Konkurrencedygtige Transplantationer at vurdere hæmatopoietisk stamcelletransplantation Fitness
Summary
This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.
Abstract
The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.
Introduction
Hæmatopoiese er en regenerativ proces, der sikrer påfyldning af blodlegemer, der er gået tabt gennem skade, stråling og celledød. Denne proces sikres ved hæmatopoietiske stamceller (HSC), der stort set er bosiddende i den voksne knoglemarven. Desuden kan hæmatopoietiske stamceller anvendes til terapeutiske formål i autoimmune sygdomme, hæmatologiske maligniteter og immundefekter 1. Der er derfor behov for bedre at forstå de mekanismer, der regulerer HSC funktion, herunder deres proliferative ekspansion og deres evne til at nå og indpode modtageren knoglemarv efter transplantation. Selv om de seneste undersøgelser har rapporteret adskillige celleoverflademarkører, herunder SLAM familiemedlemmer CD150 og CD48, til fremadrettet berige voksne HSC'er og føtale HSC'er til ca. 50% renhed 2-4, guldstandarden foranstaltning til funktionel HSC'er forbliver et in vivo repopulating assay til bestemme deres evne til at genetablere blod cell produktion i en bestrålet vært fem.
In vivo klonal repopulation assay blev oprindeligt udviklet af Till og McCulloch 6 og er siden blevet forfinet og udvidet. Som oprindeligt defineret, HSC'er sikre produktionen livslang blodlegemer gennem selvstændig fornyelse og differentiering. Overførslen af HSC'er i en bestrålet recipient tillader således os at vurdere: deres evne til at differentiere gennem en analyse af de forskellige blodcellelinjer (T-lymfocytter, B-lymfocytter, granulocytter, monocytter) og deres evne til selvfornyelse via seriel transplantation. Analysen vil normalt indebære en sammenligning af funktionalitet og / eller mængden af to populationer af HSC'er, fx celler, der kommer fra to mus af forskellige genotyper eller celler, der er blevet behandlet eller ubehandlet med forskellige faktorer, der kan påvirke vedligeholdelse eller udvidelse af HSC'er i kultur. Donorkimærisme, eller bidrag overført donor HSC'er to blodlegemer kan derefter bestemmes ved hjælp af flowcytometri analyse i det perifere blod og knoglemarv under anvendelse celleoverflademarkører eller andre metoder, der vil skelne donorceller fra modtageren, eller vært. De mest anvendte markører er helt klart de to alleler for det gen Ptprc eller CD45 leukocyt antigen 7, som vi har valgt til eksempler nedenfor.
Den klonale repopulation analysen kan enten være konkurrencedygtige eller ikke-konkurrencedygtige. I en ikke-kompetitiv indstilling, er kontrol- og test- HSC'er overført til separate recipientmus og resultaterne for hver celletype vil være uafhængigt af den anden. I en konkurrencepræget indstilling, er funktionen af både test og kontrol HSC'er målt mod en befolkning på konkurrent HSC'er. Den her beskrevne protokollen bruger den konkurrencemæssige indstilling, men kan også tilpasses til ikke-konkurrencesituationer. Begge metoder har deres fordele og begrænsninger, og vi vil sammenligne dem i detaljer idiskussion. Vi beskriver også forskellige strategier for at sikre lighed i antallet af transplanterede HSC'er, forklare, hvordan at tilpasse analysen til kvantificering af HSC'er ved at begrænse fortynding assay (LDA), og give eksempler på både vellykkede og mislykkede transplantationer for fortolkning af resultaterne.
Protocol
Representative Results
Discussion
Den her beskrevne protokollen er designet til at evaluere den relative egnethed af donor (test) HSC'er mod kendt konkurrent HSC'er. Situationen for konkurrence øger den relative sensitivitet (mere tilbøjelige til at opdage moderate fald i stamceller fitness) og giver en intern teknisk kontrol for effekten af bestråling og injektion. Det bør dog ikke anvendes som et absolut mål for HSC fitness; et fald i konkurrencemæssig rekonstituering betyder ikke automatisk, at HSC'er ikke ville klare sig godt…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi er taknemmelige for Roxann Hetu-Arbour for hjælp med figuren design og demonstration af procedurerne. Forskning i laboratoriet blev understøttet af en Transition pris fra Cole Foundation, Discovery give no. 419226-2012 fra naturvidenskab og teknik Forskningsråd of Canada (NSERC) og Canada Foundation for Innovation (CFI Leaders Fund give nr. 31377). KMH er en chercheur-Boursier Junior til Fonds de recherche du Québec – Santé (FRQS).
Materials
| Microtainer tubes with K2EDTA | BD Biosciences | 365974 | ||
| 20G needle | BD Syringe | For blood sampling from the mandibular vein | ||
| LabQuake Shaker rotisserie | Thermo Scientific | C415110 | Any other rotating mixer will work as well to prevent coagulation of blood samples | |
| Purified anti-mouse CD16/CD32 (clone 2.4G2, Fc Block) | BD Biosciences | 2.50 | 553142 | Alternatively use clone 93 from eBioscience (cat # 14-0161) or Biolegend (cat# 101310) |
| Pe-Cy7-conjugated anti-mouse CD3e (clone 145-2C11) | eBioscience | 0.25 | 25-0031 | For most flow cytometry antibodies, the clone is important but the colours and companies can vary depending on the available equipment |
| PE-conjugated anti-mouse CD19 (clone 1D3) | eBioscience | 0.25 | 12-0193 | |
| APC-eFluor780 (APC-Cy7 equivalent)-conjugated anti-mouse GR1 (clone RB6-8C5) | eBioscience | 0.25 | 47-5931 | |
| FITC-conjugate anti-mouse CD45.1 (clone A20) | eBioscience | 2.50 | 11-0453 | |
| eFluor450-conjugated anti-mouse CD45.2 (clone 104) | eBioscience | 1.00 | 48-0454 | |
| Biotinylated anti-human/mouse CD45R (B220) (clone RA3-6B2) | eBioscience | 1.25 | 13-0452 | |
| Biotinylated anti-mouse CD3e (clone 145-2C11) | eBioscience | 1.25 | 13-0031 | |
| Biotinylated anti-mouse CD11b (clone M1/70) | eBioscience | 1.25 | 13-0112 | |
| Biotinylated anti-mouse GR1 (clone RB6-8C5) | eBioscience | 1.25 | 13-5931 | |
| Biotinylated anti-mouse TER119 (clone TER119) | eBioscience | 0.63 | 13-5921 | |
| V500 streptavidin | BD Biosciences | 0.50 | 561419 | |
| PE-conjugated anti-mouse CD117 (clone 2B8) | BD Biosciences | 0.25 | 553355 | |
| PE-Cy7-conjugated anti-mouse Ly6A/E (Sca1) (clone D7) | BD Biosciences | 0.25 | 558162 | |
| PerCP-eFluor710-conjugated anti-mouse CD135 (clone A2F10) | eBioscience | 0.50 | 46-1351 | |
| Alexa fluor 647-conjugated anti-mouse CD150 (clone TC15-12F12.2) | Biolegend | 0.63 | 115918 | BD Biosciences and eBioscience do not carry the same clone |
| 1ml tuberculin syringe with 27G needle | BD Syringe | 309623 | ||
| 1ml tuberculin syringe with 25G needle | BD Syringe | 309626 | ||
| 70 um cell strainer | BD Falcon | 352350 |
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