JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Bioengineering

Protokol for mikroRNA overførsel i knoglemarv-afledte hæmatopoietisk stamceller til at aktivere celle Engineering kombineret med magnetisk målretning

Published: June 18, 2018
doi:
10.3791/57474
, , , ,
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,Rostock University

Summary

Denne protokol illustrerer en sikker og effektiv procedure for at ændre CD133+ hæmatopoietisk stamceller. Den præsenteres ikke-virale, magnetiske polyplex-baserede tilgang kan danne grundlag for optimering af terapeutisk stamceller følger så godt som overvågning administreret celle produkt via magnetisk resonans.

Abstract

Mens CD133+ hæmatopoietisk stamceller (SCs) har vist sig for at give store potentiale inden for regenerativ medicin, deres lave opbevaring satser efter indsprøjtning i skadet væv samt de observerede massive celle død priser føre til meget begrænset terapeutiske virkninger. For at overvinde disse begrænsninger, forsøgte vi at etablere et ikke-virale baseret protokol for egnet celle engineering forud for deres administration. Ændring af menneskelig CD133+ udtrykker SCs ved hjælp af mikroRNA (miR) indlæses magnetiske polyplexes blev behandlet med hensyn til optagelsen effektivitet og sikkerhed samt målretning potentialet i cellerne. Afhængige af vores protokol, vi kan opnå høj miR optagelse priser af 80-90%, mens CD133+ stamceller egenskaber forbliver upåvirket. Desuden tilbyder disse modificerede celler mulighed for magnetisk målretning. Vi beskriver her en sikker og meget effektiv procedure for ændring af CD133+ SCs. Vi forventer, at denne fremgangsmåde til at give en standardteknologi for optimering af terapeutisk stamceller effekter og for overvågning af administreret celle produktet via magnetisk resonans imaging (MR).

Introduction

CD133+ SCs repræsenterer en heterogen stilk og stamfader celle population med lovende potentiale for regenerativ medicin. Deres hæmatopoietisk, endotel og myogenic differentiering potentielle1,2,3 sætter CD133+ -celler, f.eks., at bidrage til neovascularization processer gennem differentiering i nyligt danner fartøjer og aktivering af pro-angiogene signalering af paracrine mekanismer4,5,6,7.

Trods deres store potentiale demonstreret i mere end 30 godkendte kliniske forsøg (ClinicalTrails.gov), er deres terapeutiske resultat stadig under kontroversiel diskussion4. Faktisk, en klinisk anvendelse af SCs er hæmmet af lav fastholdelse i orgel af interesse og massive oprindelige celle død5,8,9. Yderligere engineering af CD133+ SCs forudgående transplantation kunne hjælpe med at overvinde disse udfordringer.

En forudsætning for en effektiv celleterapi ville være en reduktion af den massive indledende celledød at forbedre engraftment terapeutiske relevante celler10. Aktuelle undersøgelser viste en enorm celletab 90-99% i stærkt perfunderet organer som hjerne og hjerte i løbet af de første 1-2 h, uafhængigt af den transplanterede celletype eller ansøgning rute11,12,13 ,14,15,16,17,18,19,20,21. SC mærkning ved hjælp af magnetiske nanopartikler (MNPs) gør det muligt for en nyskabende non-invasiv strategi til target-cellerne til webstedet af interesse22,23,24,25,26 og samtidig tillader celle overvågning ved hjælp af Mr27 og magnetiske partikel imaging (MPI). Den mest effektive i vivo undersøgelser udlignende magnetiseret celle målretning brugte celle opbevaring efter lokal administration frem for celle vejledning efter intravenøs injektion23,24,28 . Derfor designet vores gruppe en levering system bestående af superparamagnetisk jernoxid nanopartikler29. Med denne teknik, CD133+ SCs og menneskelige umbilical vene endothelial celler (HUVECs) kunne effektivt være målrettet, som det fremgår af in vitro- forsøg30,31.

En anden hurdle for SC terapier er fjendtlige inflammatoriske miljøet i de berørte væv efter transplantation, som bidrager til den første celle død32. Ud over flere præ conditioning undersøgelser blev anvendelsen af terapeutiske relevante miRs testet33; Det er blevet med held påvist, at anti-apoptotiske miRs hæmme apoptose i vitro og øge celle engraftment i vivo33. Disse små molekyler, der består af 20 – 25 nukleotider, spiller en afgørende rolle som posttranskriptionelle modulatorer af messenger RNA’er (mRNAs), og dermed påvirke stamcelle skæbne og opførsel34. Derudover eksogen indførelsen af miRs undgå uønskede stabile integration i vært genom34.

