Name: Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods (Video) | JoVE Journal
Uploaded: 2012-11-24T14:00:00.000+00:00
Duration: 14 min 52 s
Description: Learn to isolate and characterize human dental pulp stem cells from permanent teeth using enzymatic dissociation and direct outgrowth methods. Compare their differentiation into odontoblasts for regenerative dental research.

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5 minutes

We gratefully acknowledge Dr. Leila Shakeri &amp; Dr. Aref Dournaei for their critical discus and Mr. Mohammad Reza Khadem Sharif for his technical supports.

1. Prepare the Enzyme Solution and Proliferation Medium (PM)
<ol>
 <li>Make Collagenase Type I Solution: Weigh out collagenase type I (12 mg/ml) and dissolve in 1 ml PBS and filter using a 0.2 &mu;m syringe filter. Then place it 15 ml tube and keep it at -20 &deg;C until needed.</li>
 <li>Make dispase Solution: Weigh out dispase (16 mg/ml) and dissolve in 1 ml PBS and filter using a 0.2 &mu;m syringe filter. Then place it 15 ml tube and keep it at 4 &deg;C until needed. </li>
 <li>Make enzyme solution: Add 1 ml collagenase type I solutions (12 mg/ml) and 1 ml dispase solutions (16 mg/ml) into the 2 ml sterile PBS containing 100 mg/ml penicillin, 100 mg/ml streptomycin. Total concentration of dispase and Collagenase I in final volume should be 4 mg/ml and 3 mg/ml, receptively. Then, aliquot this volume in to four 15 ml tube, each containing 1 ml enzyme solution. Each tube could be used for one pulp digestion. </li>
 <li>Make washing solution: Add 100 mg/ml penicillin, 100 mg/ml streptomycin into PBS. </li>
 <li>Make basic media: Alpha modification of Eagle's medium (&alpha;-MEM) supplemented with 10% FBS &amp; 100 units/ml penicillin, 100 mg/ml streptomycin.</li>
 <li>Make the Proliferation Media (PM): Alpha modification of Eagle's medium (&alpha;-MEM) supplemented with 10% FBS, 100 &mu;M L-ascorbic acid 2-phosphate, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin B. </li>
 <li>Make Odontogenic media: Alpha modification of Eagle's medium (&alpha;-MEM) supplemented with 10% FBS, 100 &mu;M L-ascorbic acid 2-phosphate, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.01 &mu;M Dexamethasone, 5 mM &beta;-Glycerol phosphate, 1.8 mM Monopotassium phosphate. </li>
</ol>
2. Prepare Human Dental Pulp Tissue for Dental Pulp Stem Cell Isolation
<ol>
 <li>Normal human third molars were collected from adults (21-29 years of age) at the Dental Clinic of the Imam Ali under approved Institutional Review Board (IRB). </li>
 <li>Teeth were placed into the basic medium (&alpha;-MEM supplemented with 10% FBS) and were transferred into laboratory at 4 &deg;C. </li>
 <li>Under the sterile condition, working within a biohazard laminar flow hood, set-up was done one 100 mm Petri dishes for each tooth to be processed. Tooth surfaces were cleaned by 70% ethanol. </li>
 <li>While working in one of the Petri-dishes, tooth was kept by dental forceps and a scalpel blade was used to clean off debris and periodontal ligament on the roots. </li>
 <li>Cemento-enamel junction was slowly cut by using sterilized dental disc to reveal the pulp chamber. It should be considered that cutting process must be performed slowly to reduce overheating of the dental tissue. </li>
 <li>Pulp tissue was gently separated from the crown and root. </li>
 <li>Pulp tissue was cut into small pieces with the aid of a scalpel blade. </li>
</ol>
3. Human Dental Pulp Isolation
<ol>
 <li>Pulp tissues underwent either enzymatic dissociation of pulp tissue or direct cell outgrowth from pulp tissue explants for isolation of hDPSCs. </li>
 <li>Enzymatic dissociation of pulp tissue: 
 
