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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

Published: August 31st, 2016

DOI:

10.3791/54345

1INRS-Institut Armand-Frappier, Université du Québec, Institut National de la Recherche Scientifique

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.

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.

Hematopoiesis is a regenerative process that ensures the replenishing of blood cells that have been lost through injury, radiation and cell death. This process is ensured by hematopoietic stem cells (HSC) that largely reside in the adult bone marrow. In addition, hematopoietic stem cells can be used for therapeutic purposes in autoimmune disorders, hematological malignancies and immunodeficiencies1. There is thus a need to better understand the mechanisms that regulate HSC function, including their proliferative expansion and their ability to reach and engraft the recipient bone marrow after transplant. Although recent studies have reported several cell sur....

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All procedures described in this protocol have been approved by the institutional animal ethics committee and follow the Canadian Council on Animal Care guidelines.

Note: To maintain sterile/specific pathogen-free housing conditions, conduct all procedures involving direct handling of live mice inside a biological safety cabinet or a laminar flow hood. Clean or sterilize cages, restraining devices, housing materials, chow and water provided to the animals appropriately. Use only sterile, dispo.......

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A general description of the competitive transplant setting, including secondary transplants (discussed further below) can be found in Figure 1. A representative analysis for pre-transplant bone marrow HSCs can be found in Figure 2. More detailed information on the exclusion of doublets and dead cells can be found elsewhere9.

Figures 3 and 4 provide e.......

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The protocol described here is designed to evaluate the relative fitness of donor (test) HSCs against known competitor HSCs. The situation of competition increases the relative sensitivity of the assay (more likely to detect moderate decreases in stem cell fitness) and provides an internal technical control for the efficacy of irradiation and injection. However, it should not be used as an absolute measure of HSC fitness; a decrease in competitive reconstitution does not automatically mean that the HSCs would not perform.......

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We are grateful to Roxann Hétu-Arbour for assistance with the figure design and demonstration of the procedures. Research in the lab was supported by a Transition award from the Cole Foundation, Discovery grant no. 419226-2012 from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canada Foundation for Innovation (CFI Leaders Fund grant no. 31377). KMH is a Chercheur-Boursier Junior for the Fonds de recherche du Québec - Santé (FRQS).

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Name Company Catalog Number Comments
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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