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Génétique

Isolering og karakterisering MicroRNA-baserte genetisk modifisering av menneskelig Dental hårsekken stamceller

Published: November 16, 2018
doi:
10.3791/58089
, , , , ,
1Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), Department of Cardiac Surgery,Rostock University Medical Center, 2Department Life,Light and Matter of the Interdisciplinary Faculty at Rostock University, 3Department of Operative Dentistry and Periodontology,Rostock University Medical Center

Summary

Denne protokollen beskriver forbigående genteknologi av dental stamceller hentet fra menneskelige dental hårsekken. Brukes ikke-viral endring strategi kan bli grunnlag for forbedring av terapeutiske stamcelleforskningen produkter.

Abstract

Hittil er forskjellige på forskjellige utviklingsstadier stilk cellen i fokus for behandling av degenerative sykdommer. Likevel, enkelte aspekter som første massive celledød og lav terapeutiske effekter, nedsatt deres omfattende kliniske oversettelse. Genteknologi av stamceller før transplantasjon dukket opp som en lovende metode for å optimalisere terapeutisk stamcelleforskningen effekter. Sikker og effektiv genet leveringssystemer mangler imidlertid fortsatt. Utviklingen av passende metoder kan derfor gi en tilnærming for å løse dagens utfordringer i stilk cellen-basert behandling.

Nåværende protokollen beskriver utvinning og karakterisering av menneskelig dental hårsekken stamceller (hDFSCs) og deres ikke-viral genmodifisering. Postnatal dental hårsekken avduket som en lovende og lett tilgjengelig for høsting voksen multipotent stamceller besitter høy spredning potensial. Prosedyren beskrevet isolasjon presenterer en enkel og pålitelig metode for å høste hDFSCs fra påvirket visdom tennene. Denne protokollen omfatter også metoder for å definere stamcelleforskningen egenskaper isolert celler. Genetisk engineering av hDFSCs presenteres en optimalisert kationisk lipid-baserte transfection strategi muliggjør svært effektiv microRNA Introduksjon uten forårsaker cytotoksiske effekter. MicroRNAs er egnet kandidater til forbigående celle manipulasjon, disse små translasjonsforskning regulatorer kontrollere skjebne og oppførsel av stamceller uten fare stabil genomet integrering. Derfor representerer denne protokollen en sikker og effektiv fremgangsmåte for utvikling av hDFSCs som kan bli viktig for å optimalisere deres terapeutisk effekt.

Introduction

Menneskelige dental hårsekken er en løs ectomesenchymally-avledet bindevev omgir utvikle tann1,2. Foruten sin funksjon å koordinere osteoclastogenesis og osteogenesis for tann utbruddet prosessen, havner Dette vevet stammen og stamfar celler spesielt for utviklingen av periodontium3,4,5. Derfor regnes dental hårsekken som en alternativ kilde å høste humant voksne stamceller6,7.

Flere studier vist at menneskelige dental hårsekken stamceller (hDFSCs) er i stand til å skille i periodontal slektslinje inkludert osteoblasts, leddbånd fibroblaster og cementoblasts8,9,10 . Videre disse cellene ble vist å matche alle karakteristikkene av mesenchymal stromal celler (MSCs) inkludert selv fornyende kapasitet, plast etterlevelse, uttrykk for bestemte overflate markører (f.eks, CD73, CD90, CD105) så vel som osteogenic, adipogenic og chondrogenic differensiering potensielle11,12,13. Andre studier viser også en neural differensiering potensialet i hDFSCs2,14,15,16,17,18.

Deres lovende egenskaper og enkel tilgang ble hDFSCs nylig relevante for tissue engineering19,20,21. Den første studier konsentrert om potensialet i DFSCs å regenerere beinet, periodontal og tann røtter19,22,23,24,25,26, 27,28,29,30. Kunnskap om neurogenic evnen til hDFSCs, deres program som potensielle behandling for nevrodegenerative sykdommer har vært undersøkt31,32,33. HDFSCs har også fått betydning med hensyn til den fornyelse av andre vev (f.eks hornhinnen epitel)34,35. Terapeutiske potensialet i hDFSC er ikke bare basert på deres direkte differensiering potensial men også på sin paracrine aktivitet. Nylig har hDFSCs vist seg å skille ut en rekke bioaktive faktorer, for eksempel matrise metalloproteinases (MMPs), insulin-lignende vekstfaktor (IGF), vaskulær endotelial vekstfaktor (VEGF), grunnleggende fibroblast vekstfaktor (bFGF) og hepatocyte vekst faktor (HGF), som spiller en avgjørende rolle for angiogenese, immunomodulation, ekstra mobil matrix remodeling og reparative prosesser36.