 <ol>
 <li>Small pieces of pulp tissues were transferred into Enzyme solution for 1 hr at 37 &deg;C. Vortex every 30 min to help breaking up tissue.</li>
 <li>Afterwards, large cell aggregates were removed and Single-cell suspensions were obtained by passing cells through a 70-&mu;M cell strainer.</li>
 <li>Single-cell suspensions were centrifuged at 1,200 rpm for 5 min at room temperature.</li>
 <li>Supernatants were carefully pipetted off and pellet was re-suspended in 1 ml proliferation medium (PM). (Note: FBS in proliferation medium terminate enzymatic dissociation.) </li>
 <li>Single-cell suspensions of dental pulp was seeded into 25 cm2 culture flask with PM and then incubated at 37 &deg;C in 5% CO2. </li>
 <li>The culture medium was changed every three days until the cell confluency was achieved. </li>
 </ol>
 </li>
 <li>Direct cell outgrowth from pulp tissue explants:
 <ol>
 <li>Pulp tissue was minced into 1-2-mm fragments, and each piece was placed in 25-cm2 culture flasks with PM and then incubated at 37 &deg;C in 5% CO2. It should be considered that total volume of the PM for outgrowth of cell must support the attachment of pulp pieces for further cell outgrowth (2-3 ml per flask). </li>
 <li>Medium was changed after outgrowth was observed. </li>
 <li>The outgrown cells at confluence were sub-cultured at ratio of 1:4 (passage 1). </li>
 </ol>
 </li>
</ol>
4. Immunophenotyping
<ol>
 <li>250,000 cells/tube of both types of cells in passage 3 were incubated by PE or FITC-conjugated anti-human antibodies with their isotypes for 30 min at 4 &deg;C in dark place. (Note: the concentration of antibodies was applied according to the manufacture protocol.) </li>
 <li>After the incubation time, 100 &mu;l PBS or FACS dilution solution was added to each tube and centrifuged at 1,200 rpm for 5 min in dark place. </li>
 <li>Supernatants were removed &amp; pellets were re-suspended in 100 &mu;l PBS or FACS dilution in dark place. </li>
 <li>Targeted surface markers (CD markers) were evaluated by BD FACSCalibur. </li>
</ol>
5. Induction of Differentiation of DPSCs into Mineralized Cells &amp; Quantified Alizarin Red S Assay 
<ol>
 <li>DPSCs that were obtained by either enzymatic dissociation of pulp tissue (known as DPSC-ED) or direct cell outgrowth from pulp tissue explants (DPSC-OG) were sub-cultured for 3 passages. </li>
 <li>At Passage 3, cells were seed into a 6 well plate at 5,000 cell/well. </li>
 <li>At confluence of 60%, odontogenic medium were replaced by conventional PM. Three wells were remained as a negative control by adding growth medium. </li>
 <li>Medium change every 3 days until day 21. </li>
 <li>At day 21, Cells were washed with PBS and were fixed by 1 ml/well 10% paraformaldehyde for 15 min at room temperature. </li>
 <li>Then paraformaldehyde was pipetted off, carefully and cells were washed 3 times (5-10 min for each time) with distilled H2O. (Note: Avoid disturbing the monolayer.)</li>
 <li>After washing, 1 ml/well Alizarin red S were added for 20 min at room temperature. </li>
 <li>Cells were washed by deionized H2O four times (5 min for each time with gentle rocking) to remove any additional Alizarin red.</li>
 <li>1 ml/well H2O were added to prevent the cells from drying.</li>
 <li>The plates were assumed to visual inspection and/or image acquisition.</li>
 <li>For Alizarin Red S quantification assay, H2O were replaced by 1 ml 10% acetic acid to each well of 6-well plate &amp; incubate for 30 min at room temperature with shaking. </li>
 <li>Then, cell monolayer/well were scrape &amp; both acetic acid &amp; cells were transferred into the separate 1.5 ml micro-centrifuge tubes. (Note: each well should be transfer into a separate tube i.e. three test wells &amp; three control wells for each ED &amp; OG groups.)</li>
 <li>Tubes were vortexed vigorously for 30 sec. </li>
 <li>Heat to 85 &deg;C for 10 min. (Note: to avoid evaporation, tubes were sealed with parafilm.)</li>
 <li>After heating, tubes were transferred into ice for 5 min. (Note: Tubes should not be opened until become cooled.)</li>
 <li> The slurries were centrifuged at 20,000 x g for 15 min. While centrifuging, Alizarin Red standards were made up.</li>
 </ol>
 Alizarin Red S standard were prepared by first diluting the dilution buffer &amp; used for making 2-fold serial dilutions based on the table. 
 <table border="1">
 <tbody>
 <tr>
 <td colspan="7">High Range concentration of dye </td>
 <td colspan="7">Low Range concentration of dye </td>
 </tr>
 <tr>
 <td>2 mM</td>
 <td>1 mM</td>
 <td>500 &mu;M</td>
 <td>250 &mu;M</td>
 <td>125 &mu;M</td>
 <td>62.5 &mu;M</td>
 <td>31.3 &mu;M</td>
 <td>15.6 &mu;M</td>
 <td>7.8 &mu;M</td>
 <td>3.9 &mu;M</td>
 <td>1.9 &mu;M</td>
 <td>0.9 &mu;M</td>
 <td>0.4 &mu;M</td>
 <td>Blank</td>
 </tr>
 </tbody>
 </table>
 <ol start="16">
 <li>After the centrifugation, 1 ml of supernatant/tube were transferred into new separate 1.5 ml micro-centrifuge tubes.</li>
 <li>The pH was neutralized with ~375 &mu;l 10% ammonium hydroxide per tube. (Note: pH rage should be 4.1-4.5.)</li>
 <li>150 &mu;l of standard &amp; samples (includes separate tests &amp; controls) were added into an opaque-walled, transparent bottom 96-well plate.</li>
 <li>The absorbance was read at 405 nm and OD values from samples were compared with standard plot for further analysis.</li>
</ol>

