Inherent Differences in SDF-1α-Induced CXCR4 Expression Patterns on MPB, BM and CB CD34+ Cells.

We have previously shown that in vitro migration and NOD/SCID engraftment potential of mobilized peripheral blood (MPB) CD34+ cells can be significantly increased 2–3 fold by preincubation of graft cells with 100 ng/ml of SDF-1α (CXCL-12) for 30mins at 4°C. However, bone marrow (BM) and cord blood (CB) CD34+ cells did not exhibit similar increases in migration or engraftment after SDF-1α preincubation and, in the case of CB, preincubation somewhat reduced in vitro migration. We believe that these biological differences in SDF-1α responses may begin to explain some of the reported differences in hematopoietic potential among these three sources of CD34+ cells, and may be related to rate and degree of CXCR4 receptor recycling after SDF-1α exposure. In the present work, we examined patterns of CXCR4 expression both before (part 1 studies) and after (part 2 studies) SDF-1α pretreatment, and found that, in general, MPB CD34+ cells appear to possess an inherent ability to mobilize internal CXCR4 stores with greater efficiency than BM and CB CD34+ cells, while CB appears most sensitive to SDF-1α-induced CXCR4 downmodulation. This conclusion is supported by the following results: In part 1 studies, when untreated CD34+ cells were incubated with SDF-1α (100 ng/ml, 37°C), CXCR4 expression initially decreased in all tissues, but returned to basal levels in BM and MPB cells, while re-expression on CB cells remained below baseline for the duration of the 2hr assay. In parallel cultures where SDF-1α was washed out after 60mins, CXCR4 re-expression was most rapid in MPB CD34+ cells, reaching 4.5±2.7-fold higher levels of expression after 30mins, compared to 1.8±1.1-fold for BM (p<0.05) and 3.4±2.0 for CB. After SDF-1α pretreatment (part 2 studies) and subsequent 37°C culture, CXCR4 expression declined initially in all three tissues, but was rapidly regained in MPB such that the fold increase above basal levels was 2-fold higher compared to BM or CB. CB CD34+ cells again exhibited a sustained decline of CXCR4 expression below baseline for up to 2hr. In the presence of SDF-1α, preincubated CB CD34+ cells again showed the greatest reduction in CXCR4 expression to <50% of baseline levels compared to BM and MPB, where re-expression reached basal values (p<0.05). Additionally, CXCR4 re-expression after SDF-1α wash-out was significantly increased more than 3-fold in MPB CD34+ cells compared to BM. The lower percentage of fresh MPB CD34+ cells expressing surface CXCR4 compared to BM and CB (%CXCR4+ = 9.6±3.5, 28.4±5.9 and 28.1±6.9, respectively, p<0.05, n=6–8), may contribute to the pronounced effect of SDF-1α on MPB, considering that the activity level of the recycled receptor may differ from that on fresh cells, a topic currently under study in our laboratory. Taken together, these results suggest that after SDF-1α preincubation, MPB CD34+ cells retain their ability to rapidly mobilize CXCR4 from internal stores to the cell surface, while BM and CB responses are more blunted than prior to SDF-1α pretreatment. In addition, exposure of CB CD34+ cells to SDF-1α results in prolonged down-modulation of the SDF-1α/CXCR4 axis manifested by internalization of CXCR4. These data highlight key biological differences in the SDF-1α/CXCR4 axis in CD34+ cells from MPB, BM, and CB that may begin to explain some of the observed differences in their hematopoietic potential. Furthermore, these findings may be important in the design of clinical transplantation protocols utilizing specific SDF-1α-pretreatment protocols to maximize the clinical benefit of transplanted HSC from different sources.