Bred klinisk oversettelse av stilk cellen terapi er imidlertid fortsatt nedsatt av flere utfordringer, som massiv første celledød og lav gunstig stamcelleforskningen effekter37,38. Genteknologi gir en lovende strategi for å møte disse utfordringene, og derfor kan øke den terapeutiske effekten av stamceller38,39,40. Forbigående celle manipulering er microRNAs (miRs) egnede kandidater, som disse små translasjonsforskning regulatorer kontrollere skjebne og oppførsel av stamceller uten fare for stabil genomet integrering41,42, 43. hittil har flere nyttige miRs er identifisert fremme stem celle spredning, overlevelse, homing, paracrine aktivitet samt deres differensiering inn flere overleveringslinjer44. For eksempel utviklet miR-133a MSCs viste en økt overlevelse og engraftment i infarcted rotten hjerter som resulterer i en bedre hjertefunksjon sammenlignet med uforandret MSCs45. Likeledes, miR-146a overexpressing MSCs ble vist å skille ut høyere mengder VEGF som i sin tur førte til en forbedret effektivitet i iskemiske vevet46.

Dette manuskriptet presenterer en detaljert protokoll for selektiv utvinning og genteknologi av hDFSCs. For dette formålet beskrev vi høsting og enzymatiske fordøyelsen av menneskelig dental follicles og påfølgende isolering av hDFSCs. For å karakterisere isolert celler, har viktige instruksjoner for verifikasjon av MSC egenskaper blitt inkludert i henhold til veiledning av det internasjonale samfunnet for mobilnettet terapi13. I tillegg gir vi en detaljert beskrivelse for generering av miR-modifisert hDFSCs ved å bruke en kationisk lipid-baserte transfection strategi og evalueringen av hva effektivitet og cytotoksisitet.

Protocol

HDFSCs er isolert fra dental follikler av utdraget visdomstennene tilbys av Institutt for Oral og Maxillofacial plastikkirurgi i Rostock University Medical Center. Informert samtykke og skriftlig godkjennelse er Hentet fra alle pasienter. Denne studien ble godkjent av den lokale etikk av universitetet i Rostock (tillatelse No. A 2017-0158). 1. isolering av hDFSCs Merk: For å hindre bakteriell forurensning, bør visdomstennene ikke være utbrudd før …

Representative Results

Her presenterer vi en detaljert isolasjon instruksjon å høste hDFSCs fra menneskelige dental hårsekken vev. På grunn av den lette tilgangen til dental hårsekken under rutinemessig kirurgi er det en lovende kilde for utvinning av voksne stamceller. Den isolerte hDFSCs viste alle kjennetegn på definisjonen av MSCs13. Faktisk celler var plast-tilhenger under beskrevet oppdrettsforholdene og vist en fib…

Discussion

Voksen stilk celler er i fokus for behandling av flere degenerative sykdommer. Spesielt bein margtransplantasjon (BM)-avledet stilk celler, inkludert blodkreft stamceller (HSCs) og MSCs, er under intens klinisk undersøkelse47. Men BM høsting er en invasiv prosedyre forårsaker smerte på stedet av donasjon og kan føre til uønskede hendelser48. Postnatal dental vevet har nylig dukket opp som en roman og lett tilgjengelig kilde til stamceller. Disse dental stamceller ble …

Divulgations

The authors have nothing to disclose.

Acknowledgements

Dette arbeidet ble støttet av FORUN programmet i Rostock medisinske sentrum (889018) og fuktig Foundation (2016-11). I tillegg PM RD støttes av BMBF (VIP + 00240).