<ol><li>Volponi, A. A., Pang, Y., Sharpe, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cell-based+biological+tooth+repair+and+regeneration%5BTitle%5D)+AND+%22Trends+Cell+Biol%22%5BJournal%5D)">Stem cell-based biological tooth repair and regeneration.</a> Trends Cell Biol. 20, 715-722 (2010).</li>
<li>Nosrat, I. V., Widenfalk, J., Olson, L., Nosrat, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dental+pulp+cells+produce+neurotrophic+factors%2C+interact+with+trigeminal+neurons+in+vitro%2C+and+rescue+motoneurons+after+spinal+cord+injury%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury.</a> Dev. Biol. 238, 120-132 (2001).</li>
<li>Gandia, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+dental+pulp+stem+cells+improve+left+ventricular+function%2C+induce+angiogenesis%2C+and+reduce+infarct+size+in+rats+with+acute+myocardial+infarction%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction.</a> Stem Cells. 26, 638-645 (2008).</li>
<li>Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postnatal+human+dental+pulp+stem+cells+%28DPSCs%29+in+vitro+and+in+vivo%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A.%22%5BJournal%5D)">Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.</a> Proc. Natl. Acad. Sci. U. S. A.. 97, 13625-13630 (2000).</li>
<li>Graziano, A., d'Aquino, R., Laino, G., Papaccio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dental+pulp+stem+cells%3A+a+promising+tool+for+bone+regeneration%5BTitle%5D)+AND+%22Stem+Cell+Rev%22%5BJournal%5D)">Dental pulp stem cells: a promising tool for bone regeneration.</a> Stem Cell Rev. 4, 21-26 (2008).</li>
<li>Kerkis, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+transplantation+of+human+immature+dental+pulp+stem+cells+from+baby+teeth+to+golden+retriever+muscular+dystrophy+%28GRMD%29+dogs%3A+Local+or+systemic%5BTitle%5D)+AND+%22J.+Transl.+Med%22%5BJournal%5D)">Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic.</a> J. Transl. Med. 6, 35(2008).</li>
<li>Onyekwelu, O., Seppala, M., Zoupa, M., Cobourne, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tooth+development%3A+2.+Regenerating+teeth+in+the+laboratory%5BTitle%5D)+AND+%22Dent.+Update%22%5BJournal%5D)">Tooth development: 2. Regenerating teeth in the laboratory.</a> Dent. Update. 34, 20-29 (2007).</li>
<li>Cordeiro, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dental+pulp+tissue+engineering+with+stem+cells+from+exfoliated+deciduous+teeth%5BTitle%5D)+AND+%22J.+Endod%22%5BJournal%5D)">Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth.</a> J. Endod. 34 (08), 962-969 (2008).</li>
<li>Pierdomenico, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multipotent+mesenchymal+stem+cells+with+immunosuppressive+activity+can+be+easily+isolated+from+dental+pulp%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp.</a> Transplantation. 80, 836-842 (2005).</li>
<li>de Mendonca Costa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconstruction+of+large+cranial+defects+in+nonimmunosuppressed+experimental+design+with+human+dental+pulp+stem+cells%5BTitle%5D)+AND+%22J.+Craniofac.+Surg%22%5BJournal%5D)">Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells.</a> J. Craniofac. Surg. 19, 204-210 (2008).</li>
<li>Tirino, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+the+identification%2C+characterization+and+banking+of+human+DPSCs%3A+current+strategies+and+perspectives%5BTitle%5D)+AND+%22Stem+Cell+Rev%22%5BJournal%5D)">Methods for the identification, characterization and banking of human DPSCs: current strategies and perspectives.</a> Stem Cell Rev. 7, 608-615 (2011).</li>
