CD27 marks murine embryonic hematopoietic stem cells and type II prehematopoietic stem cells

To the editor:

Cell surface markers of adult hematopoietic stem cells (HSCs) are well established, but fewer markers are available for embryonic HSCs and their immediate precursors (pre-HSCs). HSCs differentiate from hemogenic endothelial cells in the major arteries of the embryo [dorsal aorta, umbilical, vitelline (A+U+V)]. They briefly reside in hematopoietic clusters attached to the arterial wall, then are released into the circulation to colonize the fetal liver (FL). All of the >500 hematopoietic cluster cells (HCCs) express Kit, a marker of FL and bone marrow (BM) HSCs, and endothelial markers, such as vascular endothelial cadherin (CD144) and CD31.¹  In mouse embryos, the few (1-3) adult repopulating HSCs within the clusters are Kit⁺, CD31⁺, CD144⁺, CD45⁺, CD43⁺, CD41^low, CD201^high, and Ly6a-GFP⁺ and lack expression of the BM and FL HSC markers Sca1 and CD150.^2-7  Clusters also contain ∼65 immature HSCs called pre-HSCs that cannot engraft adult recipients directly, but can be matured ex vivo into HSCs that can engraft.⁸  Pre-HSCs [and less mature precursors (pro-HSCs)] are CD144⁺CD41⁺Kit⁺ and can be subdivided based on CD43 and CD45 expression into (from least to most mature) pro-HSCs (CD43^–CD45^–), type I pre-HSCs (CD43⁺CD45^–), and type II pre-HSCs (CD43⁺CD45⁺).^6,9  Also in the major arteries are ∼500 to 1000 progenitors with lymphoid potential, which share markers with type II pre-HSCs/HSCs.^10,11 

The transgene Ly6a-GFP marks all progenitors with lymphoid potential, pre-HSCs, and HSCs within HCCs in the major arteries (A+U+V)^11,12  (Marijke Maijenburg, Joanna Tober, et al, unpublished data), and HSCs differentiate from Ly6a-GFP⁺ endothelial cells in vivo.¹³  To identify novel cell surface markers that could be used in lieu of the Ly6a-GFP transgene to further enrich pre-HSCs and HSCs, we determined the gene expression profile of embryonic day (E) 11.5 Ly6a-GFP⁺ HCCs (CD31⁺CD144⁺ESAM⁺Kit⁺) by RNA sequencing (GEO accession number GSE97509), and compared it to profiles of Ly6a-GFP^- HCCs, Ly6a-GFP⁺ endothelial cells (CD31⁺CD144⁺ESAM⁺Kit^–), and Ly6a-GFP^– endothelial cells (Figure 1A-B; supplemental Table 1, available on the Blood Web site). A total of 1380 genes were differentially expressed across the 4 populations. We performed consensus clustering of these genes (Figure 1C). Cluster 3, which consists of 173 genes with higher expression in Ly6a-GFP⁺ HCCs compared with the other 3 populations, generated enriched gene ontology terms associated with leukocyte functions, such as “regulation of defense response,” “leukocyte migration,” and “B-cell activation” (Figure 1C-D; supplemental Table 2). Among cluster 3 genes were 14 encoding cell surface proteins, including known markers of embryonic HSCs, such as Spn (encoding CD43)⁶  and Gpr56¹⁴  (Figure 1E).

