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B. Nowlan et al.
Identifying HSC in inbred mouse strains that either do not or poorly express SCA1, such as BALB/c or non-obese diabetic (NOD) mice,7,8 or when treatments affect SCA1 expression is challenging. The SCA1 antibody detects LY6A and LY6E, which are two similar proteins of the LY6 phosphatidylinositol-anchored membrane proteins antigen family encoded by two different genes.9 LY6E is expressed by 10-15% of blood leukocytes, whereas LY6A is expressed by 50-70% of leukocytes.8 Inbred strains with the LY6.1 haplotype (e.g., BALB/c, C3H, DBA/1, CBA, FVB/N) do not express LY6A. This causes reduced SCA1 expression, thus compromising the classical method of identifying the HSC population based on the LSK pheno- type.3,8 Furthermore, even though the NOD strain and other immunodeficient strains on the NOD background are from the LY6.2 haplotype, they also express low levels of SCA1.10 In addition, SCA1 expression can be affected by treatments such as irradiation, bacterial infections, and interferons which cause a transient increase in SCA1 expression in Lin- KIT+ (LK) cells in C57BL/6 mice11,12 fur- ther questioning the suitability of SCA1 antigen to charac- terize HSC in challenged mice.
The combination of CD27 and CD201 (endothelial pro- tein C receptor – EPCR) has been proposed as an alterna- tive to SCA1/c-kit staining for HSC identification in mouse strains with low expression of SCA1 or following irradia- tion.13 It was demonstrated that Lin- CD27+ CD201+ cells contained all HSC activity tested in a long-term competi- tive repopulation assay in lethally irradiated recipient mice and this HSC phenotype remained consistent in several mouse strains, including BALB/c and NOD, or following irradiation.13
Several reports suggest that mouse HSC express both CD27 and CD201.14,15 CD27 is a member of the tumor necrosis factor receptor family expressed on T, B, and nat- ural killer (NK) cells, involved in proliferation, differentia- tion, and IgG production. CD27 was detected on 90% of LSK cells in C57BL/6 mice.15 Likewise, high expression of CD201 was also observed on 90% of LSK cells.14 CD201+ cells are multipotent in both colony assays and mouse transplant reconstitution. CD201 and CD150 are co- expressed in the embryonic mouse hematopoietic develop- ment of a long-term reconstituting population of HSC throughout life.16,17 In addition, CD201 is also expressed on multipotent human CD34+ HSC,18 showing that the pat- tern of CD201 expression is conserved between human and mouse HSC, unlike that of the CD34 antigen.6 As few HSC markers are shared between both species, this is becoming a significant cross-species HSC marker.
Recently, the use of NOD.CB17-prkdcscid il2rgtm1Wj1/Sz (NSG) mice for human xenografts has increased19-21 relative to the parental (NOD.CB17-prkdcscid/Sz, NOD-scid) mice. NSG mice do not express functional interleukin-2 receptor and therefore lack NK cells in addition to their lack of B and T cells from the parental NOD-scid strain, resulting in more profound immunosuppression and making the animals more amenable to human xenograft engraftment.21
Metastatic cancer cells and human HSC can hijack the mouse BM HSC niche,22 thus any treatments affecting xenografts should also be examined for the drugs’ effects on the host mouse HSC content in order to detect potential adverse effects of the drugs. However, there are no reliable flow cytometry methods to assess the impact of human xenografts or prototype anti-cancer therapies on the host mouse HSC in these strains.
In this study, we examined CD27 and CD201 expression on BM cells in NOD-scid and NSG mice. We demonstrate that staining protocols using CD27 and CD201 with FLT3, CD48, and CD150 are complementary to enrich functional HSC in these strains. These antibodies could be combined to prospectively enrich HSC as validated by serial dilution transplantations in recipient mice. We also investigated the overexpression of CD48 in NOD-scid and NSG mice. Furthermore, we identified that low SCA1 expression was limited to hematopoietic cells, whereas BM mesenchymal stromal cells (MSC) and endothelial cells expressed SCA1 at levels similar to those in C57BL/6 mice.
Methods
Mice
Mouse experiments were approved by both the University of Queensland and Queensland University of Technology Animal Ethics Committees. C57BL/6 and NOD-scid mice were purchased from the Australian Resource Centre (Cannin Vale, WA, Australia). NSG mice (Jackson Laboratories, Bar Harbor, ME, USA), were bred at the Translational Research Institute Biological Research Facility (Brisbane, Australia). Mice were 7-8 weeks old at the time of the experiments.
Sample isolation
BM was flushed from femurs using phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS). Spleens were har- vested from mice and processed in PBS and 2% FBS using the Miltenyi gentleMACS and a C-type tube (Bergisch Gladbach, Germany). Blood was collected via cardiac puncture into 3.2% sodium citrate. Each fraction was counted using a Coulter AcT Diff Analyzer (Beckman Coulter).
To isolate BM stromal/endothelial cells, bones were harvested from NSG and C57BL/6 mice. BM was flushed and discarded and the bones were treated with 1 mg/mL collagenase type-1 (Worthington) as previously described.23 Blood cells were depleted using the EasySepTM Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (Cat. n. 19771 StemCell Technologies) following the manufacturer’s protocol.
BM-MSC were isolated from NSG femurs using a modification of a previously described protocol24 (see Online Supplementary Methods).
Flow cytometry
All antibodies and stains used are described in Online Supplementary Table S1.
HSC stains were applied to 5x106 BM cells, while lineage stains were applied to 106 BM or spleen cells. Cells were stained in PBS and 2% FBS containing 0.1 mg/mL purified rat anti-CD16/CD32 (Fc Block) (BD Bioscience), with the appropriate antibody cocktail. The cells were then washed and resuspended in PBS plus 2% FBS containing 2 mg/mL dead cell discriminator dye 7-amino-actino- mycin D (7-AAD) (Invitrogen) and analyzed on a CyAn flow cytometer (Beckman Coulter).
Stromal and endothelial cells were stained with an “endosteal” stain (Online Supplementary Methods and Online Supplementary Table S1). Samples were analyzed on a Fortessa flow cytometer (BD Bioscience).
Flow cytometry data were analyzed with FlowJo v10 software (FlowJo LLC, Ashland, OR, USA).
Transplantations
Male donor BM cells were enriched for c-KIT by magnetic-acti-
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haematologica | 2020; 105(1)