<li>Tirino, V., Paino, F., De Rosa, A., Papaccio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification%2C+isolation%2C+characterization%2C+and+banking+of+human+dental+pulp+stem+cells%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Identification, isolation, characterization, and banking of human dental pulp stem cells.</a> Methods Mol. Biol. 879, 443-463 (2012).</li>
<li>Eslaminejad, M. B., Vahabi, S., Shariati, M., Nazarian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+Growth+and+Characterization+of+Stem+Cells+from+Human+Dental+Pulp+of+Deciduous+Versus+Permanent+Teeth%5BTitle%5D)+AND+%22J.+Dent.+%28Tehran%29%22%5BJournal%5D)">In vitro Growth and Characterization of Stem Cells from Human Dental Pulp of Deciduous Versus Permanent Teeth.</a> J. Dent. (Tehran). 7, 185-195 (2010).</li>
<li>Wei, X., Ling, J., Wu, L., Liu, L., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+mineralization+markers+in+dental+pulp+cells%5BTitle%5D)+AND+%22J.+Endod%22%5BJournal%5D)">Expression of mineralization markers in dental pulp cells.</a> J. Endod. 33, 703-708 (2007).</li>
<li>Nombela-Arrieta, C., Ritz, J., Silberstein, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+elusive+nature+and+function+of+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Nat.+Rev.+Mol+Cell+Biol%22%5BJournal%5D)">The elusive nature and function of mesenchymal stem cells.</a> Nat. Rev. Mol Cell Biol. 12, 126-131 (2011).</li>
<li>Huang, G. T., Gronthos, S., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+derived+from+dental+tissues+vs.+those+from+other+sources%3A+their+biology+and+role+in+regenerative+medicine%5BTitle%5D)+AND+%22J.+Dent.+Res%22%5BJournal%5D)">Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine.</a> J. Dent. Res. 88, 792-806 (2009).</li>
<li>Graziano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scaffold%27s+surface+geometry+significantly+affects+human+stem+cell+bone+tissue+engineering%5BTitle%5D)+AND+%22J.+Cell+Physiol%22%5BJournal%5D)">Scaffold's surface geometry significantly affects human stem cell bone tissue engineering.</a> J. Cell Physiol. 214, 166-172 (2008).</li>
<li>d'Aquino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+dental+pulp+stem+cells%3A+from+biology+to+clinical+applications%5BTitle%5D)+AND+%22J.+Exp.+Zool.+B.+Mol.+Dev.+Evol%22%5BJournal%5D)">Human dental pulp stem cells: from biology to clinical applications.</a> J. Exp. Zool. B. Mol. Dev. Evol. 312, 408-415 (2009).</li>
<li>Mitsiadis, T. A., Feki, A., Papaccio, G., Caton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dental+pulp+stem+cells%2C+niches%2C+and+notch+signaling+in+tooth+injury%5BTitle%5D)+AND+%22Adv.+Dent.+Res%22%5BJournal%5D)">Dental pulp stem cells, niches, and notch signaling in tooth injury.</a> Adv. Dent. Res. 23, 275-279 (2011).</li>
<li>Shi, S., Gronthos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perivascular+niche+of+postnatal+mesenchymal+stem+cells+in+human+bone+marrow+and+dental+pulp%5BTitle%5D)+AND+%22J.+Bone+Miner.+Res%22%5BJournal%5D)">Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp.</a> J. Bone Miner. Res. 18, 696-704 (2003).</li>
<li>Tomic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunomodulatory+properties+of+mesenchymal+stem+cells+derived+from+dental+pulp+and+dental+follicle+are+susceptible+to+activation+by+toll-like+receptor+agonists%5BTitle%5D)+AND+%22Stem+Cells+Dev%22%5BJournal%5D)">Immunomodulatory properties of mesenchymal stem cells derived from dental pulp and dental follicle are susceptible to activation by toll-like receptor agonists.</a> Stem Cells Dev. 20, 695-708 (2011).</li>
<li>Tsukamoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mineralized+nodule+formation+by+cultures+of+human+dental+pulp-derived+fibroblasts%5BTitle%5D)+AND+%22Arch.+Oral+Biol%22%5BJournal%5D)">Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts.</a> Arch. Oral Biol. 37, 1045-1055 (1992).</li>
<li>About, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+dentin+production+in+vitro%5BTitle%5D)+AND+%22Exp.+Cell+Res%22%5BJournal%5D)">Human dentin production in vitro.</a> Exp. Cell Res. 258, 33-41 (2000).</li>
<li>Couble, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Odontoblast+differentiation+of+human+dental+pulp+cells+in+explant+cultures%5BTitle%5D)+AND+%22Calcif.+Tissue+Int%22%5BJournal%5D)">Odontoblast differentiation of human dental pulp cells in explant cultures.</a> Calcif. Tissue Int. 66, 129-138 (2000).</li>
<li>Onishi, T., Kinoshita, S., Shintani, S., Sobue, S., Ooshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulation+of+proliferation+and+differentiation+of+dog+dental+pulp+cells+in+serum-free+culture+medium+by+insulin-like+growth+factor%5BTitle%5D)+AND+%22Arch.+Oral+Biol%22%5BJournal%5D)">Stimulation of proliferation and differentiation of dog dental pulp cells in serum-free culture medium by insulin-like growth factor.</a> Arch. Oral Biol. 44 (99), 361-371 (1999).</li>
<li>Huang, G. T., Sonoyama, W., Chen, J., Park, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+characterization+of+human+dental+pulp+cells%3A+various+isolation+methods+and+culturing+environments%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">In vitro characterization of human dental pulp cells: various isolation methods and culturing environments.</a> Cell Tissue Res. 324, 225-236 (2006).</li>
<li>Bakopoulou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+the+impact+of+two+different+isolation+methods+on+the+osteo%2Fodontogenic+differentiation+potential+of+human+dental+stem+cells+derived+from+deciduous+teeth%5BTitle%5D)+AND+%22Calcif.+Tissue+Int%22%5BJournal%5D)">Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth.</a> Calcif. Tissue Int. 88, 130-141 (2011).</li>
<li>Curtis, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EF1alpha+and+RPL13a+represent+normalization+genes+suitable+for+RT-qPCR+analysis+of+bone+marrow+derived+mesenchymal+stem+cells%5BTitle%5D)+AND+%22BMC+Mol.+Biol%22%5BJournal%5D)">EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.</a> BMC Mol. Biol. 11, 61(2010).</li>
<li>Suchanek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dental+pulp+stem+cells+and+their+characterization%5BTitle%5D)+AND+%22Biomed.+Pap.+Med.+Fac.+Univ.+Palacky+Olomouc+Czech+Repub%22%5BJournal%5D)">Dental pulp stem cells and their characterization.</a> Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 153, 31-35 (2009).</li>
<li>d'Aquino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+postnatal+dental+pulp+cells+co-differentiate+into+osteoblasts+and+endotheliocytes%3A+a+pivotal+synergy+leading+to+adult+bone+tissue+formation%5BTitle%5D)+AND+%22Cell+Death+Differ%22%5BJournal%5D)">Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation.</a> Cell Death Differ. 14, 1162-1171 (2007).</li>
<li>Atari, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+pluripotent+stem+cells+from+human+third+molar+dental+pulp%5BTitle%5D)+AND+%22Histol.+Histopathol%22%5BJournal%5D)">Isolation of pluripotent stem cells from human third molar dental pulp.</a> Histol. Histopathol. 26, 1057-1070 (2011).</li>
</ol>

No conflicts of interest declared.

This protocol describes the process of isolation and characterization of hDPSCs from dental pulp using two methods, enzymatic dissociation and direct outgrowth of stem cells from pulp tissue explants. In addition, in vitro differentiation of these cells into odontoblasts, was assessed by quantitative Alizarin Red S assay &amp; QPCR.
Existing protocols for isolating pulp tissue from human tooth had been used various instruments such as pliers (bone forceps)9, extirpation ...

Stem cells are clonogenic cells which possess two remarkable features, known as multi-potency and self-renewal15. Among all stem cells with different replicative potencies, dental stem cells as the postnatal stem cells have drawn attention in recent years because of their accessibility, plasticity, and high proliferative ability in comparison with other adult stem cells16. Characteristically, similar to the mesenchymal stem cells, dental pulp stem cells are adherent clonogenic cells which have multiple differentiation capacity into mesenchyme and/or non-mesenchyme lineages, both in vitro and in vivo.1-8,17,18 DPSCs are identified by their negative expression of hematopoietic antigens (e.g., CD45, CD34, CD14), and positive expression of CD90, CD29, CD73, CD105, CD44 and STRO-1.19,20
Easy obtained potential with minimum pain &amp; morbidity make human DPSCs as a valuable source of MSCs compared to the ordinary sources, such as bone marrow mesenchymal stem cells21. In general, DPSCs have been isolated by either outgrowth method, i.e., migration of stem cells from pulp tissue explants (DPSC-OG)22-24, and/or by enzymatic digestion (DPSC-ED)4,6,25. Previous studies have shown that the isolation method and culture conditions can induce different populations or lineages under in vitro passages26,27. In the case of permanent teeth (pDPSCs), Huang et al revealed that enzymatic digested pDPSCs have higher proliferation potential compared to the outgrown DPSCs.26 Moreover, in the case of deciduous teeth (dDPSCs), it was demonstrated that STRO-1 &amp; CD34 markers expressed more in dDPSC-ED in comparison with dDPSC-OG. In addition, dDPSC-ED displayed higher mineralization rate in defined osteo/odonto medium.27 Therefore, due to the outstanding potential of DPSCs in regenerative medicine, more studies will be required for better understanding of possible various populations which are derived from different isolation methods.
Here, it was attempt to introduce easy way of pulp extraction, by using one-step dental diamond disk to facilitate the process of pulp extraction. Moreover, after the isolation of human pulp-derived stem cells by applying ED or OG methods, general properties &amp; differentiation capacity between two groups were also investigated.

<table><tbody><tr><td>&amp;alpha;-MEM</td><td>GIBCO</td><td>11900-073</td><td></td></tr><tr><td>Collagenase type I</td><td>Sigma-Aldrich</td><td>C0130-100MG</td><td></td></tr><tr><td>Dispase</td><td>GIBCO</td><td>17105-041</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>GIBCO</td><td>15140-122</td><td></td></tr><tr><td>Amphotericin B</td><td>GIBCO</td><td>15290-018</td><td></td></tr><tr><td>Fetal Bovine serum defined (FBS)</td><td>HyClone</td><td>SH30070.03</td><td></td></tr><tr><td>L-ascorbic acid 2-phosphate</td><td>Sigma</td><td>A8960-5G</td><td></td></tr><tr><td>L-glutamine</td><td>GIBCO</td><td>25030-024</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td></td></tr><tr><td>&amp;#946;-Glycerol phosphate disodium salt hydrate, BioUltra</td><td>Sigma</td><td>G9422-100G</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Osteogenesis Assay Kit</td><td>Millipore</td><td>PS01802031</td><td></td></tr><tr><td>Mouse IgG2b-PE isotype control</td><td>BD pharmingen</td><td>50808088029</td><td></td></tr><tr><td>FITC mouse IgG2b isotype control</td><td>BD pharmingen</td><td>559532</td><td></td></tr><tr><td>FITC mouse IgG1 &amp;#954; isotype</td><td>BD pharmingen</td><td>11471471</td><td></td></tr><tr><td>FITC/PE mouse anti-human CD34/CD45</td><td>BD pharmingen</td><td>341071</td><td></td></tr><tr><td>PE anti-human CD146</td><td>BD pharmingen</td><td>550315</td><td></td></tr><tr><td>Monoclonal mouse anti-human CD90/FITC</td><td>Daka</td><td>00034418</td><td></td></tr><tr><td>PE mouse anti-human CD73</td><td>BD pharmingen</td><td>550257</td><td></td></tr><tr><td>Anti-h CD105/Endoglin PE</td><td>BD pharmingen</td><td>FAB10971P</td><td></td></tr><tr><td>PE mouse anti-human CD11b/Mac1</td><td>BD pharmingen</td><td>5553888</td><td></td></tr><tr><td>CD44 PE mouse anti human</td><td>BD pharmingen</td><td>555479</td><td></td></tr><tr><td>Phosphate buffer Solution (PBS)</td><td>GIBCO</td><td>003000</td><td></td></tr><tr><td>70-&amp;mu;m cell strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>0.2 &amp;mu;m syringe filter</td><td>Millex-GV</td><td>SLGV033RB</td><td></td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; culture flask</td><td>Sigma-Aldrich</td><td>Z707481</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>BD FACSCalibur</td><td>BD</td><td>342975</td><td></td></tr><tr><td>multiskan microplate spectrophotometer </td><td>Thermo scientific</td><td>51119200</td><td></td></tr><tr><td>Fleurcense Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Flowing Software version 2.3.1</td><td></td><td></td><td></td></tr></tbody></table>

isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods

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

<ol>
 <li>DPSCs that were obtained by enzymatic dissociation (DPSC-ED) are shown here at day 10, 15,18 (Figure 1). The colonies initiated to form on almost 3 to 5 days after the isolation. </li>
 <li>Outgrown DPSCs (DPSC-OG) are shown in Figure 2 on day 5, 10, 13 &amp; 18. Fibroblast-like cells began to migrate from pulp tissue into the flask by parallel ordering by almost 5 days after seeding. </li>
 <li>DPSCs at passage 3 using both methods are shown in Figure 3. Both types of DPSCs ...

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 ...

The overall goal of this procedure is to isolate, characterize, and differentiate human gentile pulse stem cells from permanent teeth by using two methods. This is accomplished by collecting healthy impacted wisdom teeth, cutting them around cement to enamel junction at the beginning. Dental poly stem cells D PSCs are isolated by two different methods.In the first method, polyp tissues are enzymatically digested by incubating in collagenase type one plus disc space solution. Here we call them ed. Considering the isolation method in the second method, palp tissues are only placed into the flask without any digestion.In this way, dpss begin to migrate from tissue into the flask. These cells named OG refer to the outgrowth isolation method. The second step of the procedure is the identification of stem cells using fall cytometry.The third step of the procedure is the induction of odontoblast differentiation, and the final step of the procedure is the comparative evaluation of odontoblast differentiation between two groups by in red staining and QBCR. I'm RA Kde from Department of Stem Cells and Developmental Biology at Rian Institute. Today I'm going to show you the isolation characterization and comparative differentiation of human mesenchymal stem cells by using two methods.Hello, I am Dr.Rezaian from Rio Institute. We are working on a project related to human dental pulp stem cells. These cells are kind of mechy stem cells, which are easy to obtain with minimum pain and morbidity.Mechy stem cells specify with plastic aran ability, formation of colonies, and multiple differentiation capacity. Hello, I'm Dr.Ami from the Department of Stem Cells. At institute, we use this procedure in our lab to isolate stem cells for the purpose of research studies and future applications.In regenerative medicine, the very first step for using this source of stem cells for future stem cell based therapy is to choose the best protocol for isolating stem cells from human part tissue. In order to achieve this goal, it is crucial to investigate certain aspects of cellular behavior under different stem cell isolation conditions. First, I'm going to isolate human dental pulp stem cells by using either enzymatic dissociation of pulp or outgrowth of stem cells from tissue implants.Then characterize and differentiate them into ogen blast. So let's get started. First way, space and collagenase Type one dissolve both in PBS.Then filter them using oh 0.2 micron syringe filter. Pull both into conical tube. Then add pener and PBS to achieve final concentration.Transfer healthy wisdom teeth into the lab in the cold basic medium under the steel condition. Clean surface of the tooth by 70%ethanol before studying. Make sure to clean up the breeze from the surface of the tooth.Cut tooth around the cement enamel junction by using sterilized dental disc to reveal pulp chamber. It should be considered that cutting process must be performed slowly to reduce overheating of dental tissue. Then gently separate pulp tissue from the crown means pulp tissue into one to two millimeter pieces with the Scarpa blade.Transfer small pieces of pulp tissues into one milliliter enzyme solution for one hour at 37 degrees Celsius vortex every 30 minute to help breaking up pulp tissues. Afterwards, remove large aggregates by passing them through a 70 micron cell strain. Then add PBS containing PENER centrifuges in 1, 200 RPM for five minutes.Remove the supernat carefully. Then we suspend the plate in the proliferation medium pm transfer it into the culture flask and add medium. Then incubate incubated.Change the medium every three days until the cell confluence is achieved for the outwork method. Repeat the cutting