Delineating the relationship between immune system aging and myogenesis in muscle repair

Recruited immune cells play a critical role in muscle repair, in part by interacting with local stem cell populations to regulate muscle regeneration. How aging affects their communication during myogenesis is unclear. Here, we investigate how aging impacts the cellular function of these two cell types after muscle injury during normal aging or after immune rejuvenation using a young to old (Y‐O) or old to old (O‐O) bone marrow (BM) transplant model. We found that skeletal muscle from old mice (20 months) exhibited elevated basal inflammation and possessed fewer satellite cells compared with young mice (3 months). After cardiotoxin muscle injury (CTX), old mice exhibited a blunted inflammatory response compared with young mice and enhanced M2 macrophage recruitment and IL‐10 expression. Temporal immune and cytokine responses of old mice were partially restored to a young phenotype following reconstitution with young cells (Y‐O chimeras). Improved immune responses in Y‐O chimeras were associated with greater satellite cell proliferation compared with O‐O chimeras. To identify how immune cell aging affects myoblast function, conditioned media (CM) from activated young or old macrophages was applied to cultured C2C12 myoblasts. CM from young macrophages inhibited myogenesis while CM from old macrophages reduced proliferation. These functional differences coincided with age‐related differences in macrophage cytokine expression. Together, this study examines the infiltration and proliferation of immune cells and satellite cells after injury in the context of aging and, using BM chimeras, demonstrates that young immune cells retain cell autonomy in an old host to increase satellite cell proliferation.     

In vivo GDF3 administration abrogates aging related muscle regeneration delay following acute sterile injury

Tissue regeneration is a highly coordinated process with sequential events including immune cell infiltration, clearance of damaged tissues, and immune‐supported regrowth of the tissue. Aging has a well‐documented negative impact on this process globally; however, whether changes in immune cells per se are contributing to the decline in the body’s ability to regenerate tissues with aging is not clearly understood. Here, we set out to characterize the dynamics of macrophage infiltration and their functional contribution to muscle regeneration by comparing young and aged animals upon acute sterile injury. Injured muscle of old mice showed markedly elevated number of macrophages, with a predominance for Ly6C^high pro‐inflammatory macrophages and a lower ratio of the Ly6C^low repair macrophages. Of interest, a recently identified repair macrophage‐derived cytokine, growth differentiation factor 3 (GDF3), was markedly downregulated in injured muscle of old relative to young mice. Supplementation of recombinant GDF3 in aged mice ameliorated the inefficient regenerative response. Together, these results uncover a deficiency in the quantity and quality of infiltrating macrophages during aging and suggest that in vivo administration of GDF3 could be an effective therapeutic approach.     

Adult Sox2+ stem cell exhaustion in mice results in cellular senescence and premature aging

Aging is characterized by a gradual functional decline of tissues with age. Adult stem and progenitor cells are responsible for tissue maintenance, repair, and regeneration, but during aging, this population of cells is decreased or its activity is reduced, compromising tissue integrity and causing pathologies that increase vulnerability, and ultimately lead to death. The causes of stem cell exhaustion during aging are not clear, and whether a reduction in stem cell function is a cause or a consequence of aging remains unresolved. Here, we took advantage of a mouse model of induced adult Sox2+ stem cell depletion to address whether accelerated stem cell depletion can promote premature aging. After a short period of partial repetitive depletion of this adult stem cell population in mice, we observed increased kyphosis and hair graying, and reduced fat mass, all of them signs of premature aging. It is interesting that cellular senescence was identified in kidney after this partial repetitive Sox2+ cell depletion. To confirm these observations, we performed a prolonged protocol of partial repetitive depletion of Sox2+ cells, forcing regeneration from the remaining Sox2+ cells, thereby causing their exhaustion. Senescence specific staining and the analysis of the expression of genetic markers clearly corroborated that adult stem cell exhaustion can lead to cellular senescence induction and premature aging.     

Generation of pluripotent stem cells from eggs of aging mice

Oocytes can reprogram genomes to form embryonic stem (ES) cells. Although ES cells largely escape senescence, oocytes themselves do senesce in the ovaries of most mammals. It remains to be determined whether ES cells can be established using eggs from old females, which exhibit reproductive senescence. We attempted to produce pluripotent stem cell lines from artificial activation of eggs (also called pES) from reproductive aged mice, to determine whether maternal aging affects pES cell production and pluripotency. We show that pES cell lines were generated with high efficiency from reproductive aged (old) mice, although parthenogenetic embryos from these mice produced fewer ES clones by initial two passages. Further, pES cell lines generated from old mice showed telomere length, expression of pluripotency molecular markers (Oct4, Nanog, SSEA1), alkaline phosphatase activity, teratoma formation and chimera production similar to young mice. Notably, DNA damage was reduced in pES cells from old mice compared to their progenitor parthenogenetic blastocysts, and did not differ from that of pES cells from young mice. Also, global gene expression differed only minimally between pES cells from young and old mice, in contrast to marked differences in gene expression in eggs from young and old mice. These data demonstrate that eggs from old mice can generate pluripotent stem cells, and suggest that the isolation and in vitro culture of ES cells must select cells with high levels of DNA and telomere integrity, and/or with capacity to repair DNA and telomeres.     

Long-term self-renewal of postnatal muscle-derived stem cells

The ability to undergo self-renewal is a defining characteristic of stem cells. Self-replenishing activity sustains tissue homeostasis and regeneration. In addition, stem cell therapy strategies require a heightened understanding of the basis of the self-renewal process to enable researchers and clinicians to obtain sufficient numbers of undifferentiated stem cells for cell and gene therapy. Here, we used postnatal muscle-derived stem cells to test the basic biological assumption of unlimited stem cell replication. Muscle-derived stem cells (MDSCs) expanded for 300 population doublings (PDs) showed no indication of replicative senescence. MDSCs preserved their phenotype (ScaI+/CD34+/desmin(low)) for 200 PDs and were capable of serial transplantation into the skeletal muscle of mdx mice, which model Duchenne muscular dystrophy. MDSCs expanded to this level exhibited high skeletal muscle regeneration comparable with that exhibited by minimally expanded cells. Expansion beyond 200 PDs resulted in lower muscle regeneration, loss of CD34 expression, loss of myogenic activity, and increased growth on soft agar, suggestive of inevitable cell aging attributable to expansion and possible transformation of the MDSCs. Although these results raise questions as to whether cellular transformations derive from cell culturing or provide evidence of cancer stem cells, they establish the remarkable long-term self-renewal and regeneration capacity of postnatal MDSCs.     

Behaviour of telomere and telomerase during aging and regeneration in zebrafish

Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio) offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration.     

Proliferative feeder cells support prolonged expansion of human embryonic stem cells

Long-term proliferation of human embryonic stem (hES) cells is currently achieved by co-culturing with mitotically inactive primary mouse embryonic fibroblasts (mEFs), which serve as feeder cells. This study explores the possibility that proliferative mEFs can be used as feeder cells to maintain the prolonged expansion of hES cells. All undifferentiated hES cell clumps were re-plated on six different densities of proliferative mEFs. hES colonies cultured on 1 x 10(5) - 5 x 10(5) proliferative mEFs amplified over 130 days of continuous culture and remained undifferentiated, as did those cultured on mitotically inactive mEFs. This suggests that certain densities of proliferative mEFs can maintain the propagation of hES cells, which may be helpful for identifying the cytokines and adhesion molecules that are required for their self-renewal.     

Comparative angiogenic activities of induced pluripotent stem cells derived from young and old mice

