Hematopoietic origin of most CD45– bone marrow cells

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 show, unexpectedly, that the vast majority of TNCs (~85%) have a hematopoietic rather than mesenchymal origin. Single cell RNA-sequencing reveals erythroid and lymphoid progenitor signatures among CD51– TNCs. When cultured with BM-derived stromal cells, Ly6D+ CD44+ CD51–TNCs give rise to B-lymphoid cells, whereas Ly6D–CD44+ CD51–TNCs generate erythroid cells. In addition, CD44+ CD51– TNCs contribute to repopulate B-lymphoid and erythroid cells after transplantation in mice. The CD44+ CD51– TNC population also expands during phenylhydrazine-induced acute hemolysis or in a model of sickle cell anemia. These findings thus uncover physiologically relevant, yet unappreciated, classes of stromal-associated CD45– hematopoietic progenitors. Single CD44+ CD51- and CD44- CD51- TNCs were sorted and sequenced on an Illumina Nextseq500