Name: isolation, characterization and microrna-based genetic modification of human dental follicle stem cells
Uploaded: 2018-11-16T14:00:00.000+00:00
Duration: 7 min 56 s
Description: Watch this Scientific Journal Video about Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells at JoVE.com

This work was supported by the FORUN Program of the Rostock University Medical Centre (889018) and the DAMP Foundation (2016-11). In addition, P.M. and R.D. are supported by the BMBF (VIP+ 00240).

HDFSCs are isolated from the dental follicles of extracted wisdom teeth provided by the Department of Oral and Maxillofacial Plastic Surgery of the Rostock University Medical Center. Informed consent and written approval was obtained from all patients. This study was authorized by the local ethics committee of the University of Rostock (Permission No. A 2017-0158).
1. Isolation of hDFSCs
NOTE: To prevent bacterial contamination, wisdom teeth should not be erupted before extraction
<ol>
	<li>Preparation of required solutions
	<ol>
		<li>Prepare phosphate-buffered saline (PBS)/Penicillin-Streptomycin-Glutamine (P-S-G) solution: mix 495 mL of PBS with 5 mL of P-S-G solution. Store aliquots of 50 mL at 4&#160;&#176;C.</li>
		<li>Prepare hDFSC culture medium: mix 445 mL of basal medium with 5 mL of antibiotic agent and 50 mL of fetal bovine serum (FBS). Store at 4&#160;&#176;C.</li>
		<li>Prepare Collagenase type I stock solution (30 mg/mL): dilute 300 mg of Collagenase type I in 10 mL of basal medium supplemented with 1% antibiotic agent. Vigorously mix the solution. Filter the solution using a 0.2 &#181;m filter. Store aliquots of 500 &#181;L of Collagenase type I stock solution at -20&#160;&#176;C.</li>
		<li>Prepare Dispase II stock solution (40 mg/mL): dilute 40 mg of Dispase II in 10 mL of basal medium supplemented with 1% antibiotic agent. Vigorously mix the solution. Filter the solution using a 0.2 &#181;m filter. Store aliquots of 500 &#181;L of Dispase II stock solution at -20&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Surgical procedure
	<ol>
		<li>Inject a sufficient amount (maximum: 2 mL) of local anesthetic.</li>
		<li>Create and raise a three-sided mucoperiosteal flap. Raising a lingual flap is not required.</li>
		<li>Protect the lingual aspect with a periosteal elevator.</li>
		<li>Gently mill buccal and distal bone with a round hard metal burr.</li>
		<li>Loosen the tissue of the follicle with a periosteal elevator. Carefully remove the complete tooth (germ) and follicle by pulling out the crown (and follicle) with posterior (third-molar) forceps.</li>
		<li>Debride and irrigate the socket thoroughly with NaCl solution.</li>
		<li>Replace the mucoperiosteal flap with vicryl sutures (see Table of Materials).</li>
		<li>Remove the tooth follicle from the oral cavity and place them into an aliquot of 50 mL of PBS/P-S-G solution. 
		NOTE: The sample can be stored at 4&#160;&#176;C until further processing.</li>
	</ol>
	</li>
	<li>Enzymatic digestion of the tooth follicle
	<ol>
		<li>Pre-warm hDFSC culture medium and PBS/P-S-G solution to room temperature (RT).</li>
		<li>Thaw one aliquot of each Collagenase type I and Dispase II stock solutions. Prepare a digestion solution of 3 mg/mL Collagenase type I and 4 mg/mL Dispase II by adding 500 &#181;L of each Collagenase type I and Dispase II stock solution to 4 mL of basal medium containing 1% antibiotic agent.</li>
		<li>Place extracted follicle in a sterile Petri dish and add 10 mL of PBS/P-S-G solution to wash the extracted tissue. Repeat this washing step twice.</li>
		<li>Mince the extracted follicle to pieces of about 1 mm x 1 mm with a sterile scalpel within the Petri dish containing 10 mL of PBS/P-S-G solution.</li>
		<li>Transfer minced tissue and PBS/P-S-G solution from the Petri dish into a 50 mL conical centrifuge tube. Wash the Petri dish with 10 mL of PBS/P-S-G solution and transfer the solution into the same tube.</li>
		<li>Centrifuge the conical centrifuge tube for 10 min at 353 x g at RT and discard the supernatant.</li>
		<li>Add 5 mL of digestion solution to the pelleted tissue. Gently mix the solution and the tissue. Incubate the mixture at 37&#160;&#176;C and 5% CO2 for 2 h in a shaking incubator.</li>
		<li>Centrifuge digested cell/tissue suspension at 353 x g for 10 min at RT. Discard the supernatant and re-suspend the obtained pellet in 6 mL of hDFSC culture medium.</li>
		<li>Seed cell suspension in a 25 cm2 cell culture flask and incubate cells at 37&#160;&#176;C, 5% CO2 and 20% O2. 
		NOTE: If tissue is not completely digested, transfer the remaining tissue to the cell culture flask as well.</li>
