Hematopoietic recovery following PBSC is dependent on progenitor-cell number infused and affect of previous chemotherapy on progenitor quality. Progenitor-cell mobilization is adversely affected by prior radiotherapy. The minimum threshold of GM-CFC required is achieved in most patients with a single apheresis, but an optimal collection usually requires at least two harvests.

This study shows that delayed hematologic recovery is frequent if less than 1 x 10(6)/kg CD34+ cells are infused after high-dose therapy, particularly with GM-CFC less than 1 x 10(5)/kg. The procedure-related mortality in this latter group is high. In most patients whose PBSC collection contains less than 1 x 10(5)/kg GM-CFC, the use of bone marrow cells does not improve engraftment, which suggests that poor PBSC mobilization usually indicates poor marrow function.

The transmigration of hematopoietic progenitor cells (HPCs) across vascular endothelium is a critical step in the homing of transplanted stem cells, but the molecular basis for this is unknown. We used mobilized peripheral blood CD34+ selected cells and cultured bone marrow microvascular (BMECs) and human umbilical vein endothelial cells (HUVECs) to investigate the adhesion and transendothelial migration of HPCs. Colony-forming cells (CFCs) in freshly isolated CD34+ cells showed high levels of adhesion to both forms of endothelium (28% ± 4% and 38% ± 6% of granulocyte-macrophage colony-forming cells [GM-CFCs] adhering to HUVECs and BMECs, respectively), but were unable to migrate to any significant extent across either (1.0% ± 0.3% and 1.1% ± 0.6% of GM-CFCs migrating across HUVECs and BMECs, respectively). Greater than 95% of peripheral blood CD34+ cells are in G0/G1 of the cell cycle, but after 48 to 72 hours of stimulation with growth factors (interleukin-3 [IL-3] 12 ng/mL, stem cell factor 10 ng/mL, and IL-6 10 ng/mL), 28% ± 5% of cells were in S+G2/M. Growth factor stimulation had no effect on the adhesion of mobilized CFCs but resulted in enhanced migration of these cells (9.8% ± 1.6% and 12.6% ± 3.1% of GM-CFCs migrating across HUVECs and BMECs, respectively; P < .01, n = 6). Assessment of cell proliferation by the3H-thymidine suicide method showed that, whereas 11.7% ± 3.3% of proliferating CFCs transmigrated across endothelium, only 1.3% ± 0.3% of nonproliferating CFCs did so (P < .05, n = 5). Transmigration of growth factor-activated CFCs was inhibited by anti-CD18 monoclonal antibody (MoAb; 50% ± 18% inhibition) and by anti–platelet endothelial cell adhesion molecule-1 (PECAM-1) MoAb (70.8% ± 7.1% inhibition; P < .05, n = 3). IL-1 stimulation of HUVECs had no significant effect on CD34+cell transmigration, but caused marked enhancement of neutrophil migration. Stem cell homing may depend, in part, on the ability of local cytokines to upregulate the transmigratory ability of these cells. The transmigration of HPCs shares at least some molecular pathways with that of mature cells (CD18 and PECAM-1), but is differently affected by endothelial activation.

We have investigated whether a state of tolerance toward EGFP-expressing skin tissue can be induced by prior establishment of EGFP molecular chimerism by transplant of gene-transduced bone marrow in mice. Irradiated (10 Gy) C57BL/6J mice were transplanted with bone marrow cells transduced with two different retroviral vectors encoding EGFP. EGFP-transduced, mock-transduced, and age-matched control mice received skin grafts from both C57BL/6 EGFP-transgenic (B6-EGFP. Tg) and MHC-mismatched B10.A donor mice at 8, 29, or 39 weeks after bone marrow transplantation. Although 14 of 17 control mice rejected EGFP.Tg skin grafts within 100 days, 24 of 25 mice receiving EGFP-expressing bone marrow cells accepted their B6-EGFP.Tg grafts out to 200 days after skin grafting, including animals with undetectable levels of EGFP expression in blood cells. The EGFP-transduced animals rejected third-party grafts from MHC-mismatched mice within 20 days, indicating that acceptance of the EGFP-expressing skin grafts was the result of the induction of specific and operational immune tolerance. Thus, our data indicate that (a) EGFP-expressing tissue elicits an immunological rejection in C57BL/6 mice and (b) tolerance can be induced by engrafting relatively small numbers of EGFP-transduced hematopoietic cells. These experiments utilizing EGFP as an immunogen point to the wider therapeutic potential of employing transplantation of gene-transduced hematopoietic cells for establishing immunological tolerance and thereby preventing rejection of gene-corrected cells and tissues.

We have performed a pilot study of MDR-1 gene transfer chain reaction (PCR) for vector-derived MDR-1 cDNA in patients receiving CD34-selected peripheral blood stem sequence in all cases. Analysis of peripheral blood and cell (PBSC) transplant for lymphoma. To ensure minimum bone marrow cells after transplant has, however, shown engraftment thresholds and facilitate CD34 purification, no evidence of in vivo gene transfer with a follow-up of 12, mobilisation of Ͼ2 × 10 6 CD34 cells/kg was a condition for 15 and 18 months. The effect of MDR-1 substrate drugs recruitment. Of 11 patients counselled for study entry, onlyhas not yet been tested as all patients remain in clinical five achieved this target in a single apheresis. In three conand radiological remission of their lymphoma. These senting patients, purified CD34 cells were exposed to results confirm the difficulty of achieving in vivo gene trans-A12M1 MDR-1 retroviral supernatant for 6 h, cryoprefer in human haemopoietic cells and indicate major logisserved then thawed and readministered following ablative tical constraints in PBSC mobilisation in patients with chemotherapy. No delay in engraftment was observed, relapsed and resistant disease in whom initial studies are although one patient received additional back-up cells.appropriate. Gene transfer was demonstrated by polymerase