Advanced age is associated with decreased stem cell activity. However, the effect of aging on the differentiation capacity of induced pluripotent stem (iPS) cells into cardiovascular cells has not been fully clarified. We investigated whether iPS cells derived from young and old mice are equally capable of differentiating into vascular progenitor cells, and whether these cells regulate vascular responses in vivo. iPS cells from mouse embryonic fibroblasts (young) or 21 month-old mouse bone marrow (old) were used. Fetal liver kinase-1 positive (Flk-1(+)) cells, as a vascular progenitor marker, were induced after 3 to 4 days of culture from iPS cells derived from young and old mice. These Flk-1(+) cells were sorted and shown to differentiate into VE-cadherin(+) endothelial cells and α-SMA(+) smooth muscle cells. Tube-like formation was also successfully induced in both young and old murine Flk-1(+) cells. Next, hindlimb ischemia was surgically induced, and purified Flk-1(+) cells were directly injected into ischemic hindlimbs of nude mice. Revascularization of the ischemic hindlimb was significantly accelerated in mice transplanted with Flk-1(+) cells derived from iPS cells from either young or old mice, as compared to control mice as evaluated by laser Doppler blood flowmetry. The degree of revascularization was similar in the two groups of ischemic mice injected with iPS cell-derived Flk-1(+) cells from young or old mice. Transplantation of Flk-1(+) cells from both young and old murine iPS cells also increased the expression of VEGF, HGF and IGF mRNA in ischemic tissue as compared to controls. iPS cell-derived Flk-1(+) cells differentiated into vascular progenitor cells, and regulated angiogenic vascular responses both in vitro and in vivo. These properties of iPS cells derived from old mice are essentially the same as those of iPS cells from young mice, suggesting the functionality of generated iPS cells themselves to be unaffected by aging.     

Lasting effects of an impairment of Th1-like immune response in γ-irradiated mice: A resemblance between irradiated mice and aged mice

Although one of the several chronic effects of ionizing radiation is aging, there are no experimental data on radiation-induced immunological aging. The most interesting change in aging was a helper T (Th) 1/Th2 imbalance. We investigated chronic effect on immune responses after ionizing radiation and its effects in irradiated mice were compared with those of aged mice. The 2-month-old mice received a whole-body irradiation of 5 Gy. At 6 months after irradiation, we compared the immune functions of the irradiated mice with those of normal mice of the same age and with those of older. Interferon (IFN)-γ and antigen-specific immunoglobulin (Ig)G2a level were lower in the irradiated mice than in normal mice of same age, showing similar levels to those of old normal mice. In contrast, interleukin (IL)-4 and IL-5 and antigen-specific IgG1 level were increased in irradiated mice when compared with the same aged-normal mice. Next, we investigated the low expression of IL-12p70, IL-12 receptors and IL-18 receptors in irradiated and old mice. Also, the decrease of natural killer cell activity was intensified in the irradiated mice, showing lower than values to those of old mice. Interestingly, in irradiated mice, the absolute numbers and the percentages of natural killer (NK) cells was extremely decreased. But the absolute numbers of Th cells and cytotoxic T (Tc) cells in old mice were significantly decreased. In conclusion, an immunological imbalance by the whole-body irradiation of 5 Gy induces to persist in the long term, resulting in the similar results with aging. Our results suggest that the downregulation of the Th1-like immune response shown in old mice rapidly occurred through exposure of ionizing radiation.     

Temporal regulation of Cre recombinase activity in neural stem cells

Neural stem cells are known to give rise to distinct subtypes of neurons and glial cells over time by changing their competency. However, precise characterization of neural stem cells at various developmental stages remains to be performed. For such analysis, a tool to manipulate neural stem cells at different time points is necessary. Here, we generated transgenic mice that express Cre-ER(T2) in the ventricular zone of the developing nervous system under the control of the nestin promoter and enhancer (Nes-CreER(T2)). In mice expressing Cre-ER(T2) at appropriate levels, Cre recombinase activity was mostly inactive but efficiently activated by tamoxifen within 1 day. When such mice were crossed with the ROSA-26 or Z/EG reporter mice, neural stem cells were permanently labeled after administration of tamoxifen. Thus, Nes-CreER(T2) mice offer a powerful tool to manipulate neural stem cells genetically at desired time points.     

