Isolering af Perivaskulær multipotente precursorcelle populationer fra humant hjertevæv
Summary
Humant hjertevæv huser multipotente perivaskulære precursor cellepopulationer, som kan være egnede til myocardial regenerering. Den her beskrevne teknik muliggør den samtidige isolering og oprensning af to multipotente stromale cellepopulationer forbundet med native blodkar, dvs. CD146 + CD34 – pericytter og CD34 + CD146 – adventitiaceller celler, fra den humane myocardium.
Abstract
Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34– pericytes and vascular CD34+CD146– adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.
Introduction
Hjertet har længe været betragtet som et post-mitotisk organ. Imidlertid har nyere undersøgelser vist, at tilstedeværelsen af begrænset cardiomyocyte omsætning hos voksne menneskelige hjerter 1. Native stængel / stamceller med cardiomyocyte differentiering potentiale er også blevet identificeret i myokardiet hos voksne gnavere og menneskelige hjerter, herunder Sca-1 +, c-kit +, cardiosphere-dannende, og senest, perivaskulære præcursorceller 2,3. Disse celler udgør attraktive kandidater til behandlinger, der skal forbedre hjerte-reparation / regeneration gennem celle transplantation eller stimulering af in-situ spredning.
Mesenkymale stamceller / stromale celler (MSC) er blevet isoleret fra næsten alle humant væv 4,5 Kliniske forsøg med de terapeutiske anvendelser af MSC er blevet udført for flere patologiske tilstande, såsom kardiovaskulær reparation 6, graft-versus-host-sygdom 7 </sup>, Og levercirrose 8. Gavnlige virkninger er blevet tilskrevet evne MSC til: hjem til inflammationssteder 9; differentiere til forskellige celletyper 10; udskiller pro-reparative molekyler 11; og modulere værtsimmunresponser 12. Isoleringen af MSC'erne har traditionelt påberåbt sig deres fortrinsret overholdelse plastik substrater. Men den resulterende population af celler er typisk markant heterogen 13. Ved at bruge fluorescerende cellesortering (FACS) med en kombination af centrale perivaskulære cellemarkører, har vi været i stand til at isolere og oprense en multipotente MSC-lignende precursor population (CD146 + / CD31 – / CD34 – / CD45 – / CD56 -) fra multiple humane væv, herunder voksne skeletmuskulatur og hvid fedt 14.
Perivaskulære cellepopulationer i forskellige ikke-hjertevæv er blevet vist at have stamceller / stamceller egenskaber and bliver undersøgt til klinisk anvendelse i det kardiovaskulære indstilling. Pericyter, en af de mest kendte perivaskulære celle-undergrupper, er en heterogen population, der spille flere patofysiologiske roller, herunder i udviklingen af nye skibe 15, regulering af blodtrykket 16, og vedligeholdelse af vaskulær integritet 17,18. Som vist i flere væv, specifikke delmængder af pericytter indbygget udtrykke MSC-antigener og opretholde deres MSC-lignende fænotyper i primær kultur efter FACS rensning 14. Desuden er disse celler, der stabilt opretholder deres langsigtede fænotyper indenfor kultur og udviser multi-slægt differentiering potentiale, der ligner MSC'er 19,20. Disse resultater tyder på, at pericytter er en af oprindelsen af den undvigende MSC 14. Det terapeutiske potentiale af pericytter er påvist med en reduktion i myocardial ardannelse og forbedret hjertefunktion efter transplantation i iskæmisk skadethjerter 21. For nylig har vi med succes oprenset pericytter fra det menneskelige myocardium og demonstrerede deres MSC-lignende fænotyper og multipotency (adipogenese, chondrogenese og osteogenese) med fraværet af skelet myogenese 3. Desuden myocardiale pericytter udviste differentielle cardiomyogenic potentiale og angiogene kapacitet sammenlignet med modparter oprenset fra andre organer.
En anden population af multipotente perivaskulære stamceller / progenitorceller, den adventitiale celle, er blevet isoleret fra humane saphenous vener på grundlag af positive CD34-ekspression 22. Venøse adventitiaceller celler har vist sig at have klonogene potentiale, mesodermal differentiation kapacitet og proangiogen potentiale in vitro. Transplantation af disse celler i iskæmisk beskadigede hjerter mus resulterede i en reduktion i interstitiel fibrose, en stigning i angiogenese og myocardial blodgennemstrømning, reduceret ventrikulær dilation, og øget kardiel uddrivningsfraktion 23. Interessant nok har fedt-adventitiaceller celler vist sig at tabe CD34-ekspression og opregulere CD146 ekspressionen i kultur som reaktion på angiopoietin II behandling, hvilket tyder på vedtagelsen af en pericyt fænotype med stimulation 24. I hjertet, men den adventitiale cellepopulation er endnu ikke blevet prospektivt oprenset ved FACS og / eller godt karakteriseret. Ved hjælp af de celle isolation, der er beskrevet i de følgende afsnit, er vi i øjeblikket kendetegner myocardiale adventitiaceller celler og undersøge deres potentiale for regenerative applikationer.
