Differential Expression of CD49f Discriminates the Independently Emerged Hematopoietic Stem Cells and Erythroid-Biased Progenitors

While hematopoietic stem cells (HSCs) can sustain the production of all types of mature blood cells throughout the life, there also exists HSC-independent hematopoiesis, which partially supports embryonic hematopoiesis and generation of specific types of adult hematopoietic cells (e.g., macrophages). Examples of the HSC-independent hematopoiesis include (i) the primitive wave of hematopoiesis that produces unipotent progenitors for erythrocytes, megakaryocytes or macrophages, and (ii) the "pro-definitive" hematopoiesis that produces multipotent erythro-myeloid progenitors (EMPs). Given that HSCs and HSC-independent progenitors are both derived from endothelial cells in distinct or overlapping hematopoietic sites, tracing their developmental origins and clarifying the regulatory mechanism will enhance our understanding of the profound difference between them and may improve in vitro generation of HSCs.

Human HSCs have been refined based on the expression of CD49f (ITGA6). In combination with other HSC markers (CD34⁺CD38^-CD45RA^-CD43⁺CD90⁺), high expression of CD49f identifies long-term multilineage engrafting HSCs, whereas the cells with low CD49f represent a subtype of hematopoietic progenitor cells (HPCs) that possess transient engrafting activity. Meanwhile, CD49f has also been shown to be heterogeneously expressed in hemogenic endothelial cells (HECs), which give rise to both HSCs and EMPs via endothelial-to-hematopoietic transition (EHT). Thus, determining the changes (i.e., persistence, gain or loss) of CD49f expression during EHT is a key step in tracing the origins of HSCs and HSC-independent HPCs.

In this study, using an in vitro system of HSC differentiation from human embryonic stem cells (hESCs), we observed that, while CD49f is highly expressed in all hESCs, only a portion of HECs express CD49f. Importantly, live cell imaging analysis revealed that CD49f expression persists during EHT, which is accompanied by initiating CD43 expression. To test whether the differential CD49f expression is associated with HSC versus HPC functions, we sorted the CD49f^high and CD49f^low cells and performed colony forming assay and gene expression profiling. The results showed that the CD49f^high cells have multilineage potential, whereas the CD49f^low cells lack lymphoid potential but show a strong erythroid preference. Gene expression analysis confirmed that the CD49f^high and CD49f^low cells represent HSCs and erythroid-biased HPCs, respectively, and that the Wnt and Notch signaling pathways may play a role in their functions. Collectively, these observations suggest that the CD49f^high and the CD49f^low cells are concurrently derived from the CD49f^high and CD49f^low HECs, thus modeling the in vivo generation of HSCs and HSC-independent HPCs.

Based on the in vitro observations, we proposed that CD49f in vivo may also specify the distinct HSPCs emerged at different developmental stages/sites. To test this hypothesis, we isolated mouse primitive HPCs, EMPs and definitive HSCs, as well as their parental HECs, from yolk sac, embryo, and aorta-gonad-mesonephros (AGM) of different embryonic stages and determined their CD49f expression. The results showed that the primitive erythroid progenitors have lowest, whereas the definitive AGM HSCs have highest, CD49f levels; this trend was also observed in the related HECs isolated from various stages/sites. Thus, it is likely that the embryonic hematopoiesis is recapitulated, at least partially, by the in vitro system in terms of the sequential emergence of HSPCs ranging from unipotent erythroid progenitors to multipotent definitive HSCs, and this may also underlie the situation that EMPs and HSCs can be produced at the same stage/site but independently from different HECs.

In summary, using the in vitro HSC differentiation system, we found that the differential expression of CD49f discriminates HSCs and HSC-independent progenitors, which are concurrently emerged from HECs. The persistent CD49f expression during EHT suggests that the fates of HSCs and HSC-independent HPCs are pre-defined in their parental HECs. Combining our in vivo data, the differential expression of CD49f also provide a possible regulatory mechanism for the multi-wave hematopoiesis. Further exploring the function and mechanism of CD49f in these regulations should be important for fully understanding the precisely regulated HSC generation and activities.