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Génétique

Isolation, karakterisering og mikroRNA-baseret genetisk modificering af menneskelige Dental follikel 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 protokol beskriver den forbigående genteknologi af dental stamceller udvundet af menneskelige dental hårsækken. Anvendt ikke-virale modifikation-strategien kan blive grundlag for forbedring af terapeutisk stamceller produkter.

Abstract

Til dato, er flere stamcelle typer på forskellige udviklingsstadier i fokus for behandlingen af degenerative sygdomme. Men visse aspekter, såsom indledende massiv celledød og lave terapeutiske virkninger, forringet deres bred klinisk oversættelse. Gensplejsning af stamceller før transplantation fremstod som en lovende metode til at optimere terapeutisk stamceller effekter. Sikker og effektiv gen leveringssystemer mangler dog stadig. Derfor, udvikling af egnede metoder kan give en tilgang til at løse aktuelle udfordringer i stamcelle-baseret terapi.

Denne protokol beskriver udvinding og karakterisering af menneskelige dental follikel stamceller (hDFSCs) samt deres ikke-virale genetisk modifikation. Den postnatale dental follikel afsløret som en lovende og let tilgængelig kilde til høst Multipotente stamceller fra voksne besidder høj spredning potentiale. Proceduren beskrevet isolation præsenterer en enkel og pålidelig metode til at høste hDFSCs fra påvirket visdom tænder. Denne protokol omfatter også metoder til at definere stamcelle Karakteristik af isolerede celler. Genteknologi i hDFSCs præsenteres en optimeret kationiske lipid-baserede Transfektion strategi aktivering højeffektive mikroRNA Introduktion uden at forårsage cytotoksiske virkninger. MicroRNA er egnede kandidater til forbigående celle manipulation, da disse små translationel tilsynsmyndigheder styrer den skæbne og opførsel af stamceller uden fare for stabil genom integration. Denne protokol udgør således, en sikker og effektiv procedure for konstruktion af hDFSCs, der kan blive vigtigt for at optimere deres terapeutiske virkning.

Introduction

Den menneskelige dental hårsækken er en løs ectomesenchymally-afledte bindevæv omkring den tredje tand1,2. Ved siden af sin funktion at koordinere osteoclastogenesis og osteogenesis for tand vulkanen proces, havne denne væv stilk og stamfader celler især for udviklingen af parodontiet3,4,5. Derfor, dental hårsækken er betragtes som en alternativ kilde til høst menneskelige stamceller fra voksne6,7.

Flere undersøgelser viste, at menneskelige dental follikel stamceller (hDFSCs) er i stand til at differentiere i parodontale slægt herunder osteoblaster, ligament fibroblaster og cementoblasts8,9,10 . Desuden, disse celler blev vist til at matche alle Karakteristik af mesenchymale stromale celler (MSCs) herunder selvstændig fornyelse kapacitet, overholdelse af plast, udtryk for specifikke overflade markører (f.eks., CD73, CD90, CD105) så godt som osteogenic, adipogenic og chondrogenic differentiering potentielle11,12,13. Andre undersøgelser viste også et neurale differentiering potentiale af hDFSCs2,14,15,16,17,18.

På grund af deres lovende egenskaber og nem adgang blev hDFSCs for nylig relevante for tissue engineering19,20,21. De første undersøgelser koncentreret om DFSCs potentiale til at regenerere knogle, parodontale og tand rødder19,22,23,24,25,26, 27,28,29,30. Viden om neurogen evne til hDFSCs, deres anvendelse som potentiel behandling af neurodegenerative sygdomme siden undersøgte31,32,33. HDFSCs har også fået betydning med hensyn til den regenerering af andre væv (fx hornhindens epitel)34,35. Det terapeutiske potentiale i hDFSC er ikke kun baseret på deres direkte differentiering potentiale, men også på deres paracrine aktivitet. For nylig har hDFSCs vist sig at udskille et væld af bioaktive faktorer, såsom matrix metalloproteinases (MMPs), insulin-lignende vækstfaktor (IGF), vaskulær endotel vækstfaktor (VEGF), basic fibroblast vækstfaktor (bFGF) og hepatocyt vækst faktor (HGF), som spiller en afgørende rolle for angiogenese, immunomodulation, ekstra cellulære matrix remodellering og reparative processer36.

