Functional Resolution of Long-Term-and Short-Term- Hematopoietic Stem Cells

Several studies have demonstrated that the hematopoietic stem cell (HSC) compartment consists of long-term repopulating (LTR) and short-term repopulating (STR) HSC. Here we describe an improved purification approach that identifies both LTR- and STR- HSC as being lineage negative, c-kit positive, with very low Hoechst 33342 retention (Lin-, c-kit+, Holow). However, further selection of cells based on their differential retention of Rhodamine 123 resolves cells into LTR-HSC and STR-HSC. We show that our sort method highly enriches for LTR-HSC (Rhlow) and STR-HSC (Rhhigh), and demonstrate that the Rhlow cells as single transplanted cells are able to engraft 70% of mice in a competitive long-term repopulating assay. We then describe several in vitro assays that resolve Rhlow and Rhhigh cells based on the ability of single cells to survive, form clones, vary the time to their first cell division, express a high proliferative potential (HPP) or to generate HPP daughter cells at the 2- to 8-cell stage. In the presence of IL-3 alone, single Rhlow cells divided rarely and then formed only small clones (~8 cells). In contrast, Rhhigh readily divided in IL-3 alone and went on to form large clones (~10,000 cells). However, in the presence of IL-3+IL-6+SCF, both cell populations cloned in vitro with high efficiency (>90%), although the proportion of HPP clones was significantly higher in the Rhlow cell fraction (~90% vs ~40%). Furthermore, In addition, we show that the time required by Rhlow cells to undergo their first cell division in vitro is relatively non-synchronous and longer than that of Rhhigh cells. In addition, an analysis of daughter cells generated during the initial cell divsions of Rhlow or Rhhigh cells in vitro showed that expansion or maintenance of total HPP daughter cells occurred only in the Rhlow cell fraction. We measured the proliferative potential of daughter cells derived from single Rhlow and Rhhigh cells at the 2-8 cell stage. At the 2-cell stage, Rhlow cells generated an increased number of HPP daughter cells (↑1.4-fold) compared to Rhhigh cells that appeared to maintain the total number of HPP daughter cells (1.0-fold). However, by the 8-cell stage, the total number of HPP daughter cells generated by Rhlow cells expanded to nearly double that of starting HPP numbers (↑1.9 fold), compared to a decline in total HPP daughter cells in 8-cell Rhhigh clones (↓0.5 fold). Our studies at the 2-cell stage directly demonstrate symmetrical divisions (2 HPP per 2 daughter cells) that result in HPP expansion. Thus, these studies of growth factor responsivness of purified HSC (survival, cloning efficiency, time to the first cell division) and differentiation pathways (their ability to generate HPP daughter cells) identify means to differentiate LTR- and STR HSC in vitro.