Differential Expression of c-Kit Identifies Hematopoietic Stem Cells with Variable Self-Renewal Potential.

Abstract 2325

Hematopoietic stem cells (HSC) can be identified on the basis of differential cell surface protein expression, such that 10 out of 13 purified HSC (Lin⁻c-Kit⁺Sca-1⁺CD150⁺CD34⁻FLK2⁻) exhibit long-term reconstitution potential in single-cell transplants. HSCs express c-Kit, and interactions between c-Kit and its ligand, stem cell factor, have been shown to be critical for HSC self-renewal; however, HSCs express a log-fold variation in c-Kit levels. We hypothesized that differing levels of c-Kit expression on HSCs may identify functionally distinct classes of HSCs. Thus, we measured the function and cellular characteristics of c-Kit^hi HSCs and c-Kit^lo HSCs (defined as the top 30% and bottom 30% of c-Kit expressors, respectively), including colony formation, cell cycle status, lineage fates, and serial engraftment potential. In methylcellulose colony assays, c-Kit^hi HSCs formed 5-fold more colonies than c-Kit^lo HSCs (P=0.01), as well as 4-fold more megakaryocyte colonies in vitro. c-Kit^hi HSC were 2.4-fold enriched for cycling cells (G2-S-M) in comparison to c-Kit^lo HSC as assessed by flow cytometry in vivo (15.4% versus 6.4%, P=0.001). Lethally irradiated mice competitively transplanted with 400 c-Kit^lo HSCs and 300,000 competitor bone marrow cells exhibited increasing levels of donor chimerism, peaking at a mean of 80% peripheral blood CD45 chimerism by 16 weeks post-transplantation, whereas mice transplanted with c-Kit^hi HSCs reached a mean of 20% chimerism (p<0.00015). Evaluation of the bone marrow revealed an increase in HSC chimerism from 23% to 44% in mice injected with c-Kit^lo HSCs from weeks 7 to 18, while HSC chimerism decreased from 18% to 3.0% in c-Kit^hi HSC-transplanted mice (P<0.00021). Levels of myeloid chimerism in the bone marrow and peripheral blood were not significantly different during the first 4 weeks following transplantation between mice transplanted with c-Kit^hi or c-Kit^lo HSCs, and evaluation of HSC bone marrow lodging at 24 hours post-transplantation demonstrated no difference in the number of c-Kit^hi and c-Kit^lo HSCs, indicating that differential homing is not the reason for the observed differences in long-term engraftment. Donor HSCs purified from mice transplanted with c-Kit^hi HSC maintained higher levels of c-Kit expression compared to those from mice injected with c-Kit^lo HSC by week 18 post-transplantation (P=0.01). Secondary recipients serially transplanted with c-Kit^hi HSC exhibited a chimerism level of 40% to 3% from week 4 to 8 post-secondary transplant, whereas chimerism levels remained at 6% in mice injected with c-Kit^lo HSC. These results indicate that c-Kit^hi HSCs exhibit reduced self-renewal capacity compared with c-Kit^lo HSCs, and that the differences in c-Kit^hi and c-Kit^lo HSC function are cell-intrinsic. Analysis of transplanted HSC fates revealed that c-Kit^hi HSCs produced two-fold more pre-megakaryocyte-erythroid progenitors and pluriploid megakaryocytes compared to their c-Kit^lo counterparts in vivo, suggesting a megakaryocytic lineage bias in c-Kit^hi HSC. Consistent with this finding, the transplanted c-Kit^hi HSC gave rise to 10-fold more platelets and reached a maximum platelet output two days earlier than c-Kit^lo HSC. To determine the potential mechanisms underlying the transition from c-Kit^lo to c-Kit^hi HSCs, we assessed the activity of c-Cbl, an E3 ubiquitin ligase known to negatively regulate surface c-Kit expression in a Src-dependent manner. Flow cytometric analysis revealed 6-fold more activated c-Cbl in freshly purified c-Kit^lo HSC compared to c-Kit^hi HSC (P=0.02), suggesting that functional loss of c-Cbl increases c-Kit expression on c-Kit^lo HSCs. Mice treated for nine days with Src inhibitors, which inhibit c-Cbl activity, experienced a 1.5-fold and 2-fold increase in the absolute number of c-Kit^hi HSCs (P=0.067) and megakaryocyte progenitors (P=0.002), respectively. Thus, c-Cbl loss likely promotes the generation of c-Kit^hi HSCs. In summary, differential expression of c-Kit identifies HSC with distinct functional attributes with c-Kit^hi HSC exhibiting increased cell cycling, megakaryocyte lineage bias, decreased self-renewal capacity, and decreased c-Cbl activity. Since c-Kit^lo HSC represent a population of cells enriched for long-term self-renewal capacity, characterization of this cell population provides an opportunity to better understand the mechanisms that regulate HSC function.