Small-animal blood exchange is an emerging approach for systemic aging research.

We describe a small-animal blood exchange approach developed for aging research as an alternative to heterochronic parabiosis or plasma injections. In parabiosis, animals are surgically coupled, which has several disadvantages, including difficulty controlling experimental procedure, the effects of shared organs, environmental enrichment from jointly exploring the housing enclosure, involuntary exercise and an imprecise onset of blood sharing. Likewise, in plasma injections, the added volumes need to be small, and there is little flexibility in changing the relative contributions of ectopic to endogenous blood components. These factors complicate the conclusions and interpretations, including the identification of key mechanisms and molecular or cellular determinants. Our approach, where blood is exchanged between animals without them being surgically coupled, is less invasive than parabiosis. The percentage of exchanged blood or other exchanged fluids is known and precise. The age of plasma and cells can be mixed and matched at all desired relative contributions to the endogenous systemic milieu, and the onset of the effects can be accurately delineated. In this protocol, we describe the preparatory and animal surgery steps required for small-animal blood exchange in mice and compare this process with parabiosis and plasma injections. We also provide the design, hardware and software for the blood exchange device and compare automated and manual exchange methods. Lastly, we report mathematical modeling of the dilution of blood factors. The fluid exchange takes ~30 min when performed by a well-trained biomedical scientist; the entire process takes ~2 h.

Old plasma dilution reduces human biological age: a clinical study.

This work extrapolates to humans the previous animal studies on blood heterochronicity and establishes a novel direct measurement of biological age. Our results support the hypothesis that, similar to mice, human aging is driven by age-imposed systemic molecular excess, the attenuation of which reverses biological age, defined in our work as a deregulation (noise) of 10 novel protein biomarkers. The results on biological age are strongly supported by the data, which demonstrates that rounds of therapeutic plasma exchange (TPE) promote a global shift to a younger systemic proteome, including youthfully restored pro-regenerative, anticancer, and apoptotic regulators and a youthful profile of myeloid/lymphoid markers in circulating cells, which have reduced cellular senescence and lower DNA damage. Mechanistically, the circulatory regulators of the JAK-STAT, MAPK, TGF-beta, NF-κB, and Toll-like receptor signaling pathways become more youthfully balanced through normalization of TLR4, which we define as a nodal point of this molecular rejuvenation. The significance of our findings is confirmed through big-data gene expression studies.

The Microfluidic Toolbox for Analyzing Exosome Biomarkers of Aging.

As the fields of aging and neurological disease expand to liquid biopsies, there is a need to identify informative biomarkers for the diagnosis of neurodegeneration and other age-related disorders such as cancers. A means of high-throughput screening of biomolecules relevant to aging can facilitate this discovery in complex biofluids, such as blood. Exosomes, the smallest of extracellular vesicles, are found in many biofluids and, in recent years, have been found to be excellent candidates as liquid biopsy biomarkers due to their participation in intercellular communication and various pathologies such as cancer metastasis. Recently, exosomes have emerged as novel biomarkers for age-related diseases. Hence, the study of exosomes, their protein and genetic cargo can serve as early biomarkers for age-associated pathologies, especially neurodegenerative diseases. However, a disadvantage of exosome studies includes a lack in standardization of isolating, detecting, and profiling exosomes for downstream analysis. In this review, we will address current techniques for high-throughput isolation and detection of exosomes through various microfluidic and biosensing strategies and how they may be adapted for the detection of biomarkers of age-associated disorders.

Application of bio-orthogonal proteome labeling to cell transplantation and heterochronic parabiosis.

