Name: isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods
Uploaded: 2012-11-24T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods at JoV

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><...

<ol>
	<li>Volponi, A. A., Pang, Y., Sharpe, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+biological+tooth+repair+and+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+cells+produce+neurotrophic+factors,+interact+with+trigeminal+neurons+in+vitro,+and+rescue+motoneurons+after+spinal+cord+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dental+pulp+stem+cells+improve+left+ventricular+function,+induce+angiogenesis,+and+reduce+infarct+size+in+rats+with+acute+myocardial+infarction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+human+dental+pulp+stem+cells+(DPSCs)+in+vitro+and+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells:+a+promising+tool+for+bone+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+transplantation+of+human+immature+dental+pulp+stem+cells+from+baby+teeth+to+golden+retriever+muscular+dystrophy+(GRMD)+dogs:+Local+or+systemic.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+development:+2.+Regenerating+teeth+in+the+laboratory.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+tissue+engineering+with+stem+cells+from+exfoliated+deciduous+teeth.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+mesenchymal+stem+cells+with+immunosuppressive+activity+can+be+easily+isolated+from+dental+pulp.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+large+cranial+defects+in+nonimmunosuppressed+experimental+design+with+human+dental+pulp+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+identification,+characterization+and+banking+of+human+DPSCs:+current+strategies+and+perspectives.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+isolation,+characterization,+and+banking+of+human+dental+pulp+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Growth+and+Characterization+of+Stem+Cells+from+Human+Dental+Pulp+of+Deciduous+Versus+Permanent+Teeth.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mineralization+markers+in+dental+pulp+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elusive+nature+and+function+of+mesenchymal+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+derived+from+dental+tissues+vs.+those+from+other+sources:+their+biology+and+role+in+regenerative+medicine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold's+surface+geometry+significantly+affects+human+stem+cell+bone+tissue+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dental+pulp+stem+cells:+from+biology+to+clinical+applications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells,+niches,+and+notch+signaling+in+tooth+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+niche+of+postnatal+mesenchymal+stem+cells+in+human+bone+marrow+and+dental+pulp.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+properties+of+mesenchymal+stem+cells+derived+from+dental+pulp+and+dental+follicle+are+susceptible+to+activation+by+toll-like+receptor+agonists.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mineralized+nodule+formation+by+cultures+of+human+dental+pulp-derived+fibroblasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dentin+production+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odontoblast+differentiation+of+human+dental+pulp+cells+in+explant+cultures.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+proliferation+and+differentiation+of+dog+dental+pulp+cells+in+serum-free+culture+medium+by+insulin-like+growth+factor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+human+dental+pulp+cells:+various+isolation+methods+and+culturing+environments.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+impact+of+two+different+isolation+methods+on+the+osteo/odontogenic+differentiation+potential+of+human+dental+stem+cells+derived+from+deciduous+teeth.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EF1alpha+and+RPL13a+represent+normalization+genes+suitable+for+RT-qPCR+analysis+of+bone+marrow+derived+mesenchymal+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells+and+their+characterization.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+postnatal+dental+pulp+cells+co-differentiate+into+osteoblasts+and+endotheliocytes:+a+pivotal+synergy+leading+to+adult+bone+tissue+formation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+pluripotent+stem+cells+from+human+third+molar+dental+pulp.">Isolation of pluripotent stem cells from human third molar dental pulp.</a> Histol. Histopathol. 26, 1057-1070 (2011).</li></ol>

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 border="1">
<tbody>
 <tr>
 <td>Name 			of the reagent</td>
 <td>Company</td>
 <td>Catalogue 			or Lot. number</td>
 <td>Comments 			(optional)</td>
 </tr>
 <tr>
 <td>&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>&beta;-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 &#x03BA; 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-&mu;m 			cell strainer</td>
 <td>Falcon</td>
 <td>352360</td>
 <td></td>
 </tr>
 <tr>
 <td>0.2 			&mu;m syringe filter</td>
 <td>Millex-GV</td>
 <td>SLGV033RB</td>
 <td></td>
 </tr>
 <tr>
 <td>25 			cm2 			culture flask</td>
 <td>Sigma-Aldrich</td>
 <td>Z707481</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</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

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 ...

Watch this Scientific Journal Video about Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods at JoV

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

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 ...

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Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...