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Abstract
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Bio-ingénierie

Protokoll for MicroRNA overføring til voksen Ben margtransplantasjon-avledet blodkreft stamceller aktivere cellen Engineering kombinert med magnetiske målretting

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 protokollen illustrerer en sikker og effektiv fremgangsmåte for å endre CD133+ blodkreft stamceller. Presentert ikke-viral, magnetiske polyplex-basert tilnærming kan gi grunnlag for optimalisering av terapeutiske stamcelleforskningen effekter så vel som for overvåking administrert celle produktet via magnetisk resonans imaging.

Abstract

Mens CD133+ blodkreft stamceller (SCs) har vist seg for å gi høyt potensial innen regenerativ medisin, deres lav oppbevaring priser etter injeksjon i skadet vev som observerte massiv celle dødelighet føre til svært begrenset terapeutisk effekt. For å overvinne disse begrensningene, forsøkte vi å etablere en ikke-viral basert protokoll egnet cellen engineering før sin administrasjon. Endring av menneskelig CD133+ uttrykke SCs benytter microRNA (miR) lastet magnetiske polyplexes ble behandlet med hensyn til opptak effektivitet og sikkerhet, samt målretting potensialet i cellene. Stole på våre protokoll, kan vi oppnå høy miR opptak priser for 80 – 90%, mens CD133+ stamcelleforskningen egenskaper forbli upåvirket. Videre tilbyr disse endrede cellene muligheten for magnetisk målretting. Vi beskriver her en trygg og svært effektiv fremgangsmåte for endring av CD133+ SCs. Vi forventer denne tilnærmingen å gi en standardteknologi for optimalisering av terapeutiske stamcelleforskningen effekter og overvåking av administrert celle produktet via magnetisk resonans imaging (MRI).

Introduction

CD133+ SCs representerer en heterogen stammen og stamfar celle befolkning med lovende potensial for regenerativ medisin. Deres blodkreft endothelial og myogenic differensiering potensielle1,2,3 gjør CD133+ celler, f.ekså bidra til neovascularization prosesser gjennom differensiering i nylig dannet fartøy og aktivering av pro-angiogenic signalering av paracrine mekanismer4,5,6,7.

Til tross for sitt høye potensial i mer enn 30 godkjente kliniske studier (ClinicalTrails.gov), er terapeutiske utfallet fremdeles under kontroversiell diskusjon4. Faktisk er en klinisk anvendelse av SCs hindret av lav oppbevaring i organ for interesse og massiv første celle død5,8,9. Ekstra prosjektering av CD133+ SCs før transplantasjon kunne hjelpe overvinne disse utfordringene.

En forutsetning for en effektiv cellen terapi ville være reduksjon av massiv første celledød å forbedre engraftment av terapeutiske relevante celler10. Aktuelle studier vist en enorm celle tap av 90-99% i svært perfused organer som hjernen og hjertet under den første 1-2 h, uavhengig av hvilken transplantert cellen eller programmet ruten11,12,13 ,14,15,16,17,18,19,20,21. SC merking bruker magnetisk nanopartikler (MNPs) gjør en nyskapende ikke-invasiv strategi til målcellene til området av interesse22,23,24,25,26 og samtidig gir cellen overvåking MRI27 og magnetiske partikler imaging (MPI). Den mest effektive i vivo studier bruk magnetisert celle målretting brukte celle oppbevaring etter lokal administrasjon fremfor celle veiledning etter intravenøs injeksjon23,24,28 . Derfor laget vårt en levering system som består av superparamagnetiske jernoksid nanopartikler29. Med denne teknikken, CD133+ SCs og menneskelige umbilical blodåre endotelceller (HUVECs) kan effektivt rettes, som demonstrert av i vitro forsøk30,31.

