Absence αof 4 Integrin in Hemopoietic Stem Cells Limits Their Self-Renewal Potential in Vivo.

We have previously shown that bone marrow (BM) cells from adult mice with conditional ablation of α4 integrin transplanted into lethally-irradiated recipients have a partial impairment in their homing and especially their short-term engraftment (MCB, 23:9349, 2003). However, the ability of α4−/− stem cells (HSC) to maintain post-transplant long-term hematopoiesis and to self renew was not tested. Therefore, we performed competitive repopulation experiments: α4+/+ cells mixed in equal proportions with α4−/− cells (verified by FACS) were given to each of 10 lethally irradiated recipients (0.5x10 ⁶/mouse). At 30, 100, 200, and 298 days post-transplant, engraftment was evaluated in blood (PB) and BM. By d. 200, 7 of the 9 surviving mice had 81.6±3% α4+/+ cells in their PB and 97.5±0.1% in their BM. In the remaining 2 mice the proportion of α4+/+ PB cells was 35.6±12%, however by d. 298 increased (93.4±2.5% in BM). To overcome a putative partial homing defect for long-term repopulating cells, similar to the one documented using a surrogate CFU-C assay, we repeated the competitive repopulation experiment using not only 1:1, but an increased ratio of α4−/− cells to 3:1 (or 6:1 by CFU-C ratio) given in splenectomized recipients. By 12 wks α4+/+ cells among Gr1+ were 77±3.7% in PB in 10 mice with 1:1 initial transplant and 79±3.8% in 10 given 3:1 cells. These results showed that 4+/+ cells greatly outcompete the α4−/−cells and contributions by α4−/− cells are lost early and late post-transplant.

Further insight was provided by transplantation of α4−/− HSC without competitor cells. 12 mice transplanted with α4−/− BM cells were sacrificed at 2 wks (6 mice), at 10 wks (3 mice) and 1 year (3 mice) later. Despite normal PB counts, evaluation of bone marrow and spleen at all times post-transplant showed subnormal values for progenitor cells vs. concurrently transplanted controls. 10 wks post-transplant 1 of the 3 mice sacrificed showed ~50% α4+/+ cells in circulation, while the other 2 had mostly α4−/− cells. From the latter (pooled BM), 2° transplants were carried out and sacrificed 14 wks later. At that time the 5 recipients had 27.5%±4.7 α4+/+ cells in their circulation. At 1 year the 3 primary transplant surviving mice had mostly α4−/− hematapoiesis and served as donors (pooled BM) for 2° transplants (n=9), evaluated 26 wks later. 5 of 9 2° recipients showed mostly α4+/+ cells, whereas 4 recipients had a mean of 6.8±1.9% α4+/+ cells in their blood. Each of these 4 recipients served as a 3° donor for 20 transplants (5/donor) which again were evaluated 25 wks later. There was a 30% survival at that time, and all 6 surviving mice were reconstituted with α4+/+ cells (multi-lineage; contributed by host and not by non-ablated donor stem cells). These data suggested that although long-term repopulation can be established with α4−/− cells in 1°recipients, hematopoiesis is quantitatively abnormal and cannot be sustained beyond a 2° transplant. Taken together, all our transplantation experiments provide compelling evidence that α4−/− HSC have a competitive disadvantage compared to +/+ cells in transplantation, and a deficit in maintaining normal hematopoiesis and stem cell self-renewal. We speculate that α4−/− HSC either are not settled to extramedullary niches supporting sustained hematopoiesis, or do not respond to signals emanating from the stem cell niche. Either way, the data underscore the requirement of α4 integrin in the interaction of HSC with the stem cell niche in order to realize their full self-renewal potential.