Studies of heterochronic parabiosis demonstrated that with age, the composition of the circulatory milieu changes in ways that broadly inhibit tissue regenerative capacity. In addition, local tissue niches have age-specific influences on their resident stem cells. Here we use bio-orthogonal proteome labeling for detecting in vivo proteins present only in transplanted myoblasts, but not in host tissue, and proteins exclusive to one young mouse and transferred during parabiosis to its old partner. We use a transgenic mouse strain that ubiquitously expresses a modified tRNA methionine synthase, metRS, which preferentially incorporates the methionine surrogate azido-nor-leucine (ANL) into newly generated proteins. Using click chemistry and a modified antibody array to detect ANL-labeled proteins, we identify several 'young' systemic factors in old regenerating muscle of the heterochronic parabiotic partners. Our approach enables the selective profiling of mammalian proteomes in mixed biological environments such as cell and tissue transplantation, apheresis or parabiosis.Clarifying the source of proteins in mixed biological environments, such as after transplantation or parabiosis, remains a challenge. Here, the authors address this need with a mouse strain that incorporates a methionine derivate into proteins, allowing for their detection using click chemistry and antibody arrays.

Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells.

The regenerative capacity of muscle dramatically decreases with age because old muscle stem cells fail to proliferate in response to tissue damage. Here, we uncover key age-specific differences underlying this proliferative decline: namely, the genetic loci of cyclin/cyclin-dependent kinase (CDK) inhibitors (CDKIs) p21 and p16 are more epigenetically silenced in young muscle stem cells, as compared to old, both in quiescent cells and those responding to tissue injury. Interestingly, phosphorylated ERK (pERK) induced in these cells by ectopic FGF2 is found in association with p21 and p16 promoters, and moreover, only in the old cells. Importantly, in the old satellite cells, FGF2/pERK silences p21 epigenetically and transcriptionally, which leads to reduced p21 protein levels and enhanced cell proliferation. In agreement with the epigenetic silencing of the loci, young muscle stem cells do not depend as much as old on ectopic FGF/pERK for their myogenic proliferation. In addition, other CDKIs, such asp15(INK4B) and p27(KIP1) , become elevated in satellite cells with age, confirming and explaining the profound regenerative defect of old muscle. This work enhances our understanding of tissue aging, promoting strategies for combating age-imposed tissue degeneration.

Heterochronic parabiosis: historical perspective and methodological considerations for studies of aging and longevity.

Pairing two animals in parabiosis to test for systemic or circulatory factors from one animal affecting the other animal has been used in scientific studies for at least 150 years. These studies have led to advances in fields as diverse as endocrinology, immunology, and oncology. A variation on the technique, heterochronic parabiosis, whereby two animals of different ages are joined to test for systemic regulators of aspects of aging or age-related diseases also has almost a century-long scientific history. In this review, we focus on the history of heterochronic parabiosis, methodological considerations and caveats, and the major advances that have emerged from those studies, including recent advances in our understanding of stem cell aging.

Age dependent increase in the levels of osteopontin inhibits skeletal muscle regeneration.

Skeletal muscle regeneration following injury is accompanied by rapid infiltration of macrophages, which play a positive role in muscle repair. Increased chronic inflammation inhibits the regeneration of dystrophic muscle, but the properties of inflammatory cells are not well understood in the context of normal muscle aging. This work uncovers pronounced age-specific changes in the expression of osteopontin (OPN) in CD11b+ macrophages present in the injured old muscle as well as in the blood serum of old injured mice and in the basement membrane surrounding old injured muscle fibers. Furthermore, young CD11b+ macrophages enhance regenerative capacity of old muscle stem cells even when old myofibers and old sera are present; and neutralization of OPN similarly rejuvenates the myogenic responses of old satellite cells in vitro and notably, in vivo. This study highlights potential mechanisms by which age related inflammatory responses become counter-productive for muscle regeneration and suggests new strategies for enhancing muscle repair in the old.

SnoN activates p53 directly to regulate aging and tumorigenesis.

