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 dental stem cells were shown to meet all MSC characteristics and showed higher proliferation capacity as BM-derived stem cells49. Here, we presented a detailed protocol for the extraction, characterization and engineering of hDFSCs.
The described isolation procedure has been developed on human dental follicles of impacted wisdom teeth, as this tissue is commonly extracted and disposed of as medical waste19. Nevertheless, other dental tissues, including dental pulp50,51, periodontal ligament52, exfoliated deciduous teeth53, and root apical papilla54, can be utilized for the extraction of dental stem cells.
Genetic engineering of stem cells by inserting miRs is a novel strategy to overcome certain difficulties in stem cell-based therapies, such as low stem cell survival43,55,56,57. This protocol presented crucial instructions for the efficient introduction of synthetic miR into hDFSCs using a commercially available cationic lipid-based transfection reagent. The application of cationic liposomal formulations for the delivery of therapeutic reagents, such as drugs and nucleic acids, has been investigated in numerous clinical trials58,59. However, cationic liposomes are potentially cytotoxic in a dose-dependent manner by causing e.g., damage to the integrity of the cell membrane or alterations in gene expression59,60,61. Therefore, particular attention must be paid to toxic effects on cells induced by transfection.
Notably, indicated transfection conditions have been optimized for miR-mediated genetic modification of hDFSCs in respect of efficiency and cytotoxicity. Nevertheless, other studies demonstrated the successful application of this transfection reagent for the delivery of additional nucleic acids, including plasmid DNA, mRNA and siRNA, into different cell types62,63,64,65,66,67,68. Results of these studies revealed that ideal delivery conditions varied significantly and have to be defined for each cell type.

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 displayed a fibroblast-like morphology (Figure 1). Flow cytometric analyses revealed that hDFSCs expressed a panel of certain surface antigens, including CD29, CD44, CD73, CD90 and CD105, while CD45 and CD117 were absent (Figure 2). Moreover, the adipogenic, osteogenic and chondrogenic differentiation potential of cells under specific in vitro culture conditions was confirmed by immunostaining of fatty acid binding protein 4 (FABP4), osteocalcin and aggrecan (Figure 3).
The described cationic lipid-based transfection strategy enabled efficient transient genetic modification of hDFSCs with a miR-uptake in ~100% of viable cells 24 h post-transfection (Figure 4B). Moreover, transfected (Figure 4A) and untransfected (Figure 4C) samples showed comparable amounts of dead cells proving gentle cell processing conditions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58089/58089fig1.jpg" /> 
Figure 1: Representative light microscope picture of hDFSCs. Cells show a fibroblast-like morphology under standard culture conditions.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58089/58089fig2.jpg" /> 
Figure 2: Representative flow cytometric immunophenotyping of hDFSCs. Flow cytometric analysis of cells after staining for specific cell surface markers (blue). Corresponding isotype controls were used as negative controls (grey). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58089/58089fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58089/58089fig3.jpg" /> 
Figure 3: Representative verification of the adipogenic, osteogenic and chondrogenic differentiation potential of hDFSCs. After differentiation, adipocytes, osteocytes and chondrocytes were identified by immunostaining of (A) fatty acid binding protein 4 (FABP4) (green), (B) osteocalcin (green) and (C) aggrecan (green). Nuclei were stained with DAPI (blue). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58089/58089fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58089/58089fig4.jpg" /> 
Figure 4: Representative gating strategy for analysis of transfection. Schematic representation of gating strategy used for the quantification of (A) cytotoxicity (blue: dead cells) and (B) Cy3-labeled miR uptake efficiency (red: Cy3+ cells) 24 h post-transfection. Untransfected cells (C,D) were used as control.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>MACS</td>
			<td>FcR</td>
			<td colspan="7">Antibodies [&#181;L]</td>
			<td colspan="7">Isotype controls [&#181;L]</td>
		</tr>
		<tr>
			<td>Sample</td>
			<td>buffer</td>
			<td>blocking</td>
			<td>CD29</td>
			<td>CD44</td>
			<td>CD45</td>
			<td>CD73</td>
			<td>CD90</td>
			<td>CD105</td>
			<td>CD117</td>
			<td>APC</td>
			<td>PerCP-Cy5.5</td>
			<td>V500</td>
			<td>PE</td>
			<td>PerCP-Cy5.5</td>
			<td>AF488</td>
			<td>PE-Cy7</td>
		</tr>
		<tr>
			<td></td>
			<td>[&#181;l]</td>
			<td>reagent [&#181;l]</td>
			<td>APC</td>
			<td>PerCP-Cy5.5</td>
			<td>V500</td>
			<td>PE</td>
			<td>PerCP-Cy5.5</td>
			<td>AF488</td>
			<td>PE-Cy7</td>
			<td>IgG1</td>
			<td>IgG2b</td>
			<td>IgG1</td>
			<td>IgG1</td>
			<td>IgG1</td>
			<td>IgG1</td>
			<td>IgG1</td>
		</tr>
		<tr>
			<td>1</td>
			<td>30</td>
			<td>10</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>37.5</td>
			<td>10</td>
			<td></td>
			<td>2.5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3</td>
			<td>37.5</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td>2.5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4</td>
			<td>30</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5</td>
			<td>35</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6</td>
			<td>35</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7</td>
			<td>37.5</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>2.5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8</td>
			<td>30</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>9</td>
			<td>30</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10</td>
			<td>37.5</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>2.5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>11</td>
			<td>30</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12</td>
			<td>40</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>13</td>
			<td>30</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>5</td>
			<td></td>
		</tr>
		<tr>
			<td>14</td>
			<td>37.5</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>2.5</td>
		</tr>
	</tbody>
</table>
Table 1: Pipetting layout for immunophenotyping of hDFSCs.

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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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

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

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

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Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells