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

Vi takker Dr. Leila Shakeri &amp; Dr. Aref Dournaei for deres kritiske discus og Mr. Mohammad Reza Khadem Sharif for hans tekniske hjælpemidler.

1. Forbered enzymopløsningen og spredning Medium (PM) <ol><li> Gør Collagenase Type I Løsning: Afvej collagenase type I (12 mg / ml) og opløses i 1 ml PBS og filtreres under anvendelse af en 0,2 um sprøjtefilter. Derefter placere det 15 ml rør og holde det ved -20 ° C indtil brug. </li><li> Gør dispase Løsning: Afvej dispase (16 mg / ml) og opløses i 1 ml PBS og filtreres under anvendelse af en 0,2 um sprøjtefilter. Derefter placere det 15 ml rør og holde det ved 4 ° C in...

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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. 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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. 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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. 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Denne protokol beskriver processen med isolering og karakterisering af hDPSCs fra dental pulp ved hjælp af to metoder, enzymatisk dissociation og direkte udvækst af stamceller fra pulpavæv eksplantater. Desuden, in vitro differentiering af disse celler i odontoblasts blev vurderet ved kvantitativ Alizarin Red S assay og qPCR. Eksisterende protokoller til isolering pulpavæv fra human tand var blevet brugt forskellige instrumenter, såsom tang (knogle tang) 9, udryddelse nål <sup...

Stamceller er klonogene celler, som besidder to bemærkelsesværdige features, kendt som multi-potens og selvfornyelse 15. Blandt alle stamceller med forskellige replikative kræfter, har dental stamceller som de postnatale stamceller henledt opmærksomheden i de senere år på grund af deres tilgængelighed, plasticitet og høj proliferativ evne i sammenligning med andre voksne stamceller 16. Karakteristisk lighed med de mesenkymale stamceller, dental pulp stamceller er adhærente klonogene celler, der har multiple differentiering kapacitet til mesenkym-og / eller ikke-mesenchym linier, både in vitro og in vivo. 1-8,17,18 DPSCs identificeres ved deres negative udtryk for hæmatopoietiske antigener (f.eks CD45, CD34, CD14), og positivt udtryk for CD90, CD29, CD73, CD105, CD44 og STRO-1. 19,20 Nem opnåede potentiale med mindst smerte &amp; sygelighed stille menneskelige DPSCs som et værdifuldtstand kilde MSC&#39;er sammenlignet med de almindelige kilder, såsom knoglemarv mesenchymstamceller 21. Generelt er DPSCs blevet isoleret enten ved udvækst metode, dvs, migration af stamceller fra pulpavæv eksplantater (dpsc-OG) 22-24 og / eller ved enzymatisk nedbrydning (dpsc-ED) 4,6,25. Tidligere undersøgelser har vist, at isoleringsmetoden og dyrkningsbetingelser kan fremkalde forskellige populationer eller slægter under in vitro passager 26,27. I tilfælde af blivende tænder (pDPSCs), afslørede Huang et al, at enzymatiske fordøjede pDPSCs har højere proliferation potentiale sammenlignet med de outgrown DPSCs. 26 endvidere i tilfælde af løvfældende tænder (dDPSCs), blev det påvist, STRO-1 og CD34 markører udtrykt mere i dDPSC-ED sammenlignet med dDPSC-OG. Desuden viste dDPSC-ED højere mineraliseringsgrad i definerede osteo / odonto medium. 27 På grund af den enestående potentiale DPSCs i regenerative medicin, vil flere ikke være påkrævet for bedre forståelse af mulige forskellige populationer som er afledt af forskellige isoleringsmetoder. Her var forsøg på at indføre nem måde af papirmasse ekstraktion, ved hjælp af en-trins dental diamant disk for at lette processen med pulp ekstraktion. Endvidere, efter isolering af humane pulp-afledte stamceller ved at anvende ED eller OG metoder, blev generelle egenskaber og differentiering kapacitet mellem to grupper også undersøgt.

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Catalogue eller Lot. nummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> α-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> Kalvefosterserum defineret (FBS) </td><td> HyClone </td><td> SH30070.03 </td><td></td></tr><tr><td> L-ascorbinsyre-2-phosphat </td><td> Sigma </td><td> A8960-5G </td><td></td></tr><tr><td> L-glutamin </td><td> GIBCO </td><td> 25030-024 </td><td></td></tr><tr><td> Dexamethason </td><td> Sigma </td><td> D4902 </td><td></td></tr><tr><td> β-glycerolphosphat dinatriumsalt hydrat, BioUltra </td><td> Sigma </td><td> G9422-100G </td><td></td></tr><tr><td> Monobasisk kaliumphosphat </td><td> Sigma-Aldrich </td><td> P5655 </td><td></td></tr><tr><td> Osteogenese Assay Kit </td><td> Millipore </td><td> PS01802031 </td><td></td></tr><tr><td> Muse-IgG2b-PE isotypekontrol </td><td> BD Pharmingen </td><td> 50808088029 </td><td></td></tr><tr><td> FITC muse-IgG2b isotypekontrol </td><td> BD Pharmingen </td><td> 559.532 </td><td></td></tr><tr><td> FITC muse IgG1 κ isotype </td><td> BD Pharmingen </td><td> 11471471 </td><td></td></tr><tr><td> FITC / PE muse-anti-humant CD34/CD45 </td><td> BD Pharmingen </td><td>341.071 </td><td></td></tr><tr><td> PE anti-human CD146 </td><td> BD Pharmingen </td><td> 550.315 </td><td></td></tr><tr><td> Monoklonalt muse-anti-humant CD90/FITC </td><td> Daka </td><td> 00034418 </td><td></td></tr><tr><td> PE muse-anti-humant CD73 </td><td> BD Pharmingen </td><td> 550.257 </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 muse-anti-humant CD11b/Mac1 </td><td> BD Pharmingen </td><td> 5553888 </td><td></td></tr><tr><td> CD44 PE muse anti human </td><td> BD Pharmingen </td><td> 555.479 </td><td></td></tr><tr><td> Phosphatpufferopløsning (PBS) </td><td> GIBCO </td><td> 003.000 </td><td></td></tr><tr><td> 70-um cell strainer </td><td> Falcon </td><td> 352.360 </td><td></td></tr><tr><td&gt; 0,2 um sprøjtefilter </td><td> Millex-GV </td><td> SLGV033RB </td><td></td></tr><tr><td> 25 cm2 dyrkningskolbe </td><td> Sigma-Aldrich </td><td> Z707481 </td><td></td></tr><tr><td></td><td></td><td></td><td> UDSTYR </td></tr><tr><td> BD FACSCalibur </td><td> BD </td><td> 342.975 </td><td></td></tr><tr><td> Multiskan mikroplade spektrofotometer </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

<p class="jove_content"&gt; Udvikling visdomstænder er let tilgængelig kilde til stamceller i voksenalderen, som kan opnås ved rutinemæssige ortodontisk behandling. Humane pulp-afledte stamceller (hDPSCs) besidder høj proliferation potentiale med multi-slægt differentiering kapacitet sammenlignet med den almindelige kilde til voksne stamceller<sup&gt; 1-8</sup&gt;, Og derfor kunne hDPSCs være gode kandidater til autolog transplantation i vævsteknologi og regenerativ medicin. Sammen med disse fordele, har de mesenkymale stamceller (MSC) funktioner, såsom immunolodulatory effekt gøre hDPSCs mere værdifuldt, selv i tilfælde af allotransplantation<sup&gt; 6,9,10</sup&gt;. Derfor er det primære trin til anvendelse af denne kilde til stamceller er at vælge den bedste protokol til isolering hDPSCs fra pulpavæv. For at nå dette mål, er det afgørende at undersøge effekten af ​​forskellige isolation forhold på forskellige cellulære adfærd, såsom deres fælles overflademarkører &amp; også deres differentiering kapacitet.</p&gt;<p class="jove_content"&gt; Således her adskiller vi menneskelig pulpavæv fra påvirket tredje kindtand tænder, og derefter bruges både eksisterende protokoller baseret på litteratur, til isolering hDPSCs,<sup&gt; 11-13</sup&gt;<em&gt; Dvs</em&gt; Enzymatisk dissociation af pulpavæv (dpsc-ED) eller udvækst fra væv eksplantater (dpsc-OG). I denne forbindelse forsøgte vi at lette isoleringsmetoder ved hjælp af dental diamant disk. Derefter disse celler karakteriseres i form af stromal-associerede markører (CD73, CD90, CD105 og CD44), hæmatopoietiske / endothelial markører (CD34, CD45 og CD11b), perivaskulær markør, såsom CD146 og også STRO-1. Derefter blev de to protokoller i forhold baseret på differentiering styrken i odontoblasts af både kvantitativ polymerase chain reaction (qPCR) og Alizarin Red-farvning. QPCR blev brugt til vurdering af ekspressionen af ​​mineralisering-relaterede gener (alkalisk phosphatase, ALP, matrix ekstracellulære phosphoglycoprotein, MEPE &amp; dentin sialophosphoprotein, DSPP).<sup&gt; 14</sup</p

<ol><li> DPSCs, der blev opnået ved enzymatisk dissociation (dpsc-ED) er vist her på dag 10, 15,18 (fig. 1). Kolonierne indledt til dannelse på næsten 3-5 dage efter isolering. </li><li> Vokset DPSCs (dpsc-OG) er vist i figur 2 på dag 5, 10, 13 &amp; 18. Fibroblastlignende celler begyndte at migrere fra pulpavæv til kolben ved parallelle bestilling med næsten 5 dage efter podning. </li><li> DPSCs ved passage 3 ved hjælp af begge fremgangsmåder er vist i figur 3.. Begge typer DPSCs var ...

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

Fremgangsmåden beskrevet isolering og karakterisering af humane dental pulp stamceller (hDPSCs) ved anvendelse af enten<strong&gt; Enzymatisk dissociation af papirmasse (dpsc-ED)</strong&gt; Eller<strong&gt; Direkte udvækst af stamceller fra pulpavæv eksplantater (dpsc-OG). Derefter fulgt af</strong&gt;<em&gt; In vitro</em&gt; Sammenlignende differentiering af begge typer hDPSCs i odontoblasts.

Isolation, karakterisering og sammenlignende differentiering af humane Dental Pulp stamceller fra blivende tænder ved hjælp af to forskellige metoder

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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Medicine

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Afledt Cellekultur og isolering af CD133<sup&gt; +</sup&gt; Formodede kræftstamceller fra melanom

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

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

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Isolering af kræft stamceller fra humane Prostata Cancer Prøver

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

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To metoder til fastsættelse primære humane Endometriecancer stromacellers fra Hysterektomi Prøver

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 ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Berigelse for kemoterapi kræft i æggestokkene stamceller fra humane cellelinier

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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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

Karakterisering af immunceller i menneskers fedtvæv ved hjælp af flowcytometri

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

Mundtlige Biofilmdannelse på forskellige materialer til tandimplantater

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

En enzym-fri metode til isolering og udvidelse af humane Adipose-afledte mesenchymale stamceller

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

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