Abstract
Abstract 640
Phenotypic definition of murine and human hematopoietic stem cells (HSC) relies on a large number of markers. Few surface antigens with functional importance have been identified as key common markers on adult murine and human HSC. Intimate interactions between HSC and elements of the hematopoietic niche (HN) depend on ligand-counter ligand molecules co-expressed independently on niche components or putative HSC. To date, not a single surface marker has been identified as a common marker expressed on murine and human HSC and on cells of the HN. We previously demonstrated that CD166 is expressed on osteoblasts (OB) that mediate a hematopoiesis enhancing activity (HEA). Given that CD166 is a member of the immunoglobulin superfamily that can mediate homophilic cell-cell interactions, we investigated the role of CD166 in identifying HSC and the impact of CD166 on hematopoiesis, stem cell engraftment, and the HN. Interestingly, CD166+, but not CD166- fractions of murine and human repopulating HSC identified by a rigorous hierarchical classification for each species mediated robust long-term engraftment. In the murine system, 25 sorted Lineage- Sca1+ ckit+ (LSK) CD48-CD150+CD166+ cells mediated 69.5 ± 7.3% chimerism 4 months post-transplantation (PT) while donor-derived chimerism supported by 25 sorted LSKCD48-CD150+CD166- cells was only 13.6 ± 11.6% (p<0.01) suggesting that CD166 identifies long-term repopulating cells beyond what is possible with SLAM markers. In the human system, 1000 cord blood-derived Lin-CD34+CD38-CD49f-CD166+ cells and Lin-CD34+CD38-CD49f+CD166+ cells engrafted at 44.5 ± 9.7% and 38.4 ± 8.9%, respectively 16 weeks PT in conditioned NSG mice. More importantly, chimerism derived from Lin-CD34+CD38-CD49f+CD166- cells was 1.6 ± 0.1% (p<0.01 vs both fractions) demonstrating that the CD166+ but not the CD166- fraction of CD34+Lin-CD38- cells (regardless of the status of CD49f expression) contains long-term engrafting human HSC. In CD166 knockout (KO) mice, numbers of LSKCD48-CD150+ cells in the bone marrow and Lin-CD48- cells in the peripheral blood were significantly reduced relative to wild-type (WT) controls although other hematopoietic parameters in KO mice were within normal ranges. Phenotypically defined HSC from CD166−/− mice failed to engraft in lethally irradiated WT recipients. Levels of engraftment 4mo PT of 1,000 LSK cells from WT donors into WT recipients was 71.8% ± 8.3% while that obtained from a similar number of KO cells was 5.8% ± 2.8% (p<0.01). To permit direct comparison of KO and WT mice as recipients, both genotypes were transplanted with purified LSK cells from GFP C57BL/6 mice. While short-term repopulating GFP cells engrafted efficiently in KO mice 1mo PT (66.0% ± 6.5%), reconstitution declined substantially 2mo PT and was 10.3% ± 2.7% at 3mo PT (compared to 52.6% ± 10.4% in WT hosts, p<0.01) and less than 5% at 4mo PT, demonstrating that the CD166−/− hematopoietic niche can not support long-term repopulating cells. We used our previously described co-culture system to assess the impact of homophilic CD166 interactions on the HEA of OB. The highest HEA was reached when both OB and LSK cells expressed CD166. However, when either or both cell types lacked CD166 expression, the degree of HEA was significantly lower demonstrating that homophilic CD166 interactions are critical to maintaining HSC function. Since Stat3 has 3 binding sites on the CD166 promoter, we examined the relationship between expression of Stat3 and CD166. HSC from Stat3−/− mice which do not engraft efficiently in WT recipients expressed very low levels of CD166. In addition, pharmacologic inhibition of Stat3 expression led to a simultaneous inhibition of CD166 expression. Reconstitution kinetics data and survival of KO mice under hematopoietic stress conditions suggested that CD166−/− HSC have an intrinsic self-renewal capacity precluding them from both rapid proliferation and expansion and maintenance of the stem cell pool in the HN. Our data illustrate for the first time, that CD166 is a universal marker of both murine and human HSC and OB within the HN and suggest that CD166 may modulate HSC-niche interactions and impact stem cell fate. The conserved homology between murine and human CD166 provides an excellent bridge between human and murine studies for efficient translational investigations and interventions for enhancing stem cell engraftment and clinical utility.
Disclosures:
Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy.
AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates
