A Specialized Polarized Membrane Domain Found On Normal but Not Leukemic Hematopoietic Progenitor Cells Is Required for Homing to the Bone Marrow Microenvironment.

Abstract 82

Hematopoietic stem/progenitor cells (HSPCs) reside in a bone marrow niche, where adhesive interactions with osteoblasts provide essential cues for their proliferation and survival. In co-cultures of osteoblasts with primary human normal CD34+ cells, CD34+CD38- cells, or the KG1a progenitor cell line, we previously showed, using live cell imaging approaches, that HSPCs made prolonged contact with the osteoblast surface via a specialized membrane domain enriched in prominin 1 (CD133), the very late antigen-4 (VLA-4), the phosphatidylethanolamine (PE) analogue rhodamine-PE, and the tetraspanins CD63 and CD81. At the contact site, portions of the specialized domain of the CD34+ cells containing these molecules were taken up by osteoblasts and internalized into signaling endosomes within the osteoblasts. This caused the osteoblasts to downregulate Smad signaling and to increase their production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow (Gillette J. et al, Nat. Cell Biol. 11(3): 303–311, 2009). We have now evaluated the functional significance of these specialized membrane domains for in vivo homing of normal HSPCs to the bone marrow microenvironment. G-CSF mobilized human peripheral blood CD34+ cells from two normal donors were treated for 30 minutes with the cholesterol sequestration agent methyl-β-cyclodextrin (MβCD). This treatment resulted in disruption of the CD34+ cell membrane domains but had no effect on cell viability, proliferation or colony forming capacity in vitro. However, in two independent experiments, we observed a three-fold decrease in homing of MβCD-treated CD34+ cells to the bone marrow of NOD/SCID IL2rψc^null mice 16 hours after transplantation as compared to mock-treated CD34+ cells (p=0.0002). In contrast to homing studies, long-term human cell engraftment determined by CD45 cell surface expression 2 months after transplantation in two independent experiments was not significantly different in mice transplanted with MβCD-treated CD34+ cells compared to mock-treated CD34+ cells (p=0.13). Rapid repolarization of the membrane domain after transplantation may have resulted in engraftment of MβCD-treated CD34+ cells at levels similar to those observed with mock-treated CD34+ cells. Given the known homing/engraftment defect of actively cycling HSPC, we compared membrane domains on quiescent and cycling CD34+ cells from two normal donors. At baseline, 55–68% of CD34+ cells were in the G0 phase of the cell cycle as measured by Hoechst/Pyronin Y staining and specialized membrane domains were detected on a similar percentage of CD34+ cells for both donors. After culture for 4 days in the presence of stimulatory cytokines (SCF, TPO and Flt3), less than 10% of the CD34+ cells remained in G0 and, similarly, less than 10% of the cells analyzed by microscopy had a specialized membrane domain. After 4 days, cells were continued in culture for 2 days under non-stimulatory conditions (SCF alone). Under these conditions, for one donor, the percentage of cells in G0 increased from 9% to 18% and the percentage of cells with membrane domains increased similarly from 6% to 11%. For the other donor, the percentage of cells in G0 and with membrane domains both remained unchanged after 2 days in non-stimulatory culture. Surprisingly, the polarized membrane domains detected on normal CD34+ cells were not found on peripheral blood blast cells from patients diagnosed with relapsed AML (n=2 patients) or CML (n=1 patient). Accordingly, in a preliminary experiment, no difference in homing was observed 16 hours after transplantation of MβCD- or mock-treated peripheral blood cells from an AML patient in NOD/SCID IL2rψc^null mice. Additional homing and engraftment studies using cells from leukemic patients are ongoing. In combination, these findings indicate that specialized membrane domains are found on normal but not leukemic HSPCs and that these domains are required for homing to the bone marrow microenvironment. Disruption of these domains in actively cycling progenitor cells may provide an explanation for the previously demonstrated homing/engraftment defect of cycling cells compared to quiescent cells in the G0 phase of the cell cycle.