		<li>Change medium carefully 24 h after cell seeding. Afterwards change medium every three days until confluency. 
		NOTE: HDFSCs should be plastic-adherent 24 h after cell seeding and can be separated from non-adherent cells and blood components by simply changing the medium.</li>
	</ol>
	</li>
	<li>Cell harvesting
	<ol>
		<li>Pre-warm hDFSC culture medium, PBS/P-S-G solution and Trypsin/Ethylenediaminetetraacetic acid (EDTA) solution to RT.</li>
		<li>Discard the supernatant from culture flask and wash confluent cells with 5 mL of PBS/P-S-G solution.</li>
		<li>Add 1 mL of Trypsin/EDTA solution to the culture flask and incubate for 3 min at 37&#160;&#176;C. Stop the digestion process by adding 3 mL of hDFSC culture medium to the culture flask.</li>
		<li>Transfer cell suspension into a 15 mL conical centrifuge tube and centrifuge the suspension for 10 min at 353 x g at RT. Discard the supernatant and re-suspend the pellet in an appropriate amount of hDFSC culture medium.</li>
		<li>Count cells: gently mix 10 &#181;L of cell suspension with 10 &#181;L of Trypan blue solution. Apply 10 &#181;L into a counting chamber and calculate the hDFSC cell amount.</li>
	</ol>
	</li>
</ol>
2. Characterization of hDFSCs
<ol>
	<li>Immunophenotyping
	<ol>
		<li>Preparation of cells for flow cytometric analysis
		<ol>
			<li>Preparation of required solutions
			<ol>
				<li>Prepare PBS/EDTA (2 mM): mix 996 mL of PBS with 4 mL of EDTA (0.5 M).</li>
				<li>Prepare staining buffer: mix 995 mL of PBS/EDTA (2 mM) with 5 g of BSA. Filter the solution using a 0.22 &#181;m filter unit and store at 4&#160;&#176;C until usage.</li>
				<li>Prepare paraformaldehyde (PFA) stock solution (4%): dilute 4 g PFA in 100 mL of PBS and heat the solution to 80&#160;&#176;C. Mix the solution and adjust the pH value to 7.3. Aliquot obtained PFA solution in respective amounts (1.5 mL) and store at -20&#160;&#176;C until usage. 
				CAUTION: Because PFA fumes are toxic, prepare the PFA solution in a ventilated fume hood.</li>
			</ol>
			</li>
			<li>After cell harvesting (1.4), transfer 14 samples of 5 x 104 cells into 1.5 mL microcentrifuge tubes. Centrifuge the suspensions at 300 x g for 10 min at 4&#160;&#176;C. Discard the supernatant. 
			NOTE: Perform the following work in a rather shaded room. Keep cells and reagents on ice unless stated otherwise.</li>
			<li>Re-suspend cells in certain amounts of staining buffer (4&#160;&#176;C) and add FcR blocking reagent (4&#160;&#176;C) as indicated in Table 1.</li>
			<li>Add the following antibodies onto the inner side of the respective sample as indicated in Table 1: Allophycocyanin (APC) mouse anti-human CD29; APC mouse IgG1 &#954; isotype control; Peridinin-chlorophyll protein (PerCP)-Cyanine5.5 mouse anti-human CD44; PerCP-Cyanine5.5 mouse IgG2b &#954; isotype control; V500 mouse anti-human CD45; V500 mouse IgG1 &#954; isotype control; Phycoerythrin (PE) mouse anti-human CD73; PE mouse IgG1 &#954; isotype control; PerCP-Cyanine5.5 mouse anti-human CD90; PerCP-Cyanine5.5 mouse IgG1 &#954; isotype control; mouse anti-human CD105: Alexa Fluor (AF)488; mouse IgG1 negative control: AF488; PE-Cyanine7 mouse anti-human CD117; PE-Cyanine7 mouse IgG1, &#954; isotype control. After antibodies have been added to each sample, spin down antibodies and mix gently. Incubate the solutions for 10 min at 4&#160;&#176;C.</li>
			<li>Add 1 mL of PBS (4&#160;&#176;C) and centrifuge the samples at 300 x g for 10 min at 4&#160;&#176;C. Discard the supernatant.</li>
			<li>Re-suspend cells in 100 &#181;L of PBS (4&#160;&#176;C) and add 33 &#181;L of PFA (4%). Mix the solutions and store on ice or at 4&#160;&#176;C until flow cytometric analysis.</li>
		</ol>
		</li>
		<li>Flow cytometric measurement of cells 
		NOTE: Perform the following work in a rather shaded room. Keep cells on ice until measurement.
		<ol>
			<li>To examine the expression of surface antigens, transfer samples into tubes suitable for flow cytometric measurements.</li>
			<li>Measure at least 2 x 104 events using a flow cytometer. Analyze as depicted in Figure 2.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Multipotent differentiation potential of hDFSCs
	<ol>
		<li>Use a commercially available Human Mesenchymal Stem Cell Functional Identification Kit to confirm adipogenic, osteogenic and chondrogenic differentiation potential of hDFSCs. Apply donkey anti-goat AF488 secondary antibody for staining of fatty acid binding protein 4 (FABP4) and aggrecan as well as donkey anti-mouse AF488 secondary antibody for staining of osteocalcin. For staining of nuclei, use mounting medium with 4&#39;,6-Diamidino-2-phenylindole (DAPI). 
