Varying levels of aldehyde dehydrogenase activity in adult murine marrow hematopoietic stem cells are associated with engraftment and cell cycle status

Aldehyde dehydrogenase (ALDH) activity is a widely used marker for human hematopoietic stem cells (HSCs), yet its relevance and role in murine HSCs remain unclear. We found that murine marrow cells with a high level of ALDH activity as measured by Aldefluor staining (ALDHbr cells) do not contain known HSCs or progenitors. In contrast, highly enriched murine HSCs defined by the CD48−EPCR+ and other phenotypes contain two subpopulations, one that stains dimly with Aldefluor (ALDHdim) and one that stains at intermediate levels (ALDHint). The CD48−EPCR+ALDHdim cells are virtually all in G0 and yield high levels of engraftment via both intravenous and intrabone routes. In contrast the CD48−EPCR+ALDHint cells are virtually all in G1, have little intravenous engraftment potential, and yet can engraft long-term after intrabone transplantation. These data demonstrate that Aldefluor staining of unfractionated murine marrow does not identify known HSCs or progenitors. However, varying levels of Aldefluor staining when combined with CD48 and EPCR detection can identify novel populations in murine marrow including a highly enriched population of resting HSCs and a previously unknown HSC population in G1 with an intravenous engraftment defect.