Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.

TLDR

Hematopoietic stem cells are multipotent bone marrow cells that continuously replenish all adult blood lineages. Dual‑wavelength Hoechst 33342 staining identified a distinct side‑population of HSC, and blocking dye efflux enabled DNA‑content analysis of these cells. The Hoechst side‑population contains the vast majority of HSC activity, is 1,000‑fold enriched, protects recipients from lethal irradiation at low doses, contributes to lymphoid and myeloid lineages, includes 1–3% cycling cells, and its purification shows cycling and quiescent HSC have equivalent reconstitution capacity, highlighting implications for hematopoietic development and gene‑therapy.

Abstract

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the animal. While experimenting with staining of murine bone marrow cells with the vital dye, Hoechst 33342, we discovered that display of Hoechst fluorescence simultaneously at two emission wavelengths revealed a small and distinct subset of whole bone marrow cells that had phenotypic markers of multipotential HSC. These cells were shown in competitive repopulation experiments to contain the vast majority of HSC activity from murine bone marrow and to be enriched at least 1,000-fold for in vivo reconstitution activity. Further, these Hoechst-stained side population (SP) cells were shown to protect recipients from lethal irradiation at low cell doses, and to contribute to both lymphoid and myeloid lineages. The formation of the Hoechst SP profile was blocked when staining was performed in the presence of verapamil, indicating that the distinctly low staining pattern of the SP cells is due to a multidrug resistance protein (mdr) or mdr-like mediated efflux of the dye from HSC. The ability to block the Hoechst efflux activity also allowed us to use Hoechst to determine the DNA content of the SP cells. Between 1 and 3% of the HSC were shown to be in S-G2M. This also enabled the purification of the G0-G1 and S-G2M HSC had a reconstitution capacity equivalent to quiescent stem cells. These findings have implications for models of hematopoietic cell development and for the development of genetic therapies for diseases involving hematopoietic cells.