Fight Aging! Newsletter, July 17th 2017

Researchers have shown how to use a combination of human endothelial cells and mesenchymal stem cells to initiate a process called tubulogenesis that is crucial to the formation of blood-transporting capillaries. The work is an important step with fragile endothelial cells (ECs) made from "induced pluripotent stem cells," or iPSCs, a type of cell that can potentially be made from the cells of any human patient. Because iPSCs can be patient-specific, researchers hope to find ways of using them to generate tissues and replacement organs that can be transplanted without risk of rejection by a patient's immune system. But the fragility of endothelial cells during laboratory growth has limited the utilization of this critical cell type, which is found in all vasculature.

While tissue engineers have found dozens of ways to coax stems cells into forming specific kinds of cells and tissues, they still cannot grow tissues with vasculature - capillaries and the larger blood vessels that can supply the tissues with life-giving blood. Without vascularization, tissues more than a few millimeters in thickness will die due to lack of nutrients, so finding a way to grow tissues with blood vessels is one of the most sought-after advances in the field. "Ultimately, we'd like to 3-D print with living cells, a process known as 3-D bioprinting, to create fully vascularized tissues for therapeutic applications. To get there, we have to better understand the mechanical and physiological aspects of new blood-vessel formation and the ways that bioprinting impacts those processes. We are using 3-D bioprinting to build tissues with large vessels that we can connect to pumps, and are integrating that strategy with these iPS-ECs to help us form the smallest capillaries to better nourish the new tissue."

In the process of tubulogenesis - the first step to making capillaries - endothelial cells undergo a series of changes. First, they form small, empty chambers called vacuoles, and then they connect with neighboring cells, linking the vacuoles together to form endothelial-lined tubes that can eventually become capillaries. The researchers investigated whether commercially available endothelial cells grown from iPSCs had tubulogenic potential. The test examined this potential in two types of semisolid gels - fibrin and gelatin methacrylate (GelMA). Finally, the researchers also investigated whether a second type of stem cell, human mesenchymal stem cells, could improve the likelihood of tubulogenesis. In fibrin, the team found robust tubule formation, as expected. They also found that endothelial cells had a more difficult time forming viable tubules in GelMA, but over several months and dozens of experiments the team developed a workflow to produce robust tubulogenesis in GelMA. This involved adding mesenchymal stem cells, another type of adult human stem cell that had previously been shown to stabilize the formation of tubules.

Vascularization is critical for the maintenance of multicellular life as blood vessels provide oxygen and nutrients to tissues while also removing waste. Beyond the diffusion limit for oxygen transport, cells in tissue cannot survive or maintain normal function. The formation of vascular networks in vitro and in vivo have applications in treating ischemic disease, elucidating vascular mechanisms, screening drug efficacy, and engineering functional tissues for regenerative medicine. Toward the creation of vascular networks, methods of creating both structurally and functionally mature and complex vasculature must be identified to address the challenge of fabricating clinically-relevant tissue models and engineered tissues.

One of the several strategies toward vascularization is to create new vessel networks through cell-mediated morphologic processes similar to those seen during embryonic development. De novo vascular formation, or vasculogenesis, occurs through assembly of populations of endothelial cells into a rudimentary capillary plexus. Subsequent steps in vivo ensure vascular stabilization and maturity by generating extracellular matrix (ECM) and recruiting supporting mural cells. Further cell-cell and cell-matrix interactions encourage or antagonize vessel sprouting (angiogenesis), branching, remodeling, and pruning.

Many groups have cultured endothelial cells in vitro to generate spontaneously self-assembled vascular networks. While some groups have modeled vasculogenesis in natural matrices such as collagen and fibrin, others have demonstrated capillary networks in more synthetic environments such as GelMA and polyethylene glycol (PEG)-based hydrogels. In addition to challenges associated with the biomaterial environment, a critical aspect for cell-based clinical translation is the selection of cell sources. Human umbilical vein endothelial cells (HUVECs) have been commonly used for studying vascular morphogenesis, and supporting mural cells also have a history of various cell sources such as smooth muscle cells, but these cell types suffer from poor batch-to-batch uniformity as well as difficulties with scale-up and in some cases poor translation potential. More recent work has demonstrated the ability of human bone marrow derived mesenchymal stem cells (hMSCs), which are multipotent even well into adulthood, to serve in a supporting mural role for endothelial cells.