		NOTE: Prepare samples without primary antibody as negative controls for microscopic analyses.</li>
		<li>Analyse expression of proteins by laser scanning confocal microscopy. Microscopy settings: 40x objective with oil immersion; excitation laser 488 nm (for AF488) and 405 nm (for DAPI).</li>
	</ol>
	</li>
</ol>
3. Transfection of hDFSCs
<ol>
	<li>After cell harvesting (1.4), seed hDFSCs on a 24-well cell culture plate 24 h prior to transfection. 
	NOTE: Starting cell density is approximately 4 x 104 cells per well. Cells should reach ~80% confluence on day of transfection. 
	Note: Seed one additional cell sample as control for flow cytometric analysis.</li>
	<li>Preparation of transfection complexes 
	NOTE: To prevent contamination from RNases, clean the workspace directly before preparation of transfection complexes using RNase decontamination solution. Use only RNase-free material and solutions. 
	NOTE: Perform the following work in a rather shaded room.
	<ol>
		<li>Prepare miR stock solution (50 &#181;M): re-suspend Cy3-labelled precursor miR (5 nmol) in 100 &#181;L nuclease-free water. Aliquot obtained miR stock solution in respective amounts (5 &#181;L) and store in the dark at -20&#160;&#176;C until usage.</li>
		<li>Dilute 40 pmol of miR (0.8 &#181;L of the 50 &#181;M miR stock solution) in 66.7 &#181;L of reduced serum medium. Mix the solution gently</li>
		<li>Dilute 0.67 &#181;L of cationic lipid-based transfection reagent in 66.7 &#181;L of reduced serum medium. Mix the solution gently and incubate for 5 min at RT.</li>
		<li>After incubation, add the pre-diluted transfection reagent to the pre-diluted miR. Mix the solution gently and incubate for 15 min at RT.</li>
	</ol>
	</li>
	<li>Add the prepared transfection complexes dropwise directly to the culture medium on cells. Mix gently by rocking the 24-well cell culture plate back and forth.</li>
	<li>Incubate the cells at 37&#160;&#176;C, 5% CO2, and 20% O2 for 24 h.</li>
</ol>
4. Analysis of Transfection
NOTE: Perform the following work in a rather shaded room.
<ol>
	<li>Cell harvesting after transfection 
	NOTE: Collect all cell solutions of one sample in the same 15 mL conical centrifuge tube.
	<ol>
		<li>24 h after transfection, collect the supernatant of samples in respective centrifuge tubes.</li>
		<li>Wash cells with 1 mL of PBS, transfer PBS into the respective centrifuge tube and add 500 &#181;L Trypsin/EDTA (RT) to cells. Incubate the solutions for 3 min at 37&#160;&#176;C.</li>
		<li>Stop trypsinization by adding 1 mL of culture medium (RT) to the cells and transfer the solution into the respective centrifuge tube. Centrifuge cells at 300 x g for 10 min at 4&#160;&#176;C. 
		NOTE: From this time, keep cells and reagents on ice unless stated otherwise.</li>
	</ol>
	</li>
	<li>Preparation of cells for flow cytometric analysis
	<ol>
		<li>Discard the supernatant. Re-suspend cells in 100 &#181;L of staining buffer (4&#160;&#176;C) and transfer cell solution to a 1.5 mL microcentrifuge tube.</li>
		<li>Add 0.5 &#181;L of amine reactive dye to the samples in order to distinguish between live and dead cells. Gently mix the solution and incubate for 10 min at 4&#160;&#176;C.</li>
		<li>Add 1 mL of PBS (4&#160;&#176;C) to cells and centrifuge at 300 x g for 10 min at 4&#160;&#176;C. Discard the supernatant.</li>
		<li>Re-suspend cells in 100 &#181;L of PBS (4&#160;&#176;C) and add 33 &#181;L of PFA (4%). Mix the solutions and store on ice or at 4&#160;&#176;C until flow cytometric analysis.</li>
	</ol>
	</li>
	<li>Flow cytometric measurement of cells 
	NOTE: Perform the following work in a rather shaded room. Keep cells on ice until measurement.
	<ol>
		<li>Transfer samples into tubes suitable for flow cytometric measurements.</li>
		<li>Examine cell viability and miR uptake efficiency using a flow cytometer. Measure at least 2 x 104 events. Use the gating strategy depicted in Figure 4. 
		NOTE: Use untransfected cells as negative control to arrange the gating for Cy3 positive cells and to calculate cell death caused by transfection.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Adult stem cells are currently in focus for the treatment of several degenerative diseases. In particular, bone marrow (BM)-derived stem cells, including hematopoietic stem cells (HSCs) and MSCs, are under intensive clinical investigation47. However, BM harvesting is an invasive procedure causing pain at the site of donation and may lead to adverse events48. Recently, the postnatal dental tissue has emerged as a novel and easily accessible source for stem cells. These denta...

