Ageing of metazoans can be generally characterized as a progressive decline of tissue and organ function, accompanied by increased oxidative damage, mitochondrial dysfunction, endocrine imbalance and genome instability.
Tissue regenerative capacity also declines with age, and in tissues such as muscle, blood, liver and brain this decline has been attributed to a diminished responsiveness of tissue-specific stem and progenitor cells.
Scientific research has demonstrated that signaling through the Notch pathway is essential for the activation, proliferation and myogenic lineage progression of satellite cells necessary for muscle repair, and that the decline in the regenerative potential of muscle with age is due to the failure of this pathway to be activated.
However, the activation of aged satellite cells and the regenerative potential of aged muscle can be restored by forced activation of Notch, demonstrating that the intrinsic regenerative capacity of aged satellite cells remains intact.
Aged muscle successfully regenerates when grafted into muscle in a young host, but young muscle displays impaired regeneration when grafted into an aged host.
It was hypothesized that there are systemic factors that support the robust regeneration of tissues in young animals and/or inhibit regeneration in old animals, and that these factors act to modulate the key molecular pathways that control the regenerative properties of progenitor cells.
The implication of this hypothesis is that old tissues might be made to regenerate as well as young tissues if, by means of systemic influences, the molecular pathways could be 'rejuvenated' from an old state to a young state.
To test this hypothesis scientists have set up an experimental system in which – in contrast to transplantation – regenerating tissues in aged animals could be exposed only to the circulating factors of young animals, and vice versa.
They established parabiotic pairings between young and old mice (heterochronic parabioses), with parabiotic parings between two young mice or two old mice (isochronic parabioses) as controls.
In parabiosis, animals develop vascular anastomoses and thus a single, shared circulatory system.
Previous work examining the effects of heterochronic parabiosis showed that such pairings may alter tissue function, but the effects on progenitor cell activity or tissue regeneration have not been examined.
The loss of muscle regeneration with age is due at least in part to an age-related impairment in the upregulation of the Notch ligand Delta after muscle injury.
Therefore, it was tested whether heterochronic parabiosis restored Delta upregulation in aged satellite cells and thus enhanced their activation and proliferation.
Using myofibre explantation to assess satellite cell activation, satellite cells for the expression of Delta has been analyzed.
In young isochronic and heterochronic parabionts there was a marked upregulation of Delta in satellite cells, whereas Delta induction was lacking in the old, isochronic parabionts (Fig. 1), typical of the response of aged muscle.
Notably, satellite cells from the aged partners of heterochronic parabionts showed a marked upregulation of Delta, comparable to that found in their young partners (Fig. 1) and in young mice not subjected to parabiotic pairings.
There was a slight inhibition of Delta upregulation in satellite cells from young mice in heterochronic parabioses compared with young isochronic parabionts (Fig. 1).
Thus, heterochronic parabiosis not only enhances the proliferative response of the resident aged progenitor cells, it also restores the key molecular signalling in these cells that is necessary for muscle regeneration.
To determine whether the rejuvenating effects of heterochronic parabiosis could be replicated in an in vitro system, satellite cells, cultured either alone or in association with myofibre explants, were prepared from young and old mice and were cultured in the presence of young or old mouse serum, thus recapitulating the humoral aspects of heterochronic parabioses.
Compared with young satellite cells cultured in either young or old serum, there was much less upregulation of Delta in old satellite cells cultured with old mouse serum (Fig. 2); however, young mouse serum restored the upregulation of Delta (Fig. 2) and the activation of Notch (Figs 2) in aged satellite cells.
There was an inhibitory effect of the old mouse serum on young satellite cells in terms of the upregulation of Delta (Fig. 2).
Serum from young mice also significantly enhanced the proliferation of myofibre-associated satellite cells in the old cultures compared with serum from old mice.
There was reduced proliferation of both young and old satellite cells cultured in young mouse serum when Notch signaling was inhibited.
Therefore, enhanced activation and proliferation of aged myogenic progenitor cells by serum from young mice is Notch dependent.
