GPI80 Distinguishes Transplantable Human Fetal Hematopoietic Stem Cells From Multipotential Progenitors

Abstract 1280

Hematopoietic stem cells (HSCs) are responsible for the lifelong maintenance of all the cells of the blood system. Self-renewal and differentiation of HSCs is tightly regulated by the complex interplay of extrinsic and intrinsic factors that work together to maintain a homeostatic balance. However, the regulatory mechanisms necessary to maintain the stemness of human HSCs are not well understood, largely due to lack of specific surface markers to purify HSCs. In order to facilitate a detailed study of the human long-term HSC and its niche environment, we performed genome-wide gene expression analysis comparing undifferentiated CD34⁺CD38⁻CD90⁺ human fetal hematopoietic stem and progenitor cells (HSPCs) and their progeny (CD34⁺CD38⁻CD90⁻) and attempted to identify novel markers specific to the long-term HSC. Expression analysis identified 58 differentially expressed candidate HSC surface markers, including GPI-80 (Vnn2), a GPI-anchored surface protein recently described in neutrophil adherence and migration. Flow cytometry revealed that only a subpopulation of CD34⁺CD38⁻CD90⁺ HSPCs expressed GPI-80. Strikingly, GPI-80 expression segregated HSPCs into two functionally distinct populations; NSG mice (NOD/Shi-scid IL2rgamma chain null mice) transplanted with CD34⁺CD90⁺GPI-80⁺ cells (herein noted as GPI-80⁺HSPCs) displayed robust, multilineage human hematopoietic reconstitution, whereas CD34⁺CD90⁺GPI-80⁻ cells (GPI-80⁻HSPCs) showed little to no hematopoietic engraftment. Furthermore, in vitro assays showed that while both populations were multipotent and generated myeloerythroid and B and T lymphoid cells in culture, only the GPI-80⁺HSPCs demonstrated the ability to self-renew on mouse mesenchymal stem cell stroma, whereas GPI-80⁻HSPCs displayed loss of self-renewal and subsequent differentiation. These results define GPI-80 as a novel HSC surface marker that can be used to purify transplantable HSCs during human development. To identify regulatory mechanisms that define the engraftable HSC, we selected differentially expressed genes between GPI-80⁺ and GPI-80⁻ HSPCs and assessed each set for enrichment of Gene Ontology (GO) categories associated with specific biological processes. There was no difference in genes regulating lineage differentiation, consistent with the finding that both populations are multipotential. However, GPI-80⁺HSPCs were enriched for genes related to cell migration, while the GPI-80⁻ progenitors were enriched for active cell cycle genes. This indicates that GPI-80 may mark a more pure population of quiescent human fetal HSCs that possess unique properties necessary for engraftment. Ongoing studies are focusing on investigating the role of GPI-80 in the homing and trafficking of fetal HSCs, and defining the mechanisms that facilitate interactions between the GPI-80⁺ HSC and its niche.