The human dental follicle is a loose ectomesenchymally-derived connective tissue surrounding the developing tooth1,2. Beside its function to coordinate osteoclastogenesis and osteogenesis for the tooth eruption process, this tissue harbors stem and progenitor cells especially for the development of the periodontium3,4,5. Therefore, the dental follicle is considered as an alternative source to harvest human adult stem cells6,7.
Several studies demonstrated that human dental follicle stem cells (hDFSCs) are capable of differentiating into the periodontal lineage including osteoblasts, ligament fibroblasts and cementoblasts8,9,10. Furthermore, these cells were shown to match all characteristics of mesenchymal stromal cells (MSCs) including self-renewing capacity, plastic adherence, expression of specific surface markers (e.g., CD73, CD90, CD105) as well as osteogenic, adipogenic and chondrogenic differentiation potential11,12,13. Other studies also revealed a neural differentiation potential of hDFSCs2,14,15,16,17,18.
Due to their promising properties and easy access, hDFSCs became recently relevant for tissue engineering19,20,21. The first studies concentrated on the potential of DFSCs to regenerate bone, periodontal and tooth roots19,22,23,24,25,26,27,28,29,30. Since the knowledge of the neurogenic capability of hDFSCs, their application as potential treatment for neurodegenerative diseases has been investigated31,32,33. HDFSCs have also gained importance with respect to the the regeneration of other tissues (e.g., corneal epithelium)34,35. The therapeutic potential of hDFSC is not only based on their direct differentiation potential but also on their paracrine activity. Recently, hDFSCs have been shown to secrete a wealth of bioactive factors, such as matrix metalloproteinases (MMPs), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF), which play a crucial role for angiogenesis, immunomodulation, extra cellular matrix remodeling and reparative processes36.
However, broad clinical translation of stem cell therapy is still impaired by several challenges, such as massive initial cell death and low beneficial stem cell effects37,38. Genetic engineering provides a promising strategy to address these challenges and therefore can greatly enhance the therapeutic efficacy of stem cells38,39,40. For transient cell manipulation, microRNAs (miRs) are suitable candidates, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration41,42,43. To date, several beneficial miRs have been identified promoting stem cell proliferation, survival, homing, paracrine activity as well as their differentiation into several lineages44. For instance, miR-133a engineered MSCs showed an increased survival and engraftment in infarcted rat hearts resulting in an improved cardiac function when compared to unmodified MSCs45. Likewise, miR-146a overexpressing MSCs were shown to secrete higher amounts of VEGF which in turn led to an enhanced therapeutic efficiency in ischemic tissue46.
This manuscript presents a detailed protocol for the selective extraction and genetic engineering of hDFSCs. For this purpose, we described the harvesting and enzymatic digestion of human dental follicles as well as the subsequent isolation of hDFSCs. In order to characterize isolated cells, important instructions for the verification of MSC properties have been included in accordance with the guidelines of the International Society for Cellular Therapy13. In addition, we provide a detailed description for the generation of miR-modified hDFSCs by applying a cationic lipid-based transfection strategy and the evaluation of transfection efficiency and cytotoxicity.