Here the potential to derive functional endothelial cells from induced pluripotent stem cells offers high batch uniformity, standardization, scale-up, and the potential for personalization. Commercially available iPS-ECs have been previously shown to form connected capillary-like networks in 2D and 3D studies with Matrigel. We sought to further validate these findings in additional matrix formulations and with live genetic reporters to provide additional tools for cell tracking and characterization. Here, we explored a cell-based strategy to form tubule networks using clinically relevant cell sources and biomaterials. We utilize iPS-ECs in monoculture or in co-culture with hMSCs in natural fibrin and semi-synthetic GelMA environments, track the progression of vasculogenesis, and quantify the network character of nascent tubules. Our results further supports the hypothesis that tubulogenesis is driven by intracellular vacuole formation and intercellular vacuole fusion. By using cell-based strategies for vascularization we allow biology to dictate the microvascular organization and exploit cellular interactions between iPSECs and hMSCs to stabilize networks when the extracellular environment cannot.

For those readers not familiar with how your technology works, could you give a brief summary of it?

The technology uses two elements. First, we build a DNA construct that contains the promoter we wish to target. This promoter controls an inducible suicide gene, called iCasp9. Next, we encapsulate that DNA in a specialized type of liposome known as a fusogenic lipid nanoparticle (LNP). The LNP protects the DNA plasmid during transit through the body's vasculature, and enables rapid fusion of the LNP with cell membranes. This LNP vector is considered "promiscuous" as it has no particular preference for senescent cells - it will target almost any cell type. Once it does, the DNA plasmid is deposited into the cytoplasm. It remains dormant unless the cell has transcription factors active that will bind to our promoter. If that happens, then the inducible iCasp9 is made. The iCasp9 doesn't activate unless a small molecule dimerizer is injected; the dimerizer causes the iCasp9 protein halves to bind together, immediately triggering apoptosis. This process ensures that the target cells are killed and that bystander cells are left unharmed. So far, we have not observed any off-target effects.

Many groups are engaged in researching small molecule drugs to remove senescent cells. What are the advantages of your system over the more traditional small molecule approach?

We've long thought that different populations of senescent cells might require different approaches to achieve sufficient clearance for effects to be apparent. So the various ventures that have begun using - in some cases - wildly differing protocols for senescent cells ablation may all have their place in the market. I personally like our approach because of its tremendous specificity without apparent off-target effects. The latter issue is one that purveyors of the small molecule approach must always be concerned with.

Cytomegalovirus (CMV) contributes to infectious burden and increases over time. It has been suggested that periodically purging these ineffective T cells may be useful. Have you considered using your technology for such a purpose?

Yes. We've made some initial efforts in this direction, and it is a favorite project of Aubrey de Grey at the SENS Research Foundation, but we don't have any experiments currently planned. It is on our "to do" list along with several other immune system-related experiments.

We have seen increased interest lately in increasing the ratios of H1 and H2 macrophages to treat conditions. Could your system be used to selectively destroy the H1 macrophages to favor a more healing environment?

So long as there is a promoter to be targeted, we could very likely achieve this goal. The beauty of our approach is that it is easy to try various types of promoter targets, and once we have resources to do so, we will expand our repertoire of targets. I'm not an immunologist, so someone with the necessary expertise would have to identify promoter targets and then we could have a go at it.

Have you started a mouse lifespan study to see if increased lifespan is observed with senescent cell clearance, and what sort of mice are being used?

We would like to conduct a lifespan study but haven't begun one as yet. First, lifespan studies are relatively expensive, for obvious reasons. Second, we hope to enlist an academic collaborator to participate in managing the study but we haven't located one yet. Finally, we are really focused on getting the treatment to the clinic, and through phase 1/2 studies in man. Doing anything that detracts from that goal means clinical delay.

Because cells are the fundamental building blocks of humans and animals, it is clear that cellular changes contribute to the ageing process. A major open question, however, is the nature of those changes and how exactly they contribute to degeneration and disease in old age. In 1961, it was discovered that human cells can only divide a finite number of times in culture. The limited proliferative ability of human cells in vitro, known as replicative senescence (RS), has since become a major focus of research in biogerontology. In addition to RS, a number of factors can accelerate and/or trigger cell senescence, including various forms of stress like oxidative stress.

For a long time it was debated whether the discovery of cellular senescence had any physiological relevance or was merely an artefact of cells grown in relatively artificial culture conditions. It was proposed that senescence may represent ageing, however, recent data has revealed that this view is too simplistic, since senescence has been shown to play multiple important physiological roles, such as: tumour suppression, tissue repair and wound healing, embryonic development, and age-related degeneration. In addition, senescent cells have been detected in the context of many different age-related diseases, including atherosclerosis, lung disease, diabetes, and many others.