Materials

Mouse anti Human CD105 Antibody: Alexa Fluor 488 Bio-Rad MCA1557A488 Clone SN6, monoclonal
Mouse IgG1 Negative Control Antibody: Alexa Fluor 488 Bio-Rad MCA928A488 monoclonal
APC Mouse Anti-Human CD29 Antibody BD Biosciences 559883 Clone MAR4, monoclonal
APC Mouse IgG1, κ Isotype Control Antibody BD Biosciences 555751 Clone MOPC-21, monoclonal
PE Mouse Anti-Human CD73 Antibody BD Biosciences 550257 Clone AD2, monoclonal
PE Mouse IgG1, κ Isotype Control Antibody BD Biosciences 555749 Clone MOPC-21, monoclonal
PE-Cy7 Mouse Anti-Human CD117 Antibody BD Biosciences 339217 Clone 104D2, monoclonal
PE-Cy7 Mouse IgG1, κ Isotype Control Antibody BD Biosciences 557872 Clone MOPC-21, monoclonal
PerCP-Cy5.5 Mouse Anti-Human CD44 Antibody BD Biosciences 560531 Clone G44-26, monoclonal
PerCP-Cy5.5 Mouse IgG2b, κ Isotype Control Antibody BD Biosciences 558304 Clone 27-35, monoclonal
PerCP-Cy5.5 Mouse Anti-Human CD90 Antibody BD Biosciences 561557 Clone 5E10, monoclonal
PerCP-Cy5.5 Mouse IgG1, κ Isotype Control Antibody BD Biosciences 55095 Clone MOPC-21, monoclonal
V500 Mouse Anti-Human CD45 Antibody BD Biosciences 560777 Clone HI30, monoclonal
V500 Mouse IgG1, κ Isotype Control Antibody BD Biosciences 560787 Clone X40, monoclonal
FcR Blocking Reagent, human Miltenyi Biotec 130-059-901
UltraPure EDTA Thermo Fisher Scientific 15575-020 0.5M, pH 8.0
Steritop Merck Millipore SCGPT05RE 0.22 µm, radio-sterilized, polyethersulfone
BSA Sigma-Aldrich A7906
PFA Merck Millipore 1040051000
Human Mesenchymal Stem Cell Functional Identification Kit R&D Systems SC006
RNase decontamination solution; RNaseZap RNase Decontamination Solution Thermo Fisher Scientific AM9780
Cy3-labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1 Thermo Fisher Scientific AM17120 5 nmol
Pre-miR miRNA Precursor Negative Control #1 Thermo Fisher Scientific AM17110 5nmol
Cationic lipid-based transfection reagent; Lipofectamine 2000 Transfection Reagent Thermo Fisher Scientific 11668019
Reduced serum medium; Opti-MEM I Reduced Serum Medium Thermo Fisher Scientific 31985070
Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific A-11055 polyclonal
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific A-21202 polyclonal
Mounting medium; Fluoroshield with DAPI Sigma-Aldrich F6057-20ML histology mounting medium
ELYRA PS.1 LSM 780 confocal microscope Zeiss
BD FACS LSRII flow cytometer BD Biosciences
BD FACSDiva Software 6.1.2 BD Biosciences
ZEN2011 software Zeiss
Trypsin/EDTA solution (0.05%/ 0.02%) Biochrom L2143 in PBS, w/o: Ca2+, Mg2+
Amine reactive dye; LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit Thermo Fisher Scientific L10119
PBS (1x) Thermo Fisher Scientific 10010023 pH: 7.4; w/o: Ca and Mg
P-S-G (100x) Thermo Fisher Scientific 10378016
Basal medium; Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 Thermo Fisher Scientific 11039021
Antibiotic, ZellShield Biochrom W 13-0050
FBS Thermo Fisher Scientific 10500064
Collagenase type I Thermo Fisher Scientific 17100017
Dispase II Thermo Fisher Scientific 17105041
Filter, Sterifix syringe filter 0.2 µm Braun 4099206
50 mL conical centrifuge tube Sarstedt 62,547,254
15 mL conical centrifuge tube Sarstedt 62,554,502
Cell culture flask 75 cm2 Sarstedt 833,910,002
Cell culture flask, 25 cm2 Sarstedt 833,911,002
Freezing medium, Biofreeze Biochrom F 2270
Cryotubes Thermo Fisher Scientific 377267 1.8 mL
Trypan blue solution Sigma-Aldrich T8154 0.4 %
Counting chamber Paul Marienfeld
Local anesthetic, Xylocitin (lidocaine hydrochloride) 2% with epinephrine (adrenaline) 0.001% Mibe
NaCl solution Braun 0.9 %
Vicryl satures, Vicryl rapide Ethicon 3 – 0
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Tags

Dental Follicle Stem CellsHDFSCsEnzymatic DigestionCollagenaseDispaseMicroRNAGenetic ModificationRegenerative MedicineCell IsolationCell Characterization
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Müller, P., Ekat, K., Brosemann, A., Köntges, A., David, R., Lang, H. Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells. J. Vis. Exp. (141), e58089, doi:10.3791/58089 (2018).
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