Given the growing evidence that many diseases of aging may reflect adult stem cell exhaustion, it is not surprising there is great interest in restoring adult stem cell function to ameliorate these conditions and regenerate aged tissues [
23]. Adoptive transfer of fetal MSCs into adult mice has been shown to extend median lifespan of the animals [
240]. Adult stem cell mobilization and transplant are two obvious strategies that have achieved moderate success for certain types of injury and disease in humans, and many types of adult stem cells have been utilized for this purpose [
241]. MSC cellular therapy has proven to be safe for a number of vascular disorders, such as coronary artery disease, peripheral vascular disease, erectile dysfunction, and stroke, and is an attractive option for patients who are poor surgical candidates [
242,
243,
244,
245,
246].
Despite these successes, the problem remains that adult stem cells from elderly donors, the very people who most frequently require enhanced peripheral stem cell function for tissue repair, undergo changes in their functional capacity as a result of aging (reviewed in [
104]). This decline in functional capacity, therefore therapeutic utility, has been combatted using some surprisingly simple interventions: Conditioning with hypoxia prior to transplant, for example, has been extensively documented as effective for reducing reactive oxygen species production by adult stem cells and improving their therapeutic efficacy in many
in vivo ischemia and other disease models [
247,
248,
249]. This has proven sufficient to counteract the impaired oxidative stress resistance of MSCs from elderly donors [
78].
Likewise, the use of naturally occurring antioxidant polyphenols, such as curcumin, has been documented to suppress inducible oxidative stress in human MSCs
ex vivo and may prove to be a safe method for reducing oxidative damage to the
in vivo MSC pool [
250]. Rejuvenation of aged human MSCs has been achieved by seeding cell scaffolds with proangiogenic growth factors, resulting in improved functional capacity of the aged cells after implantation into an infarcted rat heart compared to aged cells seeded on untreated scaffolds [
251]. Systemic administration of growth factors has also proven effective for restoring aged MSCs
in vivo; in the case of senile osteoporosis, intraperitoneal injections of rhBMP2 were sufficient to reverse the osteoporotic phenotype, and this effect was mediated by an expanded MSC pool displaying increased proliferation and decreased apoptosis [
252].
8.1. Transcriptional Reprogramming
The idea has been raised that it might be possible to exploit reprogramming techniques for renewal of the
in vivo stem cell pool to combat diseases of aging [
255]. While full reprogramming of stem cells
in vivo to restore tissues degenerated as a result of age is not likely to manifest clinically until highly efficient reprogramming can be achieved through delivery mechanisms other than lentiviral vectors, the idea of “direct reprogramming” of cell fate in specific tissues
in vivo has been pursued using developmental regulators that redirect a cell’s terminally differentiated state rather than returning the cell to a pluripotent state—what is known in the adult stem cell world as transdifferentiation, as opposed to dedifferentiation. This approach has been employed successfully to convert pancreatic exocrine cells to endocrine cells, rescuing the hyperglycemic phenotype in a mouse model of diabetes [
256].
Generation of stem cells resistant to the phenotypic changes that accompany replicative senescence, such as arrested proliferation and decreased differentiation potential, would create a more ideal cell type for use in stem cell-based tissue engineering and cell therapy.
Ex vivo reprogramming to achieve a kind of cell “reset” may in the future yield this improved cell source. iPS cells generated from HGPS patient fibroblasts display no evidence of progerin accumulation, nuclear envelope and epigenetic defects, or accelerated aging, suggesting this approach can in fact be used to reset an aged cell [
58]. In the case of HGPS-iPS cells, differentiation results in the rapid accumulation of progerin and restoration of the accelerated aging phenotype [
257].
However, this would not be an issue with physiologically aged donor cells. iPS cells derived from young and old non-progeroid human fibroblasts displayed no differences in mitotic activity after differentiation back to a fibroblast phenotype, suggesting that reprogramming is a successful approach to reset aged cells to a youthful phenotype in physiologically aged donors. In this study an excisable vector was used, further illustrating what might be a feasible approach to
ex vivo rejuvenation of aged cells [
232].
Stem cell rejuvenation techniques are also needed in situations where it is preferable to use cells from a specific donor who happens to be of advanced age, since HSC donor age is correlated with adverse events after infusion [
258]. Meeting this need is critical for transplants with autologous or human leukocyte antigen (HLA)-matched sibling HSCs from elderly donors, which result in better outcomes in leukemia and lymphoma than HSCs from an HLA-matched unrelated younger donor [
259]. Several groups have investigated the use of reprogramming transcription factors to restore differentiation potential and proliferative capacity of adult stem cells from aging donors. In one such study, Nanog was over-expressed in adult marrow-derived MSCs, resulting in reversal of lost myogenic differentiation potential and enhancement of proliferation comparable to that observed in neonatal marrow-derived MSCs [
260]. It remains to be seen if other approaches to dedifferentiation will restore an unblemished phenotype to cells to the same degree that reprogramming appears to.
8.2. Calorie Restriction and Pharmacologic Mimicry of Calorie Restriction
Calorie restriction as a therapeutic intervention to delay aging and extend lifespan has been extensively studied in animal models but, at levels that would confer significant clinical benefit, is unlikely to gain much traction due to low rates of adherence. Pharmacologic agents to reduce nutrient intake or absorption might be employed to this end (reviewed in [
261]). The effects of diet and exercise to reduce body weight and correct metabolic disease on adult stem cell populations are unknown, although reduction in visceral fat has been shown to correct endocrine functions of adipocytes. Enhancement of PPARδ signaling has been suggested as an adjunct therapy to boost catabolism in visceral adipose tissue, perhaps in part through differentiation of adipose-resident MSCs to mitochondria-enriched small adipocytes [
261]. To this end PPARδ agonists have been tested in clinical trials, but despite protective effects against obesity and diabetes, development was discontinued due to multi-organ cancer formation in animal models [
262,
263].
Other studies have investigated the use of pharmacologic agents to mimic the molecular benefits of calorie restriction for extending lifespan and healthspan (
Figure 4, [
264]). A recent report described extension of lifespan and healthspan in male mice with administration of metformin beginning in middle age [
265]; past work has established this same phenomenon in invertebrates [
266]. At the cellular level, treated mice displayed increased AMPK activity, decreased oxidative damage, and a transcriptomic shift mimicking the effects of calorie restriction. As a result the mice maintained sensitivity to insulin and low levels of systemic inflammation into old age.
It should be noted that the dose of metformin used to achieve these effects resulted in serum drug levels an order of magnitude higher than what is typically achieved in patients when the drug is used as an antidiabetic therapy; a ten-fold higher dose proved toxic rather than beneficial in this study. As is the case for many pathways regulating longevity, cellular aging, and oncogenic resistance, the degree to which AMPK signaling is altered is likely to require a fine balance between too much and too little. This, combined with concerns about the pleiotropic effects of metformin in vivo which manifest differently with short-term versus long-term use, means that significant work is still needed before this potentially attractive therapy for systemic anti-aging can be safely employed.
Figure 4.
The growth hormone (GH)—insulin-like growth factor I (IGF1) pathway and its signaling cascade, which involves PI(3)K and Akt, can modulate longevity and cancer in model systems. Calorific restriction inhibits GH-IGF1 signaling and can also inhibit mTOR and activate AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1). Interactions between components of these pathways, and with SIRT1, remain incompletely understood. The role of SIRT1 in modulating mammalian ageing has not been demonstrated, and it seems to have a dual role in cancer. DNA repair systems and DNA damage checkpoints prevent the DNA damage accumulation that contributes to cancer and ageing, although possibly through different cellular mechanisms. Reprinted by permission from Macmillan Publishers Ltd.:
Nature Reviews Cancer [
264], copyright 2013.
Figure 4.
The growth hormone (GH)—insulin-like growth factor I (IGF1) pathway and its signaling cascade, which involves PI(3)K and Akt, can modulate longevity and cancer in model systems. Calorific restriction inhibits GH-IGF1 signaling and can also inhibit mTOR and activate AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1). Interactions between components of these pathways, and with SIRT1, remain incompletely understood. The role of SIRT1 in modulating mammalian ageing has not been demonstrated, and it seems to have a dual role in cancer. DNA repair systems and DNA damage checkpoints prevent the DNA damage accumulation that contributes to cancer and ageing, although possibly through different cellular mechanisms. Reprinted by permission from Macmillan Publishers Ltd.:
Nature Reviews Cancer [
264], copyright 2013.
8.3. Epigenetic Modification
The emerging field of translational epigenetics is aimed at correcting heritable but potentially reversible “epimutations” with chemical modifiers, and is gaining some traction in diseases of aging such as metabolic syndrome (reviewed in [
267]) and cancer (reviewed in [
268]) because it offers the possibility of targeting some of the same cell processes as transcriptional reprogramming without the associated risks of introducing exogenous genetic material. The treatment of epigenetically disrupted stem cells in cancer in particular may yield tremendous clinical benefit as the number of epigenetic modifiers grows, allowing for more specific targeting of known associated epimutations.
In the area of metabolic disease, epigenetic modification with nucleic acids or small molecules may allow for extending the healthspan if not the lifespan of patients. The demethylase UTX-1, the activity of which increases in mid-life, removes gene inactivating marks such as histone H3 trimethylation on lysine 27 (H3K27me3) on members of the insulin/IGF-1 signaling pathway, enhancing their activity and resulting in decreased FoxO activity and age-related cellular decline. Restoration of H3K4me3 on an insulin-like receptor gene in
C.
elegans has been shown to decrease insulin/IGF-1 signaling, resetting the cell to a more naive epigenetic state and ultimately extending the life of the animal by 30% [
139]. Although this effect was achieved through the use of RNA interference, the authors expressed optimism that in the near future small molecules might be employed to target epigenetic marks and/or modifying enzymes in a similarly specific strategy.
Similarly, the histone methyltransferase SUV39H1 is protected from proteasomal degradation by enhanced binding to progerin in
Zmpste24−/− mice, resulting in increased H3K9me3 levels and compromised genome maintenance, which leads to accelerated senescence [
269]. Targeting of SUV39H1 in this study resulted in amelioration of the progeroid phenotype in
Zmpste24−/− mice, including reduction of bone loss and extension of lifespan by 60%, suggesting a similar strategy might be useful in the context of normal aging.
In an analogous approach, H4K16 hypoacetylation was targeted in the same mouse model to ameliorate the progeroid phenotype, with overexpression of the histone acetyltransferase Mof or addition of the histone deacetylase inhibitor sodium butyrate to drinking water promoting repair of damaged DNA and resulting in reduced evidence of disease [
270]. Hypoacetylation of this mark was also found in aged wild-type mice, suggesting that aberrant histone acetylation may play a role in physiologic aging and administration of histone deacetylase (HDAC) inhibitors may have therapeutic value in disease of aging.
8.4. Strategies to Delay Senescence
Genetic modification strategies have specifically targeted known regulators of senescence and lifespan to combat diseases of aging. Preventing senescence, clearing senescent cells, or interfering with the senescence-associated secretory phenotype, in which cells release inflammatory mediators such as cytokines and matrix metalloproteinases, are all approaches that might lessen the contribution of cellular aging to chronic illness [
271]. Given the complexity of the signaling crosstalk regulating senescence and associated events, identification of therapeutically targetable elements in this network—the “senectome”—is proceeding at multiple levels, the most recent of which includes senescence-associated micro-RNAs, which could be manipulated or used as clinical biomarkers [
272].
In non-healing diabetic skin ulcers, siRNA- or
vivo-Morpholino antisense-based gene therapy targeting of
CAV1 or
PTRF, which are both turned on by oxidative stress in diabetic fibroblasts and induce p53-dependent premature senescence, inhibited senescence and accelerated ulcer repair [
273]. Atherosclerosis, especially in the context of type 2 diabetes, is related to endothelial senescence and has been reduced using a variety of interventions targeting nitric oxide levels and bioavailability in the endothelial microenvironment, including eNOS gene therapy [
274]. Gene therapy to induce telomerase activity in CD8 T cells, which undergo premature senescence in the context of HIV infection, results in enhanced proliferation and increased antiviral function [
275]. While preliminary research in this area has not resulted in karyotypic changes or wildly altered growth kinetics of the CD8 compartment, the authors of this study emphasized the need for pharmacologic approaches that would mimic these effects without the need for
TERT gene therapy due to the obvious risks.
The use of pharmacologic agents to modulate senescence-associated pathways is a promising avenue to counteract the effects of aging in the clinic. Mice of both genders treated with rapamycin starting in mid to late life display extended lifespan and reduced incidence of cancer [
276]. Treatment of cells from HGPS patients with rapamycin, results in enhanced clearance of the mutant protein progerin by autophagy and delayed onset of senescence [
277]. Conversely, activation of Akt-mTOR signaling through inhibition of isoprenylcysteine carboxyl methyltransferase, the enzyme which processes prelamin A to lamin A and enables trafficking to the nuclear rim, in
Zmpste24−/− mice also delayed onset of senescence and improved disease phenotype. This suggests the Akt-mTOR axis, like the Wnt axis, is finely tuned and must be carefully manipulated to achieve therapeutic benefit [
278]. Rapamycin is unlikely to be utilized extensively as an anti-aging therapeutic due to its side effects, which include hyperlipidemia and immunosuppression; however, newer analogs of rapamycin (rapalogs) are in development and may find use as anti-aging compounds, along with other agents that inhibit mTOR (reviewed in [
279]).
Pharmacologic activation of SIRT1 in a rat model of diabetes restored endothelial differentiation, pro-angiogenic chemokine secretion, and
in vivo angiogenic activity of bone marrow-derived early outgrowth cells to that of cells from control animals [
280]. Pharmacologic blockade of angiotensin II signaling through its type I receptor, which is used clinically to lower blood pressure and prevent insulin resistance in metabolic syndrome, also inhibits adipogenesis in adipose- and bone marrow-derived MSCs, both preventing further pathologic expansion of adipose tissue and helping to maintain an uncommitted progenitor pool for tissue homeostasis and repair [
281]. In opposition to the enhanced production of 20-HETE by the cytochrome P450 system observed in metabolic syndrome, MSCs generate P450-derived epoxyeicosatrienoic acids (EETs) from arachidonic acids, and when administered exogenously these lipid mediators have been shown to decrease adipocyte differentiation of MSCs via an increase in heme oxygenase-1 and decrease in PPARγ, C/EBPα, and Fas and to reprogram adipocyte stem cells to a new phenotype displaying a smaller cell size, increased secretion of adiponectin, and decreased secretion of inflammatory cytokines [
282]. EET agonists have also been shown to reverse a metabolic syndrome phenotype in an obese animal model, highlighting the therapeutic potential of targeting production of these molecules in the adult stem cell pool to combat this age-related disease phenotype in humans [
283].
Given the complex and sometimes unpredictable nature of these emerging pharmaceutical and genetic approaches to age-related disease therapy, sometimes the simplest approaches to maintaining health are best. In the lifelong struggle between growth-promoting signaling pathways and stress resistance pathways, sleep has a critical place in determining the balance [
171]. mTOR and FoxO signaling are turned on in distinct temporal windows during early and late sleep, respectively, in response to alterations in somatotrophic signaling, suggesting that a good night’s sleep truly does have restorative powers. Adult stem cells have been demonstrated to undergo significant circadian regulation in multiple studies, with HSCs, marrow- and adipose-derived MSCs, and cancer stem cells all subject to transcriptome modulation by core circadian regulatory proteins (reviewed in [
284]). ESCs, in contrast to adult stem cells, are not subject to circadian regulation and have been shown to acquire molecular circadian oscillation upon differentiation; subsequent transcriptional reprogramming with
Sox2,
Klf4,
Oct3/4, and
c-Myc genes was shown to suppress circadian cycling, literally resetting the internal clock in the resulting iPS cells [
285]. Interestingly, action of core circadian regulatory proteins on physiologic cellular processes is opposed by SIRT1 with aging (reviewed in [
286]), and control of central circadian cycling by SIRT1 in the brain decays over time [
287]. In general circadian rhythms break down with age, coinciding with the development of metabolic derangements and potentially contributing significantly to the organismal aging process (reviewed in [
288]). Research into the impact of macro-environmental factors, such as organismal circadian rhythms, on stem cell niches may open new therapeutic avenues for manipulating stem cell fate in diseases of aging [
289]. As therapeutic approaches go, good sleep hygiene is safe and free from side effects, cost-effective, and potentially contributes more than we currently know to the long-term maintenance of adult stem cell compartments.