We have identified SnoN as a direct activator of p53 to accelerate aging and inhibit tumorigenesis. SnoN has been shown previously to promote proliferation and transformation by antagonizing TGFß signaling. We show that elimination of this TGFß antagonistic activity of SnoN in vivo results in accelerated aging and resistance to tumorigenesis. The SnoN knockin mice display a shortened lifespan, decreased reproductivity, osteoporosis, reduced regenerative capacity, and other aging phenotypes, similar to that found in mice expressing an active p53. These activities of SnoN rely on the ability of SnoN to activate p53. SnoN can bind directly to p53 and compete with Mdm2 for binding to p53, preventing p53 ubiquitination and degradation and additionally facilitating p53 acetylation and phosphorylation. SnoN also binds to p53 on the promoter of p53 responsive genes to promote transcription activation. This activation of p53 by SnoN is necessary for its antitumorigenic and progeria activities in vivo because elimination of one copy of p53 reverses the aging phenotypes and accelerates tumorigenesis. Thus, we have revealed a novel function of SnoN in regulating aging and tumorigenesis by directly activating p53.

Embryonic anti-aging niche.

Although functional organ stem cells persist in the old, tissue damage invariably overwhelms tissue repair, ultimately causing the demise of an organism. The poor performance of stem cells in an aged organ, such as skeletal muscle, is caused by the changes in regulatory pathways such as Notch, MAPK and TGF-ß, where old differentiated tissue actually inhibits its own regeneration. This perspective analyzes the current literature on regulation of organ stem cells by their young versus old niches and suggests that determinants of healthy and prolonged life might be under a combinatorial control of cell cycle check point proteins and mitogens, which need to be tightly balanced in order to promote tissue regeneration without tumor formation. While responses of adult stem cells are regulated extrinsically and age-specifically, we put forward experimental evidence suggesting that embryonic cells have an intrinsic youthful barrier to aging and produce soluble pro-regenerative proteins that signal the MAPK pathway for rejuvenating myogenesis. Future identification of this activity will improve our understanding of embryonic versus adult regulation of tissue regeneration suggesting novel strategies for organ rejuvenation. Comprehensively, the current intersection of aging and stem cell science indicates that if the age-imposed decline in the regenerative capacity of stem cells was understood, the debilitating lack of organ maintenance in the old could be ameliorated and perhaps, even reversed.

Notch signaling pathway and tissue engineering.

The Notch pathway is a signaling network essential for proper organ development in an embryo, and is indispensable for tissue regeneration in the adult. This key regulatory signaling network is evolutionarily conserved in all metazoans and is continually utilized for the building, maintenance and repair of diverse organs and tissues. Importantly, dysfunctions in the Notch pathway have been demonstrated to result in oncogenic transformation, such as in lymphoid cancers, and have been linked to the pathogenesis of several inherited human diseases. Therefore, the ability to regulate Notch signaling intensity both positively and negatively has a very high therapeutic relevance. Adapting this pathway for tissue engineering applications has great potential to spear-head the development of smart biomaterials to deliberately control cell-fate decisions and lead to designer ex vivo morphogenesis. This review describes the components of Notch-specific signal transduction, presents the role of the Notch signaling network in constructing and repairing multiple organ systems, summarizes the Notch-related pathologies, outlines current advances in the deliberate modulation of the Notch pathway in bioengineering applications, and introduces future perspectives on the use of Notch pathway manipulations as a powerful universal tool in tissue engineering and in the orchestration of stem cell responses. This review also summarizes the existing bioengineering methods most suitable for the deliberate manipulation of Notch signaling, such as smart biomaterials able to pattern Notch ligands or to create gradients of Notch agonists and antagonists. Such methods will likely facilitate the engineering and dynamic remodeling of tissues composed of stem, progenitor and differentiated cells derived from an initially equivalent cell population.

Regulating the Notch pathway in embryonic, adult and old stem cells.

