Effects of Age and Environment on Short-Term Homing and Function of Mouse Hematopoietic Stem Cells.

Abstract 1616

The C57BL/6 mouse is one of the best studied models of hematopoietic stem cell (HSC) aging. Characteristic age-related changes include an increase in HSC frequency, a corresponding decrease in functional activity, myeloid skewing, and decreased in vivo homing ability. Studies of age-dependent differences in homing have primarily relied upon the injection of young or old bone marrow into irradiated recipients, with subsequent quantification of functional homed HSCs using secondary transplantation assays (e.g. Liang et al, Blood 2005). While this is a relevant and informative approach, it is limited because it measures homed cells indirectly, using an assay that itself is dependent on homing. To study this phenomenon more directly, we compared the short-term homing efficiency of purified HSCs from old and young mice by co-injecting them into old or young recipients. This approach enabled us to ascertain whether the previously reported decrease in homing efficiency is due to differences in physical homing to the bone marrow, or a reduced function per homed cell, or some combination of both. Second, it allowed us to determine the relationship between the decreased in vivo homing efficiency of old HSCs and their functional properties when assayed in a homing-independent stem cell assay. Third, by varying the age and irradiation status of the homing recipients, we could test directly the influence of these factors on HSC homing and function. To accomplish these objectives, we purified HSCs (CD150+CD48-LSK bone marrow cells) from old (23-26 months) GFP+ and young (3-4 months) CFP+ mice, combined them at known ratios, and injected them into non-irradiated young, non-irradiated old, or lethally irradiated young recipients. 22 hours later, these recipients were sacrificed and the ratio of GFP+ and CFP+ cells found homed to the bone marrow were compared to the ratio in which they were injected. In this way, we determined that relative to their young counterparts, the 22-hour homing efficiency of old CD150+CD48-LSK cells was consistently 2-fold lower when injected into young recipients. However, when the same cells were injected into non-irradiated old recipients, the relative homing efficiency varied from 2-fold lower to 1.1-fold higher. This suggests that the factors affecting homing are similar between individual young recipients, while the microenvironments of individual old recipients are variable in their relative ability to support homing of young and old HSCs. We recently demonstrated that the proportion of purified old HSCs that were functional when assayed clonally in vitro (cobblestone area forming cell [CAFC] assay) declined approximately 2-fold compared to young HSCs. To determine whether the non-homed HSCs were the same cells that were non-functional in the CAFC assay, we compared the CAFC seeding efficiency of old and young cells before and after homing. There was no functional improvement in homed old LSK150+48- cells, suggesting that the homing defect and reduced CAFC efficiency are non-overlapping and may be characteristics of all old HSCs. To determine if the homing microenvironment also had an effect on the function of the homed cells, CAFC assays were initiated with old and young HSCs before and after homing into each of the three recipient types. This analysis revealed that the function of the homed cells was significantly decreased for age-mismatched or irradiated recipients compared to pre-homed cells. In contrast, CAFC activity of young or old cells homed to non-irradiated age-matched recipients did not change. This suggests that the environment of an age mismatched and/or irradiated recipient can have a negative effect on the function of homed cells within a matter of hours. In conclusion, these co-homing studies reveal that compared to young HSCs, old HSCs are physically less able to home and migrate into the bone marrow within a 22-hour period post-transplantation. However, this relative homing defect is most pronounced and consistent when injected into young recipients. Second, the homing defect and reduced CAFC efficiency are non-overlapping and may be characteristics of all old HSCs. Third, old or young HSCs that do successfully home to irradiated or age-mismatched recipients become functionally compromised compared to pre-homed HSCs, suggesting that the homing defects of old HSCs can be ameliorated or exacerbated depending on the microenvironment in which they are transplanted.