<table><tbody><tr><td>Mouse anti Human CD105 Antibody: Alexa Fluor 488</td><td>Bio-Rad</td><td>MCA1557A488</td><td>Clone SN6, monoclonal</td></tr><tr><td>Mouse IgG1 Negative Control Antibody: Alexa Fluor 488</td><td>Bio-Rad</td><td>MCA928A488</td><td>monoclonal</td></tr><tr><td>APC Mouse Anti-Human CD29 Antibody</td><td>BD Biosciences</td><td>559883</td><td>Clone MAR4, monoclonal</td></tr><tr><td>APC Mouse IgG1, &amp;kappa; Isotype Control Antibody</td><td>BD Biosciences</td><td>555751</td><td>Clone MOPC-21, monoclonal</td></tr><tr><td>PE Mouse Anti-Human CD73 Antibody</td><td>BD Biosciences</td><td>550257</td><td>Clone AD2, monoclonal</td></tr><tr><td>PE Mouse IgG1, &amp;kappa; Isotype Control Antibody</td><td>BD Biosciences</td><td>555749</td><td>Clone MOPC-21, monoclonal</td></tr><tr><td>PE-Cy7 Mouse Anti-Human CD117 Antibody</td><td>BD Biosciences</td><td>339217</td><td>Clone 104D2, monoclonal</td></tr><tr><td>PE-Cy7 Mouse IgG1, &amp;kappa; Isotype Control Antibody</td><td>BD Biosciences</td><td>557872</td><td>Clone MOPC-21, monoclonal</td></tr><tr><td>PerCP-Cy5.5 Mouse Anti-Human CD44 Antibody</td><td>BD Biosciences</td><td>560531</td><td>Clone G44-26, monoclonal</td></tr><tr><td>PerCP-Cy5.5 Mouse IgG2b, &amp;kappa; Isotype Control Antibody</td><td>BD Biosciences</td><td>558304</td><td>Clone 27-35, monoclonal</td></tr><tr><td>PerCP-Cy5.5 Mouse Anti-Human CD90&amp;nbsp;Antibody</td><td>BD Biosciences</td><td>561557</td><td>Clone 5E10, monoclonal</td></tr><tr><td>PerCP-Cy5.5 Mouse IgG1, &amp;kappa; Isotype Control Antibody</td><td>BD Biosciences</td><td>55095</td><td>Clone MOPC-21, monoclonal</td></tr><tr><td>V500 Mouse Anti-Human CD45 Antibody</td><td>BD Biosciences</td><td>560777</td><td>Clone HI30, monoclonal</td></tr><tr><td>V500 Mouse IgG1, &amp;kappa; Isotype Control Antibody</td><td>BD Biosciences</td><td>560787</td><td>Clone X40, monoclonal</td></tr><tr><td>FcR Blocking Reagent, human</td><td>Miltenyi Biotec</td><td>130-059-901</td><td></td></tr><tr><td>UltraPure EDTA</td><td>Thermo Fisher Scientific</td><td>15575-020</td><td>0.5M, pH 8.0</td></tr><tr><td>Steritop</td><td>Merck Millipore</td><td>SCGPT05RE</td><td>0.22 &amp;micro;m, radio-sterilized, polyethersulfone</td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>PFA</td><td>Merck Millipore</td><td>1040051000</td><td></td></tr><tr><td>Human Mesenchymal Stem Cell Functional Identification Kit</td><td>R&amp;amp;D Systems</td><td>SC006</td><td></td></tr><tr><td>RNase decontamination solution; RNaseZap RNase Decontamination Solution</td><td>Thermo Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>Cy3-labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1</td><td>Thermo Fisher Scientific</td><td>AM17120</td><td>5 nmol</td></tr><tr><td>Pre-miR miRNA Precursor Negative Control #1</td><td>Thermo Fisher Scientific</td><td>AM17110</td><td>5 nmol</td></tr><tr><td>Cationic lipid-based