Nuværende forsøg for effektiv indførelse af nukleinsyrer (NAs) i primære SCs er hovedsagelig baseret på rekombinant virus8,35. Trods den høje Transfektion effektivitet præsenterer rekombinant virus manipulation en væsentlig hindring for en bænken til bedside oversættelse, fx, ukontrollabel genekspression, patogenicitet, immunogenicitet og pattedyrsceller mutagenese35 ,36. Ikke-viral levering systemer såsom polymer-baserede konstruktioner er derfor kritisk at udvikle. Blandt dem, polyethylenimine (PEI) repræsenterer en gyldig levering køretøj tilbyder fordele for miRs som NA kondens at beskytte mod nedbrydning, cellulære optagelse, og intracellulære frigivelse gennem endosomal undslippe37,38. Desuden demonstreret miR-PEI komplekser en høj biokompatibilitet i kliniske forsøg39. Derfor vores levering system består af en biotinylated forgrenet 25 kDa PEI bundet til en streptavidin-belagt MNP-core30,31,40.

I dette manuskript, præsenterer vi en omfattende protokol beskriver de manuelle isolation af CD133+ SC fra menneskelige knoglemarv (BM) donation med en detaljeret beskrivelse af produktets SC og (ii) en effektiv og blid Transfektion strategi af en magnetisk ikke-virale polymer-baserede levering system for gensplejsning af CD133+ SCs bruger miRs. CD133+ SCs er isoleret og magnetisk beriget fra menneskelige brystbenet BM aspirates ved hjælp af en overflade antistof-baserede magnetiske-aktiveret celle sortering (MLA) system. Bagefter, cellernes levedygtighed samt celle renhed er analyseret ved hjælp af flowcytometri. Efterfølgende, PEI-miR-MNP komplekser er forberedt og CD133+ SCs er transfekteret. 18 h efter Transfektion, optagelse effektiviteten og virkningen af Transfektion på SC markør udtryk og celle levedygtighed er analyseret. Desuden udføres evaluering af intracellulær fordelingen af Transfektion komplekse forbindelser, ved hjælp af firefarvet mærkning og struktureret belysning mikroskopi (SIM).

Protocol

Brystbenet menneskelige BM for celle isolation blev indhentet fra informeret donorerne, der gav deres skriftlige samtykke til at bruge deres prøver til forskning efter Helsinki-erklæringen. Det etiske udvalg af Universitet i Rostock har godkendt den præsenteres undersøgelse (reg. nr. A 2010 23, forlænget i 2015). 1. celle forberedelse Bemærk: Brug heparin natrium (250 IU/mL BM) at forhindre koagulation for BM undersøgelse. CD133+ …

Representative Results

Den præsenterede protokol beskriver en manuel isolation og magnetiske berigelse af menneskelige BM-afledt CD133+ SCs med en efterfølgende virus uafhængige celle engineering strategi, som en non-invasiv teknologi til i vitro celle manipulation og i vivo overvågning værktøj. Denne tre-trins afsondrethed teknologi tillader en adskillelse af multinationale selskaber fra de præ-fordøjet brystbenet…

Discussion

I de seneste år, CD133+ SCs er opstået som en lovende celle population for SC-baserede behandlinger som det fremgår af flere fase I, II og III kliniske forsøg43,44,45,46, 47 , 48 , 49 , 50 , 51 , …

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev støttet af det føderale ministerium for uddannelse og forskning Tyskland (FKZ 0312138A og FKZ 316159), den stat Mecklenburg-Vorpommern med EU ‘s strukturfonde (ESF/IVWM-B34-0030/10 og ESF/IVBM-B35-0010/12) og DFG (DA1296/2-1), den Tyske Heart Foundation (F/01/12), BMBF (VIP + 00240) og den FUGTIGE Foundation. Derudover understøttes F.H. og P.M. af FORUN Program af Rostock University Medical Centre (889001).