Dog er bred klinisk oversættelse af stamcelleterapi stadig svækket af flere udfordringer, såsom massive indledende celledød og lav gavnlige stamcelle effekter37,38. Genteknologien giver en lovende strategi for at imødegå disse udfordringer og derfor kan i høj grad øge den terapeutiske virkning af stamceller38,39,40. For forbigående celle manipulation er MicroRNA (miRs) egnede kandidater, som disse små translationel tilsynsmyndigheder styrer den skæbne og opførsel af stamceller uden fare for stabil genom integration41,42, 43. til dato, der er konstateret flere gavnlige miRs fremme stamcelle spredning, overlevelse, målsøgende, paracrine aktivitet samt deres differentiering i flere slægter44. For eksempel, manipuleret miR-133a MSCs viste en øget overlevelse og engraftment i infarcted rotte hjerter resulterer i en forbedret hjertefunktion sammenlignet med ikke-modificerede MSC’er45. Ligeledes, miR-146 overekspression MSCs blev vist til at udskille større mængder af VEGF, hvilket igen førte til en øget terapeutisk effektivitet i iskæmiske væv46.

Dette manuskript præsenterer en detaljeret protokol for selektiv ekstraktion og gensplejsning af hDFSCs. Til dette formål beskrev vi høst og enzymatiske fordøjelsen af menneskelige dental follikler og den efterfølgende isolation af hDFSCs. For at karakterisere isolerede celler, er vigtige instruktioner til verifikation af MSC egenskaber medtaget i overensstemmelse med retningslinjerne fra det internationale samfund for cellulære terapi13. Derudover giver vi en detaljeret beskrivelse af generation af miR-ændret hDFSCs ved at anvende en kationiske lipid-baserede Transfektion strategi og evalueringen af Transfektion effektivitet og cytotoksicitet.

Protocol

HDFSCs er isoleret fra dental follikler af uddraget visdom tænder fra afdeling for Oral og Maxillofacial plastikkirurgi i Rostock University Medical Center. Informeret samtykke og skriftlig godkendelse blev opnået fra alle patienter. Denne undersøgelse blev godkendt af det lokale etiske udvalg af Universitet i Rostock (tilladelse No. A 2017-0158). 1. isolering af hDFSCs Bemærk: For at forhindre bakteriel forurening, bør ikke være udbrød visdom …

Representative Results

Vi præsenterer her, en detaljeret isolation instruktion til at høste hDFSCs fra menneskelige dental hårsækken væv. På grund af den nemme adgang af dental hårsækken under rutinemæssig operation er det en lovende kilde til udvinding af stamceller fra voksne. Den isolerede hDFSCs viste alle karakteristika som beskrevet for definitionen af MSCs13. I virkeligheden var celler plast-tilhænger under bes…

Discussion

Voksne stamceller er i øjeblikket i fokus til behandling af flere degenerative sygdomme. Især knogle marv (BM)-afledte stamceller, herunder hæmatopoietisk stamceller (HSCs) og MSC’er, er under intensiv kliniske undersøgelse47. Dog BM høst er en invasiv procedure, forårsager smerter i stedet for donation og kan føre til utilsigtede hændelser48. For nylig, den postnatale dental væv er opstået som en roman og let tilgængelig kilde til stamceller. Disse dental stamce…

Divulgations

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev støttet af programmet FORUN Rostock University Medical Center (889018) og den FUGTIGE Foundation (2016-11). Derudover P.M. og R.D. understøttes af 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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