Together, these in vivo and in vitro data demonstrated that components in young serum alone are capable of reversing the molecular and cellular aspects of age-related decline in muscle stem cell activation.
In addition, it also seems that factors in old serum negatively affect the processes required for satellite cell activation and muscle repair.
According data obtained scientist have examined liver from aged mice subjected to heterochronic parabiosis to test for evidence of a more general capability to rejuvenate aged, resident progenitor cells.
In liver, there are multiple types of progenitor cells that participate in regeneration and homeostasis, depending on the physiological or pathological condition.
Proliferating hepatocytes involved in normal tissue turnover were studied, because there is a well documented decline in hepatocyte proliferation with age.
Heterochronic and isochronic parabionts were examined for hepatocyte proliferation by two independent criteria: the incorporation of BrdU or expression of the proliferation marker Ki67 in albumin positive cells.
In young isochronic parabionts (Fig. 3), the levels of basal hepatocyte proliferation were two- to threefold greater than in non-parabiosed controls, consistent with previous reports of effects of parabiosis on hepatocytes.
Also, clusters of BrdU-positive proliferating cells that were not hepatocytes were observed in aged livers.
Proliferation of albumin-positive cells in old isochronic parabionts was less than that observed in young isochronic parabionts (Fig. 3), consistent with previous reports and our observation that there is an age-related decline in the basal rate of hepatocyte proliferation in non-parabiosed animals.
However, parabiosis to a young partner significantly increased hepatocyte proliferation in aged mice (Fig. 3).
As in muscle, a reproducible reduction of progenitor cell proliferation was detected in livers of young mice after parabiotic pairing with old mice (Fig. 3).
Also as in muscle, the enhancement of hepatocyte proliferation in aged mice was due to resident cells and not the engraftment of circulating cells from the young partner.
Consistent with previous observations, less than 0.1% of hepatocytes expressed GFP in the non-transgenic, parabiotic partners.
The age-related decline in hepatocyte proliferation was due to the formation of an age-specific complex of cell cycle regulators associated with cEBP-a that inhibits E2F-driven gene expression.
One of the proteins detected in this complex in old, but not young, liver is the chromatin remodeling factor Brm.
Levels of the cEBP-a-Brm complex increase in the livers of old rodents, leading to a decline of hepatocyte proliferation.
It was tested whether the enhancement of aged hepatocyte proliferation correlated with reduced levels of cEBP-a-Brm complex formation in the aged mice subjected to heterochronic parabiosis.
The cEBP-a-Brm protein complex was detected in liver extracts from old isochronic parabionts but not from young isochronic parabionts (Fig. 3), similar to previous reports in non-parabiosed rodents.
Notably, the formation of the cEBP-a-Brm complex was diminished in liver extracts from old heterochronic parabionts (Fig. 3).
Thus, the molecular determinant had reverted to the 'youthful' phenotype, consistent with the enhanced proliferative activity of hepatocytes in these mice.
The complex was present at elevated levels in young heterochronic parabionts compared with young controls, also consistent with the modest inhibition of hepatocyte proliferation in young heterochronic parabionts.
These data showed that the young systemic environment restored a younger profile of molecular signalling to the aged progenitor cells in the liver and also enhanced their proliferation.
These observations suggest that there are systemic factors that can modulate the molecular signalling pathways critical to the activation of tissue-specific progenitor cells, and that the systemic environment of a young animal is one that promotes successful regeneration, whereas that of an older animal either fails to promote or actively inhibits successful tissue regeneration.
It will be of great interest to identify the factors that have such a critical influence on tissue-specific progenitor cells.
These studies also demonstrate that the decline of tissue regenerative potential with age can be reversed through the modulation of systemic factors, suggesting that tissue specific stem and progenitor cells retain much of their intrinsic proliferative potential even when old, but that age-related changes in the systemic environment and niche in which progenitor cells reside preclude full activation of these cells for productive tissue regeneration.