Materials

7-AAD BD Biosciences 559925
Acetic Acid with Methylene Blue Stemcell Technologies 7060 3%
anti-CD133/2-PE (clone: 293C3) Miltenyi Biotec GmbH 130-090-853
anti-CD34-FITC (clone: AC136) Miltenyi Biotec GmbH 130-081-001
anti-CD45-APC-H7 (clone: 2D1) BD Biosciences 560178
rhodamine dye; Atto 565 dye conjugated to biotin ATTO-TEC GmbH AD 565-71
BD FACS LSRII flow cytometer BD Biosciences
BD FACSDiva Software 6.1.2 BD Biosciences
BSA Sigma-Aldrich GmbH A7906
CD133 antibody-linked superparamagnetic iron dextran particles; CD133 MicroBead Kit Miltenyi Biotec GmbH 130-097-049
collagenase B Roche Diagnostics GmbH 11088831001
counting chamber Paul Marienfeld GmbH & Co. KG
Cyanine 3 dye labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1 Ambion AM17120
Cyanine 5 dye miR labelling kit; Cy5 dye Label IT miRNA Labeling Kit Mirus Bio MIR 9650
DNAse I Roche Diagnostics GmbH 10104159001 (100 U/mL)
ELYRA PS.1 LSM 780 confocal microscope Carl Zeiss Jena GmbH
FcR Blocking Reagent, human Miltenyi Biotec GmbH 130-059-901
bright green protein labeling kit; Oregon Green 488 Protein Labeling Kit Thermo Fisher Scientific O10241
aqueous mounting medium; Fluoroshield Sigma-Aldrich GmbH F6182
density gradient centrifugation tube; Leukosep Centrifuge Tube Greiner Bio-One 89048-932
MACS magnet holder; MACS MultiStand Miltenyi Biotec GmbH 130-042-303
MACS pre-separation filter Miltenyi Biotec GmbH 130-041-407 30 µm
MACS separation column (MS / LS) Miltenyi Biotec GmbH 130-042-201 / 130-042-401
MACS permanent magnet; MACS Separator Miltenyi Biotec GmbH 130-042-302
Millex-HV PVDF Filter Merck SLHV013SL 0.45 μm
mouse IgG 2b-PE Miltenyi Biotec GmbH 130-092-215
amine reactive dye; Near-IR LIVE/DEAD Fixable Dead Cell Stain Kit Thermo Fisher Scientific L10119
human lymphocyte separating medium; Pancoll Pan Biotech GmbH P04-60500 density: 1.077 g/mL
PBS Pan Biotech GmbH P04-53500 without Ca and Mg
PEI Sigma-Aldrich GmbH 408727 branched; 25 kDa
Penicillin/Streptomycin Thermo Fisher Scientific 15140122 100 U/mL, 100 μg/mL
PFA Merck Schuchardt OHG 1040051000
unlabelled precursor miR; Pre-miR miRNA Precursor Negative Control #1 Ambion AM17110
RBC lysis buffer eBioscience 00-4333-57
RNAse decontamination solution; RNaseZap Thermo Fisher Scientific AM9780
human lymphocyte medium; Roswell Park Memorial Institute (RPMI) 1640 medium Pan Biotech GmbH P04-16500
recombinant human cytokine supplement; StemSpan CC100 Stemcell Technologies 2690
serum-free haematopoietic cell expansion medium; StemSpan H3000 Stemcell Technologies 9800
Streptavidin MagneSphere Paramagnetic Particles Promega Corporation Z5481
Trypan Blue solution Sigma-Aldrich GmbH T8154 0.4 %
UltraPure EDTA Thermo Fisher Scientific 15575020 0.5 M; pH 8.0
ZEN2011 software Carl Zeiss Jena GmbH
NanoDrop 1000 Spectrophotometer Thermo Fisher Scientific
Sonorex RK 100 SH sonicating water bath Bandelin electronic Ultrasonic nominal output: 80 W; Ultrasonic frequency: 35 kHz
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Tags

MicroRNA TransferAdult Bone Marrow-derived Hematopoietic Stem CellsCell EngineeringMagnetic TargetingRegenerative MedicineHematopoietic Stem Cell ModificationMesenchymal Stem CellsBone Marrow CollectionDensity Gradient CentrifugationMononuclear Cell IsolationMagnetic Selection

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Hausburg, F., Müller, P., Voronina, N., Steinhoff, G., David, R. Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting. J. Vis. Exp. (136), e57474, doi:10.3791/57474 (2018).
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