The standard way to obtain a stem cell from dental pulp is poorly efficient, and this depends on several variable. Our protocol allowed to obtain a successful isolation of stem cell from treated teeth in 90%of the cases. Obtaining a high number of cells in a few weeks is the main advantage.
That allow us to use the cell in regenerative medicine and to create a biobank providing other scientists with this useful tool. The procedure for opening the tooth will be performed by Dr.Constantino Santacroce. Subsequently, the protocol will be shown by Stefano Martellucci, the PhD student from my lab, who is the first author of the article.
To begin, extract the third molar from the patient, quickly rinse it with PBS, put in a 15-milliliter test tube with the medium, and transfer it to the laboratory in less than two hours. Under a biohazard hood, use a cutter to open the tooth by a coronal cutting pass, parallel and tangent, through the roof of the pulp chamber. With a small excavator, gently remove the pulp, and place it in a test tube.
Then, wash with PBS three times, and centrifuge at 2, 500 times g at room temperature for 10 minutes. After the centrifugation, remove the supernatant. Resuspend the pellet in Hank's solution, and transfer the sample to a Petri dish.
Remove the Hank's solution by centrifugation at 2, 500 times g at room temperature for 10 minutes. Then, with a disposable scalpel, divide the pulp into approximately one-millimeter slices, and add one milliliter of type IV collagenase. Next, centrifuge the sample at 2, 500 times g at room temperature for 10 minutes.
Remove the supernatant, and resuspend the pellet in the medium. Culture it in a T25 flask specific for stem cells at 37 degrees Celsius in 5%carbon dioxide. During the incubation period, change the culture medium every three days.
Once the adherent cells reach confluence, add one milliliter of trypsin-EDTA solution to the cells to detach them, and incubate the cells at 37 degrees Celsius for three minutes. Next, add four milliliters of the culture medium in a one-to-five ratio to stop the trypsin action, and centrifuge the cell suspension at 259 times g for six minutes. Remove the supernatant.
Then, place the cells in a T25 flask to propagate. Then, detach the cells with one milliliter of trypsin-EDTA at 37 degrees Celsius for three minutes. Centrifuge the cell suspension at 259 times g for six minutes.
Remove the supernatant, and proceed to test the cells for cytofluorimetric analysis. To perform the transient prion protein silencing step, first culture the human dental pulp stem cells in six-well plates with two milliliters of the culture medium for 24 hours. Then, add 400 microliters of the small interfering RNA PrP medium to each sample, and incubate them at 37 degrees Celsius for six hours.
Without discarding siRNA PrP medium, add 1.6 milliliters of the culture medium in a one-to-five ratio, and leave the cells at 37 degrees Celsius for 72 hours. After the incubation period, remove the supernatant, and wash the cells with two milliliters of PBS three times at room temperature. Then, add two milliliters of the neuronal culture medium, and keep the cells for seven to 14 days in an incubator at 37 degrees Celsius.
After the incubation period, wash the cells with two milliliters of PBS three times at room temperature, and proceed to Western blot analysis to test for neuronal surface antigens. To perform the neuronal induction process, culture 2 million human dental pulp stem cells in six-well plates, up to 28 days from the pulp separation. Every three days, discard the supernatant.
Wash three times with two milliliters of PBS at room temperature, and replace two milliliters of the neuronal culture medium. After seven to 14 days, detach the cells with one milliliter of trypsin-EDTA at 37 degrees Celsius for three minutes. Then, add four milliliters of the culture medium in a one-to-five ratio to stop the trypsin action.
To characterize the human dental pulp stem cells by flow cytometry, culture 2 million per milliliter of the cells in six-well plates with two milliliters of the culture medium. At 28 days post-dental pulp separation or after an additional 14 days with neuronal culture medium, add one milliliter of the trypsin-EDTA at 37 degrees Celsius for three minutes to detach the cells. Next, add four milliliters of the culture medium in a one-to-five ratio to stop the trypsin action, and centrifugate at 259 times g at room temperature for six minutes.
Fix the untreated or treated cells with 300 microliters of 4%paraformaldehyde in PBS at four degrees Celsius for 10 minutes. Next, centrifuge, discard the supernatant, and permeabilize the cells with 1%non-ionic surfactant in PBS at room temperature for an additional 10 minutes. Then, centrifuge again, discard the supernatant, and perform the blocking with 5%nonfat dried milk in one milliliters of PBS at room temperature for one hour.
Wash three times with one milliliter of PBS, and incubate the cells with anti-CD105, anti-CD44, anti-STRO-1, anti-CD90, anti-CD73, anti-beta-three-tubulin, anti-NFH, and anti-GAP43 monoclonal antibodies at room temperature for one hour. Next, centrifuge the cells again three consecutive times with one milliliter of PBS at six-minute intervals, and then incubate with anti-mouse PE or anti-rabbit CY5 monoclonal antibodies at room temperature for one additional hour. Use the secondary antibodies for gating the immune-positive cells, and analyze all samples with a flow cytometer.
Culture 2 million cells per milliliter of the human dental pulp stem cells in six-well plates with two milliliters of the culture medium. At 21 and 28 days post-dental pulp separation or after an additional seven to 14 days with the neuronal culture medium, add one milliliter of the trypsin-EDTA at 37 degrees Celsius for three minutes to detach the cells. Then, centrifuge, discard the supernatant, and then fix the specimens with 4%paraformaldehyde in PBS at four degrees Celsius for 10 minutes.
Next, centrifuge, discard the supernatant, and then permeabilize with 1%non-ionic surfactant in PBS at room temperature for 10 minutes. Remove the supernatant, and stain the cells with rabbit anti-PrP monoclonal antibody at room temperature for one hour. Finally, centrifuge, discard the supernatant, and then incubate with anti-rabbit CY5 monoclonal antibody at room temperature for an additional hour.
Analyze all samples with a flow cytometer. After pulp separation, clusters of human dental pulp stem cells were expanded on the periphery. A comparison between the growth of these cells before and after the neuronal differentiation induced by EGF/bFGF showed significant changes in the cell morphology and neurites outgrowth.
Untreated human dental pulp stem cells expressed multipotent mesenchymal stromal specific surface antigens, such as CD44, CD90, CD105, CD73, and STRO-1. After appropriate neuronal induction stimuli, these cells expressed specific neuronal markers, such as beta-three-tubulin, NFH, and GAP43. Untreated or treated cells did not express hematopoietic markers, such as CD14 and CD19.
After 28 days, prion protein was precociously expressed in human dental pulp stem cells compared with the corresponding negative control, and its expression was increased after the neuronal differentiation process induced by EGF/bFGF. Western blot analysis showed that silencing the prion protein by sRNA before EGF/bFGF stimuli could affect the neuronal differentiation process of human dental pulp stem cells by preventing the expression of neuronal markers beta-three-tubulin and NHF. The most important thing is the choice of the enzyme to separate the cells from the pulp.
In fact, if the enzyme is too aggressive, the cells could be damaged and their growth and differentiation capacity could be compromised. The dental pulp stem cells are an excellent experimental model which can be employed in vivo and in vitro studies in the regenerative medicine field. Since PrP plays a key role in neuronal differentiation process of dental pulp stem cells, this marker may be an excellent candidate as effective target in neurodegenerative diseases.
The most important aspect to advise is the health condition of the patients, such as the absence of infectious diseases.