transfection reagent; Lipofectamine 2000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>Reduced serum medium; Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-11055</td><td>polyclonal</td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-21202</td><td>polyclonal</td></tr><tr><td>Mounting medium; Fluoroshield with DAPI</td><td>Sigma-Aldrich</td><td>F6057-20ML</td><td>histology mounting medium</td></tr><tr><td>ELYRA PS.1 LSM 780 confocal microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>BD FACS LSRII flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSDiva Software 6.1.2</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>ZEN2011 software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Trypsin/EDTA solution (0.05%/ 0.02%)</td><td>Biochrom</td><td>L2143</td><td>in PBS, w/o: Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>Amine reactive dye; LIVE/DEAD&amp;trade; Fixable Near-IR Dead Cell Stain Kit</td><td>Thermo Fisher Scientific</td><td>L10119</td><td></td></tr><tr><td>PBS (1x)</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>pH: 7.4; w/o: Ca and Mg</td></tr><tr><td>P-S-G (100x)</td><td>Thermo Fisher Scientific</td><td>10378016</td><td></td></tr><tr><td>Basal medium; Dulbecco&#039;s Modified Eagle Medium/Nutrient Mixture F-12</td><td>Thermo Fisher Scientific</td><td>11039021</td><td></td></tr><tr><td>Antibiotic, ZellShield</td><td>Biochrom</td><td>W 13-0050</td><td></td></tr><tr><td>FBS</td><td>Thermo Fisher Scientific</td><td>10500064</td><td></td></tr><tr><td>Collagenase type I</td><td>Thermo Fisher Scientific</td><td>17100017</td><td></td></tr><tr><td>Dispase II</td><td>Thermo Fisher Scientific</td><td>17105041</td><td></td></tr><tr><td>Filter, Sterifix syringe filter 0.2 &amp;micro;m</td><td>Braun</td><td>4099206</td><td></td></tr><tr><td>50&amp;nbsp;mL conical centrifuge tube</td><td>Sarstedt</td><td>62,547,254</td><td></td></tr><tr><td>15 mL conical centrifuge tube</td><td>Sarstedt</td><td>62,554,502</td><td></td></tr><tr><td>Cell culture flask 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Sarstedt</td><td>833,910,002</td><td></td></tr><tr><td>Cell culture flask, 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Sarstedt</td><td>833,911,002</td><td></td></tr><tr><td>Freezing medium, Biofreeze</td><td>Biochrom</td><td>F 2270</td><td></td></tr><tr><td>Cryotubes</td><td>Thermo Fisher Scientific</td><td>377267</td><td>1.8 mL</td></tr><tr><td>Trypan blue solution</td><td>Sigma-Aldrich</td><td>T8154</td><td>0.4 %</td></tr><tr><td>Counting chamber</td><td>Paul Marienfeld</td><td></td><td></td></tr><tr><td>Local anesthetic, Xylocitin (lidocaine hydrochloride) 2% with epinephrine (adrenaline) 0.001%</td><td>Mibe</td><td></td><td></td></tr><tr><td>NaCl solution</td><td>Braun</td><td></td><td>0.9 %</td></tr><tr><td>Vicryl satures, Vicryl rapide</td><td>Ethicon</td><td></td><td>3 - 0</td></tr></tbody></table>