The Notch pathway represents a highly conserved signaling network, which is critical to both embryonic skeletal muscle formation and regeneration in the adult. In addition to skeletal muscle, Notch also regulates the formation and maintenance of various organ systems, such as brain, blood and intestine, in evolutionary distinct vertebrate and invertebrate species. The Notch network 'cross talks' with all other key cell-fate determinants, such as the Wnt (Wingless), TGF-beta/BMP, Hh and RTK/Ras pathways. Hence, modulating the intensity of Notch resonates through multiple regulatory circuitries, and exerts profound effects on cell behaviour. Therefore, various approaches to the targeted manipulation of Notch have been developed (e.g. genetic constructs, antibodies, RNA interference, receptor decoys and gamma-secretase inhibitors). These tools might be used to broaden our understanding of this pathway in regulating responses of embryonic and adult stem cell subsets, and to develop therapeutic approaches against Notch-based diseases (e.g. Alzheimer's, Alagille Syndrome, various cancers and other disease states).

Geometric control of myogenic cell fate.

This work combines expertise in stem cell biology and bioengineering to define the system for geometric control of proliferation and differentiation of myogenic progenitor cells. We have created an artificial niche of myogenic progenitor cells, namely, modified extracellular matrix (ECM) substrates with spatially embedded growth or differentiation factors (GF, DF) that predictably direct muscle cell fate in a geometric pattern. Embedded GF and DF signal progenitor cells from specifically defined areas on the ECM successfully competed against culture media for myogenic cell fate determination at a clearly defined boundary. Differentiation of myoblasts into myotubes is induced in growth-promoting medium, myotube formation is delayed in differentiation-promoting medium, and myogenic cells, at different stages of proliferation and differentiation, can be induced to coexist adjacently in identical culture media. This method can be used to identify molecular interactions between cells in different stages of myogenic differentiation, which are likely to be important determinants of tissue repair. The designed ECM niches can be further developed into a vehicle for transplantation of myogenic progenitor cells maintaining their regenerative potential. Additionally, this work may also serve as a general model to engineer synthetic cellular niches to harness the regenerative potential of organ stem cells.

Aging, stem cells and tissue regeneration: lessons from muscle.

With age, there is a gradual decline in the regenerative properties of most tissues due to a combination of age-dependent changes in tissue-specific stem cells and in the environmental cues that promote those cells to participate in tissue maintenance and repair. In adult skeletal muscle, where the resident dedicated stem cells ("satellite cells") are capable of rapid and highly effective regeneration in response to injury, there is just such a loss of regenerative potential with age. Satellite cell activation and cell fate determination are controlled by the Notch signaling pathway that is initiated by the rapid increase in expression of the Notch ligand, Delta, following injury. In old muscle, this upregulation of Delta is blunted and thus satellite cell activation is markedly diminished. However, by indirectly inducing Notch activity, the regenerative potential of aged satellite cells can be restored. Furthermore, exposure of aged satellite cells to serum from young mice, either in vivo by heterochronic parabiotic pairings or in vitro, rejuvenates the satellite cell response. This restorative potential suggests that tissue-specific stem cells do not lose their ability to participate in tissue maintenance and repair. Therefore, it may be that even very old stem cells may be capable of maintaining and repairing aged tissues if provided with optimal environmental cues.

Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.

Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.

The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis.

We have studied the role of Notch-1 and its antagonist Numb in the activation of satellite cells during postnatal myogenesis. Activation of Notch-1 promoted the proliferation of myogenic precursor cells expressing the premyoblast marker Pax3. Attenuation of Notch signaling by increases in Numb expression led to the commitment of progenitor cells to the myoblast cell fate and the expression of myogenic regulatory factors, desmin, and Pax7. In many intermediate progenitor cells, Numb was localized asymmetrically in actively dividing cells, suggesting an asymmetric cell division and divergent cell fates of daughter cells. The results indicate that satellite cell activation results in a heterogeneous population of precursor cells with respect to Notch-1 activity and that the balance between Notch-1 and Numb controls cellular homeostasis and cell fate determination.