The Majority of CD45^– Ter119^– CD31^– Bone Marrow Cell Fraction Is of Hematopoietic Origin and Contains Erythroid and Lymphoid Progenitors

The non-hematopoietic cell fraction of the bone marrow (BM) is classically identified as CD45^– Ter119^– CD31^– (herein referred to as triple-negative cells or TNCs). Although TNCs are believed to contain heterogeneous stromal cell populations, they remain poorly defined. Here we showed that the vast majority of TNCs (∼85%) have a hematopoietic rather than mesenchymal origin. Single cell RNA-sequencing revealed erythroid and lymphoid progenitor signatures among CD51^– TNCs. Ly6D⁺ CD44⁺ CD51^– TNCs phenotypically and functionally resembled CD45⁺ pro-B lymphoid cells, whereas Ly6D^– CD44⁺ CD51^– TNCs were enriched in previously unappreciated stromal-dependent erythroid progenitors hierarchically situated between preCFU-E and proerythroblasts. Upon adoptive transfer, CD44⁺ CD51^– TNCs contributed to repopulate the B-lymphoid and erythroid compartments. CD44⁺ CD51^– TNCs also expanded during phenylhydrazine-induced acute hemolysis or in a model of sickle cell anemia. These findings thus uncover physiologically relevant new classes of stromal-associated functional CD45^– hematopoietic progenitors. Bone marrow triple-negative CD45^– Ter119^– CD31^– cells are thought to contain heterogeneous stromal cell populations. Boulais et al. show these cells are mostly hematopoietic in origin and contain previously unappreciated stromal-associated erythroid and B-lymphoid progenitor populations.