Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

This method can help answer key questions in the field of Regenerative Medicine, such as where a human dental follicle stem cells could be genetically engineered to improve their therapeutic properties. The main advantage of this technique is that stem cells can be modified by microRNA introduction without the hazard of stable genome integration. Begin the procedure for enzymatic digestion of the two follicle, by pre-warming to room temperature, human dental follicle stem cell or hDFSC culture medium and PBSPSG solution.

Thaw one aliquot each of Collagenase type I and Dispase II stock solutions. Prepare a digestion solution by adding 500 microliters of the Collagenase type I stock solution and 500 microliters of the Dispase II stock solution to 4 milliliters of Basal Medium containing 1%antibiotic agent. To avoid site contamination during human dental follicle stem cell isolation wash buffers and culture media used must contain antibiotic agents.

Place an extracted tooth follicle in a sterile Petri dish. Add 10 milliliters of PBSPSG solution to wash the extracted tissue. Aspirate and discard the solution and add fresh PBSPSG solution to repeat the wash.

Using a sterile scalpel mince the extracted follicle into pieces if about 1 by 1 millimeters. Transfer the minced tissue and PBSPSG solution from the Petri dish into a 50 milliliter conical centrifuge tube, wash the Petri dish with 10 milliliters of PBSPSG solution, and transfer the solution into the same tube. Centrifuge the conical tube at 353 x g at room temperature for ten minutes.

Add 5 milliliters of digestion solution to the palleted tissue, gently mix the solution and the tissue, incubate the mixture at 37 degrees celsius and 5%CO2 in a shaking incubator for two hours. After two hours, centrifuge the digested cell and tissue suspension at 353 g for ten minutes at room temperature. Discard the supernatant and re-suspend the obtained pallet in 6 milliliters of HDFSC culture medium.

Seed the cell suspension in a 25 square centimeter cell culture flask, and incubate the cells at 37 degrees celsius. After cell harvest and characterization of HDFSC is as described in the text protocol, seed hDFSCs in a 24 well cell culture plate. Incubate at 37 degrees celsius, 5%CO2 and 20%Oxygen for 24 hours.

On the following day dilute 40 picomoles of microRNA in 66.7 microliters of reduced serum medium, and vortex the solution. Dilute 0.67 microliters of cationic lipid based, transfection reagent in 66.7 microliters of reduced serum medium, vortex the solution, and incubate at room temperature for five minutes. After five minutes add the prediluted microRNA to the prediluted transfection reagent, mix the solution, and incubate at room temperature for fifteen minutes.

Next, add the prepared transfection complexities, dropwise, directly to the culture medium on cells. Mix gently by rocking the 24 well cell culture plate back and forth. Incubate the cells at 37 degrees celsius for 24 hours.

24 hours after transfection, collect the supernatant of samples in respective 15 milliliter conical centrifuge tubes. Wash the cells with 1 milliliter of PBS, and transfer the PBS into the respective centrifuge tube. Add 500 microliters of drips and EDTA to the cells, and incubate the 24 well plate at 37 degrees celsius, for 3 minutes.

Stop the trypsinization by adding 1 milliliter of cell culture medium for the cells, and transferring the solution into the respective centrifuge tube. Centrifuge the cells at 300 x g and 4 degrees celsius for ten minutes. From here on keep the cells and reagents on ice unless stated otherwise.

Discard the supernatant, resuspend the cells in 100 microliters of staining buffer, and transfer the cell solution to a 1.5 milliliter microcentrifuge tube. Add 0.5 milliliters of amine reactive dye to each sample in order to distinguish between live and dead cells. Gently mix the solution, and incubate at 4 degrees celsius for ten minutes.

Add 1 milliliter of PBS to the cells, and centrifuge at 300g and 4 degrees celsius for ten minutes Discard the supernatant and resuspend the cells in 100 microliters of PBS, add 33 microliters of paraformaldehyde, and mix the solutions, store the samples at 4 degrees celsius protected from light until flow cytometric measurements. Transfer the samples to tubes suitable for flow cytometric measurements, and perform flow cytometry using the gating strategy described in the text protocol. The isolated human dental follicle stem cells showed all characteristics described for the definition of mesenchymal stromal cells, cells were plastic adherent and displayed a fiberglass like morphology, under standard culture conditions.

Flow cytometric analysis revealed that hDFSCs expressed a panel of certain surface antigens, including CD29, CD44, CD73, CD90, and CD105. While CD45 and CD117 were absent. The adipogenic, osteogenic, and chondrogenic differentiation potentials of cells under specific In Vitro culture conditions was confirmed by immunostaining, a fatty acid binding protein for:Osteocalcin, and Aggrecan.

The gating strategy used for the quantification of cytotoxicity and Cy3 labeled microRNA uptake efficiency 24 hours post transfection, confirmed microRNA uptake in 100%of viable cells. Comparable amounts of dead cells in transfected and untransfected samples indicate that microRNA is introduced efficiently without cytotoxic effects. Following this procedure, other methods such as transdifferentiation can be performed in order to answer additional questions like Cytolab plasticity.