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 no...

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, &#954; 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, &#954; 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, &#954; 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, &#954; 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&#160;Antibody</td>
			<td>BD Biosciences</td>
			<td>561557</td>
			<td>Clone 5E10, monoclonal</td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5 Mouse IgG1, &#954; 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, &#954; 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 &#181;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&#38;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>5nmol</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: Ca2+, Mg2+</td>
		</tr>
		<tr>
			<td>Amine reactive dye; LIVE/DEAD&#8482; 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&#39;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 &#181;m</td>
			<td>Braun</td>
			<td>4099206</td>
			<td></td>
		</tr>
		<tr>
			<td>50&#160;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 cm2</td>
			<td>Sarstedt</td>
			<td>833,910,002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask, 25 cm2</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 ...

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.

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Genetics

6.7K visualizações.  Rostock University Medical Center. Este método pode ajudar a responder a perguntas-chave no campo da Medicina Regenerativa, como onde células-tronco de folículos dentários humanos poderiam ser geneticamente modificadas para melhorar suas propriedades terapêuticas. A principal vantagem dessa técnica é que as células-tronco podem ser modificadas pela introdução de microRNA sem o risco de integração estável do genoma. Inicie o procedimento para digestão enzimática dos dois folículos, pré-aquecimento à temperatur...