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-Kithi HSCs and c-Kitlo 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-Kithi HSCs formed 5-fold more colonies than c-Kitlo HSCs (P=0.01), as well as 4-fold more megakaryocyte colonies in vitro. c-Kithi HSC were 2.4-fold enriched for cycling cells (G2-S-M) in comparison to c-Kitlo HSC as assessed by flow cytometry in vivo (15.4% versus 6.4%, P=0.001). Lethally irradiated mice competitively transplanted with 400 c-Kitlo 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-Kithi 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-Kitlo HSCs from weeks 7 to 18, while HSC chimerism decreased from 18% to 3.0% in c-Kithi 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-Kithi or c-Kitlo HSCs, and evaluation of HSC bone marrow lodging at 24 hours post-transplantation demonstrated no difference in the number of c-Kithi and c-Kitlo 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-Kithi HSC maintained higher levels of c-Kit expression compared to those from mice injected with c-Kitlo HSC by week 18 post-transplantation (P=0.01). Secondary recipients serially transplanted with c-Kithi 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-Kitlo HSC. These results indicate that c-Kithi HSCs exhibit reduced self-renewal capacity compared with c-Kitlo HSCs, and that the differences in c-Kithi and c-Kitlo HSC function are cell-intrinsic. Analysis of transplanted HSC fates revealed that c-Kithi HSCs produced two-fold more pre-megakaryocyte-erythroid progenitors and pluriploid megakaryocytes compared to their c-Kitlo counterparts in vivo, suggesting a megakaryocytic lineage bias in c-Kithi HSC. Consistent with this finding, the transplanted c-Kithi HSC gave rise to 10-fold more platelets and reached a maximum platelet output two days earlier than c-Kitlo HSC. To determine the potential mechanisms underlying the transition from c-Kitlo to c-Kithi 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-Kitlo HSC compared to c-Kithi HSC (P=0.02), suggesting that functional loss of c-Cbl increases c-Kit expression on c-Kitlo 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-Kithi HSCs (P=0.067) and megakaryocyte progenitors (P=0.002), respectively. Thus, c-Cbl loss likely promotes the generation of c-Kithi HSCs. In summary, differential expression of c-Kit identifies HSC with distinct functional attributes with c-Kithi HSC exhibiting increased cell cycling, megakaryocyte lineage bias, decreased self-renewal capacity, and decreased c-Cbl activity. Since c-Kitlo 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. Disclosures: No relevant conflicts of interest to declare.