Mild Hyperthermia Treatment Of CD34+ Human Cord Blood Cells Increases Lipid Raft Aggregation Resulting In An Earlier Engraftment In NSG Mice Following Transplantation

Umbilical cord blood (UCB) is a viable source of hematopoietic stem cells (HSC) for transplantation to treat patients with malignant and non-malignant disease. Although the limitation of low stem cell dose in single UCB transplantation has been improved upon by the use of double UCB units, this has not resulted in a shortened time to engraftment. Therefore efforts are needed to enhance the speed of engraftment of these cells. Mild heating (39.5°C) of mature hematopoietic cells increases membrane fluidity, resulting in increased lipid raft aggregation, and priming cells for increased sensitization of responsiveness to cytokine signaling. We hypothesized that exposing CD34+ UCB cells to 39.5°C would enhance their capacity to respond to SDF-1/CXCL12, a chemokine secreted in the bone marrow microenvironment that strongly attracts HSCs and progenitor cells, by increasing lipid raft aggregation and CXCR4 expression, thus leading to better engraftment following transplantation. To determine if this mild heating had any effect on engraftment in vivo, NSG mice received 4Gy TBI followed one day later with 20,000 or 80,000 CD34+ UCB cells incubated for 4 hours at 37°C or 39.5°C prior to i.v. injection. Percent chimerism in the blood was determined through the detection of human CD45 by flow cytometry at 1, 2, 4 and 6 months following transplantation. Mild heat treatment significantly increased the percent chimerism following transplantation by 1.7 fold at 1 month (when 80,000 cells were used) and by 1.8 fold at 2 months (when 20,000 cells were used), without significant increases in percent chimerism at 4 or 6 months. This suggests that the effects of heat treatment are most likely occurring early following transplantation, potentially due to an increase in homing of the heated CD34+ UCB cells to the bone marrow microenvironment. To begin to test this hypothesis, CD34+ UCB and Mo7e cells (a human megakaryoblastic leukemia cell line) were exposed to 39.5°C for 4 hours followed by a chemotaxis assay. Exposure of the CD34+ UCB cells to 39.5°C manifested in a significant increase in the percent of cells that migrated towards 12.5 (1.8 fold increase), 25 (1.9 fold increase), 50 (2.2 fold increase), 100 (2.3 fold increase) and 200 ng/mL SDF-1 (2.5 fold increase). Mild heating also significantly increased the percent of Mo7e cells that migrated towards 12.5 (1.8 fold increase), 25 (1.4 fold increase), 50 (1.5 fold increase), 100 (1.3 fold increase), and 200 ng/mL SDF-1 (1.5 fold increase). Increases in chemotaxis towards SDF-1 following mild heating was associated with a significant, but modest, increase (1.3 fold as determined by MFI) in expression of the SDF-1 receptor, CXCR4, on the surface of CD34+ UCB cells as detected by flow cytometry. In order to optimize SDF-1 signaling, CXCR4 must be incorporated into the cholesterol-enriched lipid rafts of the cellular membrane followed by lipid raft aggregation. Mild heating increased the percent of Mo7e cells with aggregated lipid rafts from 13 percent for cells incubated for 4 hours at 37°C to 52% for cells incubated for 4 hours at 39.5C. By using a dose of MbCD (1.25mM), an agent that can deplete cholesterol in the cellular membrane thus blocking lipid raft aggregation, it was determined that the enhancement in chemotaxis seen following mild heating was dependent upon lipid raft aggregation in both CD34+ UCB and Mo7e cells. Overall, mild heating may be a simple and inexpensive novel adjuvant therapy to enhance engraftment following UCB transplantation in patients.