Isolation of CD34− and CD34+ Leukemia Stem Cells from Acute Myeloid Leukemia Blasts Using CD200
Authors: Ho Jenny M. , Dobson Stephanie M. , McLeod Jessica , Voisin Veronique , Murison Alex , Kennedy James , Zandi Sasan , Eppert Kolja , Minden Mark D. , Lupien Mathieu , Dick John E. , Wang Jean C.Y. 
Published: October 2019

Abstract
Therapy resistance and relapse in acute myeloid leukemia (AML) are driven by leukemia stem cells (LSCs). Recent evidence highlighting functional and genetic heterogeneity among LSC subclones underscores the importance of capturing the entire LSC compartment in studies of LSC biology. Although LSCs are often enriched in the CD34+CD38- cell fraction, they are frequently detected in other phenotypic fractions, and in some cases are restricted to the CD34- population. In order to discover novel LSC markers, we examined genes differentially expressed between functionally-validated LSC+ and LSC- cell fractions obtained from primary AML samples, and identified CD200 as a candidate cell surface marker for LSCs.
CD200 expression in 57 primary AML samples was analyzed by flow cytometry using anti-human CD200 clone 1B9(kindly provided by Trillium Therapeutics Inc.). CD200 was present on a greater proportion of CD45dim blasts compared to more differentiated CD45high non-blast populations (54.4% versus 21.7%, p<0.0001); CD200+ cells often represented a distinct blast population. Overall there was a positive but non-linear correlation (R2=0.46, p<0.0001) between CD34 and CD200 expression; the proportion of CD200+ blasts was significantly greater than that of CD34+ blasts in samples with low to intermediate CD34 expression. In AMLs where CD34 expression was lower than CD200, CD34 was present on a subset of CD200+ blasts; accordingly, CD200+ blasts comprised variable proportions of CD34- and CD34+ cells. These observations suggest that CD200 expression on blasts could be a better marker for LSCs than CD34.
To validate CD200 as a LSC marker, leukemic blasts were sorted from 15 primary AML samples based on CD45 and CD200 expression and transplanted into NSG mice. Samples were selected based on either the presence of both CD200+ and CD200- blasts, or CD200 expression on <5% of mononuclear cells (MNCs). In 8 of 15 AMLs, LSCs were enriched within the CD200+ fraction (termed CD200+ LSCs). In 4 of these cases, LSCs comprised both CD34- and CD34+ cells, but the entire LSC compartment was captured within the CD200+ blast population. Limiting dilution studies showed that CD200 expression enriched for CD200+ LSCs by up to 20-fold. In 6 of the remaining samples, LSCs resided in the CD200- fraction (termed CD200- LSCs), while in one sample, LSCs were present in both CD200+ and CD200- fractions. In AMLs with CD200- LSCs, CD200 was expressed on <5% of MNCs and <5% of blasts, in contrast to AMLs with CD200+ LSCs where CD200 expression on MNCs was variable (3% to 85%) and >5% on blasts. Overall, these results indicate that CD200 expression can be used to segregate LSCs from bulk leukemia cells.
CD200 expression may be a particularly useful LSC marker in cytogenetically normal AMLs with NPM1 mutation (CN-AMLNPM1c), which have low or negative CD34 expression and commonly possess CD34- LSCs. Among 20 CN-AMLNPM1c samples, the proportion of CD200+ blasts was higher than that of CD34+ blasts irrespective of FLT3-ITD status, although there was a trend towards higher CD200 expression in FLT3-ITD+ samples. In xenotransplantation assays, 7 of 8 CN-AMLNPM1c samples tested contained CD200+ LSCs while the remaining sample contained both CD200+ and CD200- LSCs. Principal component analysis of gene expression profiles demonstrated that functionally-validated CD200+ LSC-containing fractions from CN-AMLNPM1c patients clustered separately from LSC fractions from NPM1wt or cytogenetically-abnormal cases, and were enriched for stem cell genes by gene set enrichment analysis. Furthermore, ATAC-Seq analysis demonstrated greater chromatin accessibility in CD200+ LSC-containing versus CD200‒ LSC-depleted fractions from CN-AMLNPM1c patients, with unique enrichment of HOX motifs. These data validate CD200 as an LSC marker in CN-AMLNPM1c cases.
In summary, CD200 is a valuable tool for capturing heterogeneous LSC populations including both CD34+ and CD34- LSCs in many primary AML samples. It will be particularly useful for future studies of LSCs in CN-AMLNPM1c where CD34 expression does not identify LSCs.

Disclosures
No relevant conflicts of interest to declare.