Hematopoietic progenitor cells (HPC) from mobilized peripheral blood display enhanced migration and marrow homing compared to steady-state bone marrow HPC

Abstract

Objective: Faster engraftment of G-CSF-mobilized peripheral blood (MPB) transplants compared to steady-state bone marrow (ssBM) is well documented and clinically relevant. A number of different factors likely contribute to this outcome. In the present study we explored whether independent of cell number there are intrinsic differences in the efficiency of progenitor cell homing to marrow between MPB and ssBM.

Methods: Mobilization was achieved by continuous infusion of G-CSF alone or in combination with other mobilizing agents. In vivo homing assays, in vitro migration assays, gene expression analysis, and flow cytometry were utilized to compare homing-related in vivo and in vitro properties of MPB and ssBM HPC.

Results: Marrow homing of murine MPB HPC, generated by different mobilizing schemes, was reproducibly significantly superior to that of ssBM, in lethally irradiated as well as in nonirradiated hosts. This phenotype was independent of MMP9, selectins, and beta2- and alpha4-integrins. Superior homing was also observed for human MPB HPC transplanted into NOD/SCIDbeta2microglobulin(-/-) recipients. Inhibition of HPC migration abrogated the homing advantage of MPB but did not affect homing of ssBM HPC, whereas enhancement of motility by CD26 inhibition improved marrow homing only of ssBM HPC. Enhanced SDF-1-dependent chemotaxis and low CD26 expression on MPB HPC were identified as potential contributing factors. Significant contributions of the putative alternative SDF-1 receptor, RDC1, were unlikely based on gene expression data.

Conclusion: The data suggest increased motility as a converging endpoint of complex changes seen in MPB HPC which is likely responsible for their favorable homing.

Figure 1

Superior marrow homing of MPB grafts: A-D: In vivo marrow homing of murine B6/129 CFU-C from ssBM or MPB was quantified, using colony assays. A: Three hours after transplantation of lethally irradiated hosts, the mean fraction of CFU-C homed to bone marrow was 41% greater for MPB than for ssBM (*p<0.05). B: A similar marrow homing advantage as for G-CSF-MPB was observed after 18 hours for CFU-C mobilized with Cyclophosphamide+G-CSF(*p<0.001) or with Flt3L+G-CSF (not shown). C: Forty-eight hours after transplantation, recovery from recipient marrow of MPB-derived CFU-C was significantly greater compared to ssBM-derived CFU-C. This observation was made in both irradiated (left, *p<0.005) and nonirradiated (right, *p<0.005) hosts, ruling out a role of the irradiated marrow microenvironment in the differential marrow homing of ssBM and MPB. For both MPB and ssBM, the fraction of homed CFU-C was markedly increased 48 hours after transplantation compared to 18 hours, likely indicating early proliferation of the transplanted cells. Depicted in Fig. 1A-C are mean+SEM for % of injected CFU-C homed to marrow (MPB , ssBM ). D: Forty-five% more efficient mean marrow homing of MPB CFU-C compared to ssBM CFU-C was observed 18 hours after transplantation of lethally irradiated isogeneic recipients (p<0.001). Each dot represents homing in one individual animal; bold line and error bars represent mean±SEM for % of injected CFU-C homed to marrow. E: Marrow homing of human CD34+ cells from ssBM or MPB was tested in a xenotransplantation model. Eighteen hours after transplantation, the mean recovery of MPB CFU-C from recipient marrow was 3-fold increased compared to ssBM (*p<0.001). Each dot represents homing in one individual animal; bold line and error bars represent mean±SEM for % of injected CFU-C homed to marrow. These data indicate that the greater marrow homing ability of MPB CFU-C is intrinsic to a population enriched for HPC, and that it applies to human as well as to mouse CFU-C.

Figure 2

Increased migration of MPB cells: Transwell migration of MPB c-kit+ cells (A), both spontaneous and SDF-1 directed, was markedly greater than that of ssBM c-kit+ cells (*p<0.005 MPB vs. ssBM), and SDF-1 directed migration of MPB-CFU-C (B) was likewise increased (*p<0.005). CFU-C did not show appreciable spontaneous migratory activity (not shown). Superior chemotactic migration was observed despite decreased CXCR4 expression (p<0.005) on MPB c-kit+ cells. Shown is one representative histogram plot (C). Incubation with Pertussis toxin (PTX) blocked spontaneous and chemotactic migration of MPB (left) and ssBM (right) c-kit+ cells (D). Gene expression of the alternative SDF-1 receptor, RDC1, in MPB MNC was very low, since it was only detected after 40 PCR cycles, which makes a contributory role of RDC1 to MPB HPC migration unlikely (E). Migration is depicted as mean+SEM of % of total. Cell type is indicated in the upper right corner, cell source and stimulus below the column. The FACS histogram depicts expression of CXCR4 on c-kit+ cells (Isotype control lightly shaded, MPB black line, ssBM solid grey; cell counts on Y-axis, fluorescence intensity on X-axis).

Figure 3

Increased migration is responsible for superior marrow homing of MPB CFU-C: A: Cartoon depicting the experimental approach: The consequences of altered CFU-C migration for marrow homing were tested. This was done by blocking migration through inhibition of CXCR4 or Gi protein signaling (using AMD3100 or PTX), and by increasing migration (using DiprotinA, an inhibitor of the dipeptidylpeptidase CD26). B-D: AMD3100 (marrow homing assessed 3 hours after transplantation, B) and PTX (marrow homing assessed 18 hours after transplantation, C) significantly blocked marrow homing of MPB CFU-C (*p<0.005 compared to untreated MPB), but had no effect on ssBM homing. In contrast, DiprotinA significantly increased marrow homing only of ssBM CFU-C (*p<0.05 compared to untreated ssBM, marrow homing assessed 18 hours after transplantation, D). No DiprotinA effect on MPB homing was observed, likely due to loss of CD26 expression on MPB c-kit+ cells (E). These data suggest that the more avid migration of MPB cells, mediated through SDF-1, CXCR4 and Giprotein signals, guides their better marrow homing. Homing data are shown as mean+SEM for % of injected CFU-C homed to marrow, (MPB , ssBM , solid: - inhibitor, hatched: + inhibitor; * indicates significant difference (p<0.05) compared to samples from the same source that were not treated with the inhibitor). The FACS histogram depicts expression of CD26 on c-kit+ cells (Isotype control lightly shaded, MPB black line, ssBM solid grey; cell counts on Y-axis, fluorescence intensity on X-axis).

Figure 4

Favorable homing of MPB persists in the absence of MMP9, β2-integrin and selectins: Previously reported differential regulation of MMP-9, β2-integrins and selectins between MPB and ssBM HPC prompted us to test their contribution to marrow homing, using genetically deficient donors or recipients. Marrow homing of MPB or ssBM CFU-C from MMP-9-/-mice (A) CD18-/-mice (B) or CD62L-/-mice (D) in WT recipients or of WT CFU-C in CD62ELP-/-recipients (C) was entirely normal, i.e. no different than marrow homing in WT-to-WT transplantation, as shown in Fig. 1. The superior marrow homing of MPB-derived CFU-C was maintained (*p<0.005, p<0.05, p<0.001, p<0.005, respectively for ssBM vs. MPB). Eighteen hour homing data are shown as mean+SEM for % of injected CFU-C homed to marrow (MPB , ssBM ).