In the BM and thymus, lympho-hematopoietic and non-hematopoietic (stem-cell niche or stromal) cellular compartments tightly regulate competence for T-cell generation, while aging clearly induces defects in this competence [36
]. It is still controversial whether the defect originates in the LPC/HSC itself or in the supportive microenvironment. To understand the cellular mechanism of aging in the T-cell generation it is necessary to determine which cellular compartment develops the primary/intrinsic defect. We demonstrate here that this defect originates primarily from the endogenous microenvironment, as evidenced by the fact that the aged BM niches could induce the same defects in BM progenitors from young mice (Fig. ). Our findings also show that in mice, LPCs seem to lack an intrinsic defect prior to 20-22 months of age, (comparable to ~80-year-old humans), since they were able to repopulate the dGUO-treated and intact fetal thymic lobes in vivo
over either short (1 week) or long (4 weeks) time period, comparable to LPCs from young mice (Fig. ). Natural thymus-seeding LPCs from aged and young mouse BM, acquired in vivo
by a grafted fetal thymic lobe, compete well each other to generate T-lineage cells in a common young microenvironment (Fig. ). These natural thymus-seeding LPCs from aged BM were also able to compete with corresponding young LPCs even in subsequent in vitro
culture (Fig. ). These findings do not imply that LPCs do not age. In fact LPCs progressively lost their capacity to generate T-lineage cells with increasing age as demonstrated in a BMT setting, but they out-competed the ability of their niche cells to generate T-lineage with the passage of time (Fig. ).
The mechanism by which young BM progenitor cells in the old BM-niches differentiate along the same pathway as old progenitor cells to make a myeloid skew (Fig. ) is not clear. It is possible related to levels of reactive oxygen species (ROS) produced by BM niches since HSCs from a high oxygen milieu showed myeloid-skewed differentiation [37
]. However, the central role of osteoblast-rich niches in this differentiation has been defined. This similar observation was reported [38
], in which loss of osteoblasts in a gene knockout mouse model increased myelopoiesis. Another possible mechanism is hyperactivation of the mammalian target of rapamycin (mTOR) during aging [39
]. Although over-activation of mTOR in aging was found in stem cells [40
], it is possible due to defects in stem cell regulation by the niche. A recently publication demonstrated the mechanism of calorie restriction (CR) on mammalian intestinal stem cells (ISC) during aging, in which CR, similar to Rapamycin administration, inhibits activity of mechanistic target of rapamycin complex 1 (mTORC1) in Paneth cells, a key constitute of the ISC niche. It results in promoting a more favorable stem-cell microenvironment, and then the changed microenvironment normalizes the stem cells [41
Interestingly, when old BM progenitor cells were introduced into young mice, the young BM-niche did not rejuvenate differentiation profile of the old progenitor cells along the same pathway as young LPCs [23
]. However, when old LPCs were recruited into young thymic niches in vivo
(within KCT environment), the old LPCs were then directed to differentiate along the same differentiation pathway as young LPCs (Fig. ). We believe this may be accounted for in two ways. 1) The BM of aged mice may contain both defective as well as functional hematopoietic progenitors, but only the functional progenitors are able to colonize the thymus. Thus, although aged BM has a reduced functional stem cell pool, there is still sufficient population of individual normal functional LPCs on a per cell basis [21
] in aged mice. Because the thymic niche is gated [42
] and number of the LPC recruitment is limited [an estimated ~10-100 new progenitor cells enter the young adult mouse thymus per day [43
]], the normal functional LPCs from aged BM seeding fetal thymus in vivo
should be sufficient to provide the daily thymus recruitment requirement. Although the young functional BM stem cell pool is larger than the aged one, the total number of thymus-seeding LPCs from both pools should be the same at any given time. Therefore, grafted fetal thymic lobe-recruited LPCs from both aged and young mice are similar and produce the same number of T-lineage cells.
Nevertheless, when in vitro
isolated aged LPCs were introduced into the young BM microenvironment via conventional BMT, and allowed to co-exist with young LPCs [10
], the aged LPCs are unable to compete, while in a cKCT microenvironment they are able to compete with young LPCs. This finding may be accounted for by 2) the reduced transplant efficacy of aged BM progenitors. For example, when removing aged BM progenitors from osteoblastic niches, they showed decreased adhesion to stroma [31
]. In vitro
manipulation of HSCs has been suggested to undergo replicative stress [47
] or reduction of transplant efficiency [30
]. Therefore, the defect in LPCs is only encountered with transplanted aged BM cells in conventional BMT [12
], while this is not observed in physiological thymus-seeding aged BM cells (Figs. , , and ) [1
]. Thus aged BM progenitors are not likely to have an intrinsic defect, but possess the stress from aged microenvironment, since it can be avoided by the in vivo
collection (Figs. , , and ).
Although LPCs/HSCs are not exempt from aging [48
], their aging occurs at a much slower rate than the aging of their niche cells, as measured by comparing the competence of thymic T-lymphopoiesis in a time-course manner based on BM progenitor ages or niche ages in a IL7R−/−
host BMT microenvironment (Fig. ). Cells of both hematopoietic and non-hematopoietic origin undergo age-related deterioration from replicative stress and epigenetic changes [50
] that influence DNA integrity [48
], or exhaustion of their stem cell pools. However, during the process of natural aging, LPCs/HSCs outlive their niche cells, as evidenced by the onset in reduction in thymic T-lymphopoiesis of aged BM progenitors when repopulating the non-irradiated young IL7R−/−
thymus (at the age of 22 months). Concurrently, the niches lose their ability to support the production of functional T cells from the repopulating young BM progenitors in the same setting by “middle age” (~12 months of age in the mice).
In conclusion, the experiments in this study employed comprehensive in vivo
and in vitro
models to answer several pertinent questions. Which cellular component, HSC itself or HSC niche cell, determines HSCs to take age-related myeloid-skew developmental profile? Can the aged BM-niche influence competence of young BM progenitors to generate T-lineage cells by providing signals that are different from those provided by young niche cells? Why aged LPCs, derived from isolated BM progenitors, cannot compete with their young counterparts in a conventional BMT environment, while they can do so using in vivo
collected natural thymus-seeding LPCs? Which cell type (LPCs or niche cells) primarily develops age-related inability in T-cell development in a time-course manner? We have answered these questions by showing clear evidence that the dominant and primary defect arising from aging of T-lymphopoiesis lies in a dysfunction of the niche cells [51
] rather than in the T-cell progenitor pools. Further exploration of these issues will provide the foundation for gene-, pharmaceutical-, and stem-cell-based therapies by focusing on the right cellular targets and optimizing the timing for rejuvenation of reduced T-lymphopoiesis in order to treat aging-related onset of T-lymphocyte deficiency.