can become cochlear hair cells or auditory neurons. Importantly, this technique provides an in-vitro cellular system that can be used in parallel with in-vivo mammalian animal model systems.
Demonstrating the procedure today will be Azadeh Jadali, a postdoctoral fellow in my lab, as well as Zhichao Song, and Alejandra Laureano, two graduate students in the lab. After preparing the culture medium as described in the text protocol, use three milliliters of this liquid to plate one times ten to the sixth immortalized multipotent progenitor, or IMOP cells, into a 60 millimeter tissue culture dish. Incubate the cells for five to seven days at 37 degrees Celsius with five percent carbon dioxide.
Every other day, double the volume of liquid in the culture by adding fresh medium. To passage the cells, first transfer them from the culture dish to a 15 milliliter conical tube. And then place the tube, at 37 degrees Celsius, for five to ten minutes.
During this time, expect colony forming otospheres to collect at the bottom of conical via gravity sedimentation. Next, aspirate the supernatant, being careful not to disturb the palette. Resuspend the otospheres by adding 0.5 milliliters of previously prepared pre warmed, one millimolar EDTA enhanced balanced salt solution or HBSS, and then pipetting up and down two to three times.
To facilitate the dissociation of otospheres into single cells, place the conical in an incubator at 37 degrees Celsius. Periodically swirl the solution and when no otospheres are observed as sediment at the bottom of the tube, move it to room temperature. Proceed to add two milliliters of culture medium to the cells to neutralize the EDTA.
And centrifuge them at room temperature for five minutes at 200 gs. Then aspirate the diluted EDTA solution and wash the cells in five milliliters of 1x PBS. After spinning down the cells using the same settings, aspirate the 1x PBS.
Next, add 0.5 milliliters of culture medium to the conical. And resuspend the cell palette. For counting, dilute IMOP cells one to one hundred in culture medium.
Then add 75 microliters of this solution into a cassette positioned in a microfluidic particle counter and determine the cell number. Afterwards, plate one times ten to the sixth IMOP cells in a 60 millimeter tissue culture dish as previously described and repeat this passaging procedure every five to seven days. Three days after plating, use gravity sedimentation to collect otospheres as previously shown.
Proceed to aspirate the supernatant. And gently resuspend the cells in two milliliters of sensory epithelial differentiation medium. Then use a ten milliliter large pore pipette to transfer the otospheres into a new 60 millimeter dish.
Differentiate the otospheres for ten days. Adding two milliliters of fresh differentiation medium to the culture every other day. Afterwards, harvest the otospheres again through gravity sedimentation.
And aspirate the spent medium. Next, fix the otospheres in four percent paraformaldehyde in 1x PBS for 15 minutes at room temperature. Then remove the formaldehyde solution and wash the cells in 1x PBS containing 0.1 percent Triton x100.
Proceed by blocking the otospheres in 1x PBS supplemented with ten percent goat serum and 0.1 percent Triton x100. After one hour, replace the blocking buffer with that containing the appropriate dilution of Cdkn1b or CDH1 antibody. Then incubate the cells in primary antibody overnight at four degrees Celsius.
And the next day continue immunostaining using fluorescently labelled secondary antibodies, Phalloidin, and Hoechst, as described elsewhere. To prepare for imaging, place mounting medium on a glass slide. And then introduce the stained otospheres into the medium.
Next, lay a cover slip on top of the sample. After letting the mounting medium dry overnight at four degrees Celsius, place the slide on the stage of an inverted microscope equipped with a 16-bit CCD camera. Using the 20x 0.75 air objective, acquire epifluorescence images by collecting data from the blue, green, red, and infrared channels.
First, clean round glass cover slips by placing them in a sterile 100 millimeter plate and submerging them in 70 percent ethanol. Gently agitate the cover slips to ensure they are covered and leave the dish at room temperature for ten minutes. Next, rinse the cover slips three times in sterile 1x PBS and then once with sterile water.
Then let the cover slips dry by exposing them to UV light in a tissue culture hood for 15 minutes. For immediate use, place one sterile cover slip into each well of a 24 well plate. And gently shake the plate to ensure that all cover slips lie flat.
Then coat the cover slips by adding 0.25 milliliters of 1x PBS containing 10 microgams per milliliter of poly D lysine to each well. After the plate has incubated for one hour at 37 degrees Celsius, remove the poly D lysine solution and wash the wells three times with sterile 1x PBS. Once the liquid from the last wash has been aspirated, introduce 0.25 milliliters of 1x PBS containing 10 micrograms per milliliter Laminin, into each of the 24 wells.
After incubating the plate overnight at 37 degrees Celsius, aspirate the Laminin solution, then wash each well with one milliliter of 1x PBS three times, but leave the PBS from the last wash in the wells until the cells are ready to be plated. To begin neuronal differentiation, count the cells from dissociated otospheres as previously described. And then resuspend them in prewarmed neuronal differentiation medium.
Proceed by seeding between one and one point five times ten to the fifth IMOP cells into each well of the plate containing the coated cover slips. Incubate the plate at 37 degrees Celsius, with five percent carbon dioxide. Replacing the neuronal differentiation medium every other day.
After seven days, aspirate the medium, and fix the cells, which should be attached to the cover slips, by adding four percent formaldehyde into each well. And leaving them for 15 minutes at room temperature. Then remove the formaldehyde and wash the cells once in 1x PBS containing 0.1 percent Triton x100.
Next remove the wash buffer. And add blocking buffer consisting of 1x PBS containing ten percent normal goat serum, and 0.1 percent Triton x100 to each well. Leave the cells for one hour at room temperature.
And then remove the liquid and replace it with blocking buffer containing the appropriate dilution of Cdkn1b or Tubb3 antibody. Following an overnight incubation at four degrees Celsius, wash the cells once with 1x PBS containing one percent Triton x100. And then proceed with immunostaining as previously described.
After staining has been completed, perform a final wash by adding 1x PBS to the wells. To prepare them for analysis, remove the cover slips to which stained cells are attached from the 24 well plate. And place them onto mounting medium on a glass slide.
After letting the mounting medium dry overnight at four degrees Celsius, acquire epifluorescence images as previously described. Otospheres cultured for ten days in epithelial differentiation medium demonstrate increased Cdkn1b expression, a cycling dependent kinase inhibitor, and marker for inner ear differentiation, compared to undifferentiated cells. Here Hoechst staining did note cell nuclei.
These cells also exhibit morphological changes in actin filaments and regions of cell-cell adhesion characteristic of epithelia. Indicated by the appearance of Phalloidin, and Cdh1 labeling at the cell peripheries. Otospheres can also be directed to form neurons.
This bright field image shows that after seven days in neuronal differentiation medium, IMOP cells exhibit neurites. Immunostaining of these cells with a neuronal marker Tubb3 confirms their differentiation. And they also robustly express Cdkn1b.
This suggests that dual Tubb3, Cdkn1b signal can be used as an indicator of neuronal differentiation in IMOP cells. Cdkn1b fluorescence differentiated and undifferentiated cells was also normalized by determining its ratio to Hoechst signal, providing quantitative results. Histograms of this data is shown here.
The percentages of undifferentiated and differentiated IMOP cells above a predetermined threshold of normalized Cdkn1b are presented in these graphs. Both differentiated populations demonstrate a significant increase in the percentage of cells expressing Cdkn1b compared to undifferentiated controls, confirming the differentiation suggested by immunofluorescence. Once mastered, routine culture and differentiation of IMOP cells can be done in 30 minutes, if it is performed properly.
After watching this video, you should have a good understanding of how to initiate differentiation of IMOP cells. Thank you for watching this video. And good luck with your experiments.