Heri beskriver vi en fremgangsmåde til at isolere og oprense to subpopulationer af perivaskulære stamceller / progenitorceller fra human føtal eller voksen myokardiet. Denne prospektive celle isolation metode vil gøre det muligt for forskerne at opnå isogene perivaskulære stamceller / stamceller delmængder fra menneskelige hjerte biopsier for sammenlignende undersøgelser og further udforske deres terapeutiske potentiale i forskellige hjertelidelser patologiske tilstande.
Protocol
Representative Results
Discussion
Stigende beviser understøtter en begrænset regenerativ kapacitet af den voksne menneskelige hjerte efter skade. Identifikation og karakterisering af indfødte precursorceller ansvarlige for sådanne regenerative reaktioner i tilskadekomne hjerter er afgørende for både forståelsen af tilknyttede mekanismer og signalveje og udvikling af metoder til at udnytte disse celler terapeutisk.
Tidligere protokoller har beskrevet isoleringen af perivaskulære precursor celle-undergrupper fra …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors wish to thank Shonna Johnston, Claire Cryer, Fiona Rossi and Will Ramsay at the University of Edinburgh and Alison Logar and Megan Blanchard at the University of Pittsburgh for their expert assistance with flow cytometry. We also wish to thank Anne Saunderson and Lindsay Mock for their help with obtaining human tissues. Human adult and fetal heart tissue samples were procured with full ethics permission of the NHS Scotland Tayside Committee on Medical Research Ethics and the NHS Lothian Research Ethics Committee (REC08/S1101/1) respectively. This work was supported by grants from the Medical Research Council (BP), British Heart Foundation (BP), Commonwealth of Pennsylvania (BP), Children’s Hospital of Pittsburgh (BP), National Institute of Health R01AR49684 (JH) and R21HL083057 (BP), and the Henry J. Mankin Endowed Chair at University of Pittsburgh (JH). JEB was supported by a British Heart Foundation Centre of Research Excellence doctoral training award (RE/08/001/23904). WC was supported in part by an American Heart Association predoctoral fellowship (11PRE7490001).
Materials
| AbC Anti-mouse Bead Kit | Molecular Probes | A-10344 | |
| Collagenase I | Gibco | 17100-017 | Reconstitute powder as required and filter sterilise |
| Collagenase II | Gibco | 17101-015 | |
| Collagenase IV | Gibco | 17104-019 | |
| anti-human CD34-PE | BD Pharmingen | 555822 | Keep sterile |
| anti-human CD45-APC-Cy7 | BD Pharmingen | 557833 | Keep sterile |
| anti-human CD56-PE-Cy7 | BD Pharmingen | 557747 | Keep sterile |
| anti-human CD144-PerCP-Cy5.5 | BD Pharmingen | 561566 | Keep sterile |
| anti-human CD146-AF647 | AbD Serotec | MCA2141A647 | Keep sterile |
| EGM2-BulletKit | Lonza | CC-3162 | For collection of cells and culture until adhered |
| DMEM, high glucose, GlutaMAX without sodium pyruvate | ThermoFischer Scientific | 10566-016 | |
| Fetal Bovine Serum | ThermoFischer Scientific | 10500-064 | Freeze in aliquots and keep sterile |
| Gelatin | Sigma Aldrich | G1393 | Dilute with sterile water |
| IgG1k-PE | BD Pharmingen | 559320 | Keep sterile |
| IgG1k-APC-Cy7 | BD Pharmingen | 557873 | Keep sterile |
| IgG1k-PE-Cy7 | BD Pharmingen | 557872 | Keep sterile |
| IgG1k-PerCP-Cy5.5 | BD Pharmingen | 561566 | Keep sterile |
| IgG1k-647 | AbD Serotec | MCA1209A647 | Keep sterile |
| Mouse serum | Sigma Aldrich | M5905 | Keep sterile |
| Paraffin Film – Parafilm M | Sigma Aldrich | P7793 | |
| Penicillin-Streptomycin | Gibco | 15979-063 | Freeze in aliquots and keep sterile |
| Phosphate buffered saline pH 7.4 | ThermoFischer Scientific | 10010-023 | Keep sterile |
| Red Blood Cell Lysing Buffer Hybri-Max | Sigma Aldrich | R7757 | Keep sterile |
| Trypan Blue Solution | Sigma Aldrich | T8154 | |
| Trypsin-EDTA 0.5%(10X) | Invitrogen | 15400-054 | |
| FACSARIA FUSION | BD Pharmingen | Fluorescence Activated Cell Sorter |
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