En annen hurdle SC terapi er fiendtlig inflammatorisk miljøet av berørte vev etter transplantasjon, som bidrar til den første celle død32. I tillegg til flere pre condition studier var anvendelse av terapeutiske relevante miRs testet33; Det har vært vellykket demonstrert at anti-apoptotisk miRs hemmer apoptose i vitro og forbedre celle engraftment i vivo33. Disse små molekyler, består av 20-25 nukleotider, spiller en avgjørende rolle som posttranscriptional modulatorer av messenger RNAs (mRNAs), og dermed påvirke stamcelleforskningen skjebne og atferd34. Videre eksogene innføring av miRs unngå uønskede stabil integrering i vert genomet34.

Nåværende forsøk for effektiv introduksjon om nukleinsyrer (NAs) i primære SCs er hovedsakelig basert på rekombinant virus8,35. Til tross for høy transfection effektiviteten presenterer rekombinant virus manipulasjon en stor utfordring for en benken til sengen oversettelse, f.eks, ukontrollerbare genuttrykk, virusets, immunogenisitet og insertional mutagenese35 ,36. Derfor er ikke-viral leveringssystemer som polymer-baserte konstruksjoner avgjørende for å utvikle. Blant dem, polyethylenimine (PEI) representerer en gyldig levering kjøretøy tilbyr fordeler for miRs NA kondens å beskytte fra fornedrelse, mobilnettet opptak, og intracellulær utgivelse gjennom endosomal rømme37,38. Videre demonstrert miR-PEI komplekser en høy biocompatibility i kliniske studier39. Derfor levering systemet består av en biotinylated forgrenet 25 kDa PEI bundet til en streptavidin-belagt MNP kjerner30,31,40.

I dette manuskriptet presenterer vi en omfattende protokoll som beskriver (i) manuell isolering av CD133+ SC fra human benmarg (BM) donasjon med en detaljert karakterisering av SC produktet og (ii) en effektiv og skånsom transfection strategi for en magnetisk ikke-viral polymer-baserte leveringssystem for genteknologi av CD133+ SCs benytter miRs. CD133+ SCs er isolert og magnetisk beriket fra menneskelige sternal BM aspirates bruker en overflate antistoff-baserte magnetiske-aktivert celle sortering (Mac) system. Etterpå er celle levedyktigheten samt celle renhet analysert ved hjelp av flowcytometri. Deretter miR/PEI/MNP komplekser er forberedt og CD133+ SCs er transfekterte. 18 h etter hva, opptak effektiviteten og virkningen av transfection på SC markør uttrykk og celle levedyktighet analyseres. Videre utføres evaluering av intracellulær fordelingen av transfection komplekse forbindelser med firefargers merking og strukturert belysning mikroskopi (SIM).

Protocol

Sternal menneskelige BM for cellen aisolering er Hentet fra informert givere, som ga sitt skriftlige samtykke å bruke sine eksempler for forskning etter erklæring av Helsinki. Den etiske komiteen av universitetet i Rostock har godkjent presentert studien (reg. nr A 2010 23, langvarig i 2015). 1. celle forberedelse Merk: Bruk heparin natrium (250 IU/mL BM) å hindre koagulering for BM eksamen. CD133+ SC isolasjon Utar…

Representative Results

Presentert protokollen beskriver en manuell isolasjon og magnetiske anriking av menneskelig BM-avledet CD133+ SCs med en påfølgende virus uavhengig celle engineering strategi, som en ikke-invasiv teknologi i vitro celle manipulasjon og i vivo avlytting verktøyet. Denne tre-trinns aisolering teknologien tillater en separasjon av MNCs fra det pre-oversikten sternal BM gjennom tetthet gradert sentrif…

Discussion

De siste årene, CD133+ SCs har dukket opp som en lovende celle befolkningen for SC-basert behandling som dokumentert av flere fase I, II og III kliniske studier43,44,45,46, 47 , 48 , 49 , 50 , 51 , <…

Divulgations

The authors have nothing to disclose.

Acknowledgements

Dette arbeidet ble støttet av Federal Utdannings og forskning Tyskland (FKZ 0312138A og FKZ 316159), til delstaten Mecklenburg-Vorpommern med EU Structural Funds (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 fuktig Foundation. I tillegg støtter F.H. og PM av FORUN programmet i 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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