To identify new markers of embryonic HSCs and pre-HSCs, we analyzed Tnfrsf7, a gene in cluster 3 that encodes a BM HSC marker, CD27.^15,16  CD27 is a proinflammatory member of the tumor necrosis factor receptor superfamily that plays a costimulatory role in T-cell receptor–mediated immune responses^17,18  when activated by its ligand CD70. CD27 is expressed on lymphocytes, dendritic cells, on BM HSCs (in mice), and in humans on acute myelogenous leukemia (AML) blast cells.^15,16,18-20  CD27 signals through the canonical and noncanonical NF-κB pathways and the JNK pathway through TRAF family adaptors in lymphocytes and activates Wnt signaling in AML cells.^19-21 Tnfrsf7 transcripts were ∼10-fold higher in Ly6a-GFP⁺ HCCs than in the other 3 populations (Figure 1E-F), although CD27 protein was detectable on only 3.2% of the cells (Figure 2A-C). CD27 marked fewer HCCs than Ly6a-GFP; per embryo ∼560 HCCs were Ly6a-GFP⁺, 98 were CD27⁺, and 18 were Ly6a-GFP⁺CD27⁺ (Figure 2C). We fractionated CD31⁺ cells from the A+U+V based on CD27 expression and transplanted them into adult mice (Figure 2D-E). All HSCs in the A+U+V were in the CD31⁺CD27⁺ population (Figure 2E). Approximately 40% of cells in the CD48^–CD150⁺lineage^–Sca1⁺Kit⁺ (SLAM LSK) E14.5 FL population were CD27⁺ and contained most of the functional HSCs (Figure 2F-G). Back gating revealed that the CD27⁺ SLAM LSK cells were CD150^medium and clearly distinct from the CD27^– fraction, which was CD150^high (Figure 2F,H). Erythromyeloid progenitors (EMPs) emerge before HSCs²² ; the majority of CD45⁺ EMPs in the E11.5 yolk sac and FL were CD27^– (supplemental Figure 1A-C). In contrast, progenitors with lymphoid potential in the A+U+V, which emerge before HSCs,²²  were enriched in the CD27⁺ population at both E10.5 and E11.5 (supplemental Figure 1D-G), similar to the enrichment we reported for Ly6a-GFP.¹¹  In summary, CD27 marks all embryonic and FL HSCs, enriches for lymphoid progenitors in the major arteries, but is not expressed on most yolk sac–derived EMPs.

Pre-HSCs can be divided into type I and type II based on CD45 expression; type I pre-HSCs are CD144⁺CD45^–, whereas more mature type II pre-HSCs are CD144⁺CD45⁺. We fractionated type I and type II pre-HSCs based on CD27 expression and cultured them on endothelial cells transduced with a lentivirus expressing myristoylated AKT (AKT-EC), which support the maturation of pre-HSCs into HSCs^23,24  (Figure 2I-J). Cells derived from the cultures were then transplanted into adult mice. Both CD27⁺ and CD27^– type I pre-HSCs contributed to the peripheral blood (PB) of primary transplant recipients (Figure 2K), with contribution biased toward lymphoid lineages (supplemental Figure 2). HSCs matured from CD27⁺ and CD27^– type I pre-HSCs also contributed to BM SLAM LSK cells in primary transplant recipients. However, CD27⁺ type I pre-HSCs contributed to <10% of BM SLAM LSK cells and only engrafted 4 out of 10 recipients (Figure 2L). Furthermore, donor BM from primary transplant recipients did not engraft secondary recipients (Figure 2M), indicating that CD27 does not mark type I pre-HSCs. In contrast, CD27⁺ type II pre-HSCs matured into HSCs that contributed to BM SLAM LSK cells in both primary and secondary transplant recipients (Figure 2L-M). In summary, CD27 is expressed on type II but not type I pre-HSCs.

In this study, we show that CD27 expression is upregulated in a subset of HCCs, and CD27 marks HSCs, type II pre-HSCs, and progenitors with lymphoid potential within the cluster cells. Whether CD27 is simply a marker of type II pre-HSCs and embryonic HSCs, or whether it plays a functional role remains to be determined. Activation of CD27 on BM hematopoietic stem and progenitor cells by CD70 inhibits their differentiation and increases their number.¹⁸  Similarly, activation of CD27 on human AML blasts inhibited differentiation and promoted self-renewal by increasing symmetric cell divisions.¹⁹  Therefore, a possible role for CD27:CD70 signaling in the embryo may be to retain newly emerging type II pre-HSCs/HSCs in an undifferentiated state. Regardless, the demonstration that CD27 marks type II pre-HSCs and embryonic HSCs should provide an additional tool for identifying and studying these rare cells in the murine embryo.