process. After cutting tooth means pop tissues into one to two millimeter fragment.Then place them into the culture. Flash with proliferation, medium and incubated. It should be considered that total volume of the proliferation medium must be support the attachment of all pieces for further cell outgrow.Change the medium after observing outgrowth and then every three days until the cell confluence is achieved. For immunophenotyping, incubate both types of D PSCs with PE or FITC conjugated antibodies for 30 minutes at four degrees Celsius in dark. Then add PBS and centi views at 1, 200 RPM for five minutes.Remove the supernatant and resus, suspend the plate in PBS and finally evaluate surface markers by using flow cytometer subculture, both types of DPCs for three passages. Then tripsin ice and transfer them to six red culture dishes at 60%Co fluency replace PM by odontogenic medium. Three wells are remain as a negative control by adding pm.Change the medium every three days at day 21st wash cells with PBS. Then fix them by one milliliter per well. 10%formal gel height for 15 minutes at home temperature.After 15 minutes, remove the fixative carefully and wind cells three times with distilled water. Then replace water by one milliliter per red alza in red stain solution. After 20 minutes, we remove excess dye and wash cells four times with deionized water.Then add one millimeter per well water to prevent the cells from drying. After the staining, you can see three up rails turn into red compared to the controls under the microscope, you can see dis stain color absorption around the cell by large magnification for quantification of a red staining. After removing water, add one milliliter per well, 10%acidic acid, then incubated for 30 minutes with shaking.Then gently scrape the cells from the plate by using a cell scraper. Then transfer them into the separate tubes. VOR takes vigorously for 30 seconds.Then heat them to 85 degrees Celsius for 10 minutes. To avoid evaporation seal tubes with ParaView transfer tube to ice for five minutes, they incent used them at 20, 000 G for 15 minutes. Meanwhile, make Alzheimer red standard serial dilution according to the region protocol.After the centrifugation, remove the snat and transfer them into the new tubes. Neutralize the pH with 10%ammonium hydrocyte. Then add standards and also samples into 96.Well plate the then measure the absorbance at 405 nanometer and analyze data according to the standard serial dilution. Here you can see DPCs, which are isolated by enzymatic dissociation on day 10th, 15th and 18th, and also outgrown DPCs on day fifth, 10th, 13th and 18th. Both types of dpss in sase three are almost in the same size and morphology Immunophenotyping results show the presence of mesenchymal stem cell marker such as CD 44, CD 73, and CD 19, and the absence of hematopoietic and endothelial markers such as CD 34, CD 45, and CD 11 B.Interestingly, the expressions of CD 1 0 5 and CD 146 are more in outgrown d PSCs in comparison with D-P-S-C-E-D, the quantification of enzyme red staining on day 21st of odontoblast differentiation show more calcium deposition in D-P-S-C-E-D in comparison with without con dental top of stem cells.QPCR results also indicate significant higher expressions of MEP and A LP as the mineralization markers in D-P-S-C-D compared to the outgoing dental of stem cells. Meanwhile, both genes are regulated during the differentiation and also the expression of DSPP as the odontogenic marker increased during the differentiation. However, there is no significant variation in the expression of DSVP in differentiated cells between ED and OG groups.We've just showed you how to isolate human dental parts stem cells by using either enzymatic dissociation of pulp or outgrowth of stem cells from tissue explan. So that's it. I wish you good luck to try to use this procedure in your experiment.Thank you.

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Learn to isolate and characterize human dental pulp stem cells from permanent teeth using enzymatic dissociation and direct outgrowth methods. Compare their differentiation into odontoblasts for regenerative dental research.

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods (Video) | JoVE Journal

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

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