Notch信号通路参与卵巢生殖干细胞的调控及卵巢的衰老进程

卵巢生殖干细胞(ovarian germline stem cells, OGSCs)的发现,打破了生殖医学领域传统的"固定卵泡池"理论。近年来,OGSCs新的研究成果不断涌现,但关于OGSCs体内调控机制的研究仍然较少。Notch通路广泛参与多种成体干细胞不对称分裂的过程,并与细胞衰老密切相关,但其是否参与OGSCs的体内调控机制及卵巢的衰老进程尚不清楚。本研究以原代培养技术提取OGSCs,通过荧光双标染色发现,OGSCs标志基因MVH、Oct4与Notch信号通路相关分子Notch1、Hes1在OGSCs中存在共表达;抑制Notch信号通路活性后,cck-8检测发现,OGSCs的增殖活性呈下降趋势;而以免疫组化、荧光双标、Western印迹法检测性成熟期(2月龄)、不孕和衰老(20月龄)小鼠卵巢皮层中MVH、Oct4、Notch1和Hes1的表达变化,发现2月龄小鼠卵巢皮层中MVH、Oct4、Notch1和Hes1的表达量较高(P<0.05),而不孕和衰老小鼠卵巢皮层中,MVH、Oct4、Notch1和Hes1的表达量均明显下降。上述结果表明,Notch信号通路在小鼠OGSCs中高表达,并可能参与调控OGSCs的增殖机制及卵巢的衰老进程。     

Evolution of gene expression signature in mammary gland stem cells from neonatal to old mice

During the lifetime of females, mammary epithelial cells undergo cyclical expansion and proliferation depending on the cyclical activation of mammary gland stem/progenitor cells (MaSCs) in response to the change of hormone level. The structural shrink of mammary duct tree and the functional loss of mammary gland occur along with inactivation of MaSCs in old females, even leading to breast cancer occasionally. However, the gene expression signature in MaSCs across the lifespan remains unclear. Herein, we tested the tissue regeneration ability of CD24⁺CD49f^high MaSCs over six time points from neonatal (4-day-old) to aged mice (360-day-old). Further RNA-seq analyses identified four clusters of gene signatures based on the gene expression patterns. A subset of stemness-related genes was identified, showing the highest level at day 4 of the neonatal age, and the lowest level at the old age. We also identified an aging-related gene signature showing significant change in the old mice, in which an association between aging process and stemness loss was indicated. The aging-related gene signature showed regulation of cancer signaling pathways, as well as aging-related diseases including Huntington disease, Parkinson disease, and Alzheimer disease. Moreover, 425, 1056, 418, and 1107 gene variants were identified at D20, D40, D90, and D180, respectively, which were mostly reported to associated with tumorigenesis and metastasis in cancer. In summary, the current study is the first to demonstrate the gene expression shift in MaSCs from neonatal to aging, which leads to stemness loss, aging, aging-related diseases, and even breast cancer in old mice.Subject terms: Breast cancer, Ageing, Cancer stem cells, Mammary stem cells     

Differentiation rather than aging of muscle stem cells abolishes their telomerase activity

A general feature of stem cells is the ability to routinely proliferate to build, maintain, and repair organ systems. Accordingly, embryonic and germline, as well as some adult stem cells, produce the telomerase enzyme at various levels of expression. Our results show that, while muscle is a largely postmitotic tissue, the muscle stem cells (satellite cells) that maintain this biological system throughout adult life do indeed display robust telomerase activity. Conversely, primary myoblasts (the immediate progeny of satellite cells) quickly and dramatically downregulate telomerase activity. This work thus suggests that satellite cells, and early transient myoblasts, may be more promising therapeutic candidates for regenerative medicine than traditionally utilized myoblast cultures. Muscle atrophy accompanies human aging, and satellite cells endogenous to aged muscle can be triggered to regenerate old tissue by exogenous molecular cues. Therefore, we also examined whether these aged muscle stem cells would produce tissue that is “young” with respect to telomere maintenance. Interestingly, this work shows that the telomerase activity in muscle stem cells is largely retained into old age wintin inbred “long” telomere mice and in wild‐derived short telomere mouse strains, and that age‐specific telomere shortening is undetectable in the old differentiated muscle fibers of either strain. Summarily, this work establishes that young and old muscle stem cells, but not necessarily their progeny, myoblasts, are likely to produce tissue with normal telomere maintenance when used in molecular and regenerative medicine approaches for tissue repair. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Heterochronic parabiosis for the study of the effects of aging on stem cells and their niches