isolation, characterization and microrna-based genetic modification of human dental follicle stem cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

Here, we present a detailed isolation instruction to harvest hDFSCs from human dental follicle tissue. Due to the easy access of the dental follicle during routine surgery, it is a promising source for the extraction of adult stem cells.
The isolated hDFSCs showed all characteristics described for the definition of MSCs13. In fact, cells were plastic-adherent under described culture conditions and display...

Watch this Scientific Journal Video about Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells at JoVE.com

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

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

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JoVE Journal

Genetics

6.7K Views.  Rostock University Medical Center. This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

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

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Developmental Biology

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Medicine

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very important to obtain adult stem cells from different tissues such as adipose, umbilical cord, bone marrow and blood. Among the possible sources, dental pulp is particularly interesting because it is easy to obtain in respect of bioethical considerations. Indeed, human Dental Pulp Stem Cells (hDPSCs) are a type of adult stem cells able to differentiate in neuronal-like cells and can be obtained from the third molar of healthy patients (13-19 ages). In particular, the dental pulp was removed with an excavator, cut into small slices, treated with collagenase IV and cultured in a flask. To induce the neuronal differentiation, hDPSCs were stimulated with EGF/bFGF for 2 weeks. Previously, we have demonstrated that during the differentiation process the content of cellular prion Protein (PrPC) in hDPSCs increased. The cytofluorimetric analysis showed an early expression of PrPC that increased after neuronal differentiation process. Ablation of PrPC by siRNA PrP prevented neuronal differentiation induced by EGF/bFGF. In this paper, we illustrate that as we enhanced the isolation, separation and in vitro cultivation methods of hDPSCs with several easy procedures, more efficient cell clones were obtained and large-scale expansion of the mesenchymal stem cells (MSCs) was observed. We also show how the hDPSCs, obtained with methods detailed in the protocol, are an excellent experimental model to study the neuronal differentiation process of MSCs and subsequent cellular and molecular processes.

Here we present a protocol for human Dental Pulp Stem Cells isolation and propagation in order to evaluate the prion protein expression during neuronal differentiation process.

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Biology

Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which are present in both deciduous and permanent teeth, have emerged as a vital stem cell source due to their accessibility, adaptability, and innate differentiation capabilities. DPSCs offer a readily available and abundant reservoir of mesenchymal stem cells, showcasing impressive versatility and potential, particularly for regenerative purposes. Despite their promise, the main hurdle lies in effectively isolating and characterizing DPSCs, given their representation as a minute fraction within dental pulp cells. Equally crucial is the proper preservation of this invaluable cellular resource. The two predominant methods for DPSC isolation are enzymatic digestion (ED) and outgrowth from tissue explants (OG), often referred to as spontaneous growth. This protocol concentrates primarily on the enzymatic digestion approach for DPSC isolation, intricately detailing the steps encompassing extraction, in-lab processing, and cell preservation. Beyond extraction and preservation, the protocol delves into the differentiation prowess of DPSCs. Specifically, it outlines the procedures employed to induce these stem cells to differentiate into adipocytes, osteoblasts, and chondrocytes, showcasing their multipotent attributes. Subsequent utilization of colorimetric staining techniques facilitates accurate visualization and confirmation of successful differentiation, thereby validating the caliber and functionality of the isolated DPSCs. This comprehensive protocol functions as a blueprint encompassing the entire spectrum of dental pulp stem cell extraction, cultivation, preservation, and characterization. It underscores the substantial potential harbored by DPSCs, propelling forward stem cell exploration and holding promise for future regenerative and therapeutic breakthroughs.

This protocol emphasizes the extraction, culture, and preservation of multipotent stem cells from dental pulp through enzymatic digestion. Additionally, it demonstrates their potential to differentiate into osteoblasts, adipocytes, and chondrocytes, highlighting the importance of precision and consistency in the process.

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which ...

Bioengineering

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...

Establishing the Primary Culture of Dental Pulp Stem Cells

Primary Culture of Dental Pulp Stem Cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

Author Spotlight: Advancing Tissue Regeneration and Disease Modeling with Dental Pulp Stem Cells

This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an ...