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Simple efforts are needed to enhance cord blood (CB) transplantation. We hypothesized that short‐term exposure of CD34 +  CB cells to 39.5°C would enhance their response to stromal‐derived factor‐1 (SDF‐1), by increasing lipid raft aggregation and CXCR4 expression, thus leading to enhanced engraftment. Mild hyperthermia (39.5°C) significantly increased the percent of CD34 +  CB that migrated toward SDF‐1. This was associated with increased expression of CXCR4 on the cells. Mechanistically, mild heating increased the percent of CD34 +  cells with aggregated lipid rafts and enhanced colocalization of CXCR4 within lipid raft domains. Using methyl‐β‐cyclodextrin (MβCD), an agent that blocks lipid raft aggregation, it was determined that this enhancement in chemotaxis was dependent upon lipid raft aggregation. Colocalization of Rac1, a GTPase crucial for cell migration and adhesion, with CXCR4 to the lipid raft was essential for the effects of heat on chemotaxis, as determined with an inhibitor of Rac1 activation, NSC23766. Application‐wise, mild heat treatment significantly increased the percent chimerism as well as homing and engraftment of CD34 +  CB cells in sublethally irradiated non‐obese diabetic severe combined immunodeficiency IL‐2 receptor gamma chain d (NSG) mice. Mild heating may be a simple and inexpensive means to enhance engraftment following CB transplantation in patients. Stem Cells S tem  C ells   2015;33:1975–1984

Mild Heat Treatment Primes Human CD 34 + Cord Blood Cells for Migration Toward SDF ‐1α and Enhances Engraftment in an NSG Mouse Model