Aging is unmistakable and undeniable in mammals. Interestingly, mice develop cataracts, muscle atrophy, osteoporosis, obesity, diabetes and cognitive deficits after just 2–3 postnatal years, while it takes seven or more decades for the same age-specific phenotypes to develop in humans. Thus, chronological age corresponds differently with biological age in metazoan species and although many theories exist, we do not understand what controls the rate of mammalian aging. One interesting idea is that species-specific rate of aging represents a ratio of tissue attrition to tissue regeneration. Furthermore, current findings suggest that the age-imposed biochemical changes in the niches of tissue stem cells inhibit performance of this regenerative pool, which leads to the decline of tissue maintenance and repair. If true, slowing down stem cell and niche aging, thereby promoting tissue regeneration, could slow down the process of tissue and organismal aging. In this regard, recent studies of heterochronic parabiosis provide important clues as to the mechanisms of stem cell aging and suggest novel strategies for enhancing tissue repair in the old. Here we review current literature on the relationship between the vigor of tissue stem cells and the process of aging, with an emphasis on the rejuvenation of old tissues by the extrinsic modifications of stem cell niches.     

Heterochronic parabiosis for the study of the effects of aging on stem cells and their niches

Aging is unmistakable and undeniable in mammals. Interestingly, mice develop cataracts, muscle atrophy, osteoporosis, obesity, diabetes and cognitive deficits after just 2–3 postnatal years, while it takes seven or more decades for the same age-specific phenotypes to develop in humans. Thus, chronological age corresponds differently with biological age in metazoan species and although many theories exist, we do not understand what controls the rate of mammalian aging. One interesting idea is that species-specific rate of aging represents a ratio of tissue attrition to tissue regeneration. Furthermore, current findings suggest that the age-imposed biochemical changes in the niches of tissue stem cells inhibit performance of this regenerative pool, which leads to the decline of tissue maintenance and repair. If true, slowing down stem cell and niche aging, thereby promoting tissue regeneration, could slow down the process of tissue and organismal aging. In this regard, recent studies of heterochronic parabiosis provide important clues as to the mechanisms of stem cell aging and suggest novel strategies for enhancing tissue repair in the old. Here we review current literature on the relationship between the vigor of tissue stem cells and the process of aging, with an emphasis on the rejuvenation of old tissues by the extrinsic modifications of stem cell niches.     

Cell lines from old immunodeficient donors give normal responses in young recipients.

Two different immune responses were compared in spleen cells obtained from old and young CBA/HT6J mice. Spleen cells from old mice (23 to 33 months) responded about half as well as did spleen cells from young mice (4 to 10 months) in the adoptive transfer anti-sheep red blood cell (SRBC) plague-forming assay, and caused slightly less than half the uptake of tritiated thymidine in response to phytohemagglutinin (PHA) in vitro. Marrow stem cell from some of the old and young mice whose splenic immune responses were tested were transplanted into irradiated young CBA/CaJ recipients. Seven to 17 weeks later these same immune responses were tested in the spleen cells of these young recipients, and the T6 chromosome marker was used to identify donor cells. Old animals' responses varied greatly, perhaps due to suppressing cells or factors in some individuals. Therefore, cells were never pooled and the responses of receipients were compared to the responses of the donor whose marrow had populated them. The response for a particular old donor, or for the recipients of its stem cells, was divided by the response for the young control used with that donor, or for its stem cell recipients. This was called the old/young ratio. With original donors with an old/young ratio for the SRBC response of (mean +/- S.D.) 0.35 +/- 0.14, The old/young ratio for that same response in the recipients was significantly improved to 1.26 +/- 0.71. In original donors with an old/young ratio for the PHA response of 0.44 +/- 0.17, the old/young ratio in the recipients improved significantly to 0.86 +/- 0.27. Thus, little or none of the decline with age in these immune responses was intrinsic to the old lymphoid stem cells.     

Aging hair follicles rejuvenated by transplantation to a young subcutaneous environment

We demonstrate in the present study that young host mice rejuvenate aged hair follicles after transplantation. Young mice promote the hair shaft growth of transplanted old hair follicles, as well as young follicles, in contrast to old host mice, which did not support hair-shaft growth from transplanted old or young follicles. Nestin-expressing hair follicle-associated pluripotent (HAP) stem cells of transplanted old and young hair follicles remained active in young host nude mice. In contrast, the nestin-expressing HAP stem cells in young and old hair follicles transplanted to old nude mice were not as active as in young nude host mice. The present study shows that transplanted old hair follicles were rejuvenated by young host mice, suggesting that aging may be reversible.