Given the multitude of functions of senescent cells, which can be of a positive or negative nature depending on the context, it has been argued that there may be different types of senescence rather than a universal phenotype. For instance, senescence during embryonic development occurs transiently, since senescent cells are rapidly removed by the immune system after executing their role, and is not associated with the activation of a DNA damage response (DDR). In contrast, during ageing, senescent cells are thought to be persistent, induced by random molecular damage and associated with the activation of a DDR. Recent work has demonstrated that senescent cells are able to attract (potentially via the secretion of chemokines) different immune cells. It is possible that persistence of senescent cells in tissues during ageing and age-related diseases is a consequence of the inability of the immune system to clear senescent cells - in view of the well reported decline of the immune system with age - however this has not yet been experimentally tested.

Do senescent cells accumulate with age? One of the main challenges to the study of senescence in vivo has been the absence of a universal marker that can unequivocally identify senescent cells. The most widely-used marker is the presence of senescence-associated β-galactosidase (SA-β-gal) activity. Both in vitro and in vivo, the percentage of cells positive for SA β-gal increases with, respectively, population doublings and age. However, there are major limitations to the use of this marker, since SA-β-Gal staining can also be detected in immortalized cells and quiescent cells. Also, it has been suggested that a major limitation of using SA β-gal staining in vivo is a false-positive signal from macrophages and other pro-inflammatory cells. In addition, since it requires fresh tissues, its detection is not straightforward technically and has more than often generated conflicting results.

Given the challenge of identifying a specific marker able to identify senescent cells, most researchers currently rely on a multiple marker approach. Indeed, several markers have been identified which are closely associated with cellular senescence, including absence of proliferation markers, changes in heterochromatin, telomere-associated DNA damage, expression of cyclin-dependent kinase inhibitors p21, p16, and senescence-associated distension of satellites (SADS). In a variety of mouse tissues, it is clear that most of these markers increase with age; however, given the fact that most of these markers are not exclusive for senescent cells, the exact frequency of senescent cells in older tissues is still unknown. Furthermore, given the limited availability of tissues, little is known about the accumulation of senescent cells with age in healthy humans.

Interestingly, many senescence markers have also been found in post-mitotic tissues such as neurons, adipocytes, and osteocytes, which goes against the dogma that senescence is restricted to proliferating cells. It is possible that with ageing, senescence-inducing pathways (which play roles in tumour suppression and during development) can be inadvertently switched on during ageing of post-mitotic cells. However, given that the primary characteristic of senescence is a permanent cell-cycle arrest, the consequences of the activation of these pathways in post-mitotic cells are still not understood.

While there is little evidence to suggest that cells running out of divisions are a major factor in ageing, it is possible that stress and various insults are contributors to senescence in vivo. Even a small fraction of senescent cells in organs may impair tissue renewal and homeostasis, decrease organ function, and contribute to the ageing phenotype, as shown by the studies genetically ablating senescent cells. While our knowledge about senescence in vivo has increased exponentially in the last decade, this is mostly through work using laboratory mice, which have known limitations. As such, one major challenge in the field is to determine levels of senescent cells in human tissues and whether they contribute to ageing and/or pathologies in humans. Furthermore, given the diverse functions of senescent cells in processes such as repair, wound healing, cancer, development and ageing, we still need to better characterize senescence in vivo in these different contexts. Finally, we still know very little about in vivo rates of occurrence and turnover of senescent cells. Therefore, in spite of recent advances in our understanding of senescence, many questions remain and these will be timely and important areas of research for years to come.

Last June, the Methuselah Foundation and NASA officially launched the Vascular Tissue Challenge (VTC) at the White House Organ Summit, hosted by the Office of Science and Technology Policy. The VTC includes a 500,000 prize purse from NASA for the first teams that can successfully create 1cm or thicker vascularized tissues that remain functional and alive for more than 30 days. Along with this is the Center for the Advancement of Science in Space's (CASIS) "Innovations in Space Award," providing an additional 200,000 to support a research opportunity on board the International Space Station's National Laboratory. With the one year mark just behind us, we thought it was fitting to check in with the teams and see how they're doing. There's been a lot happening to advance these amazing bioengineering technologies over the last 12 months!

Since launching the Vascular Tissue Challenge, seven research organizations officially signed on to pursue the challenge of creating the thick, vascularized tissues required to win the 700,000 in awards along with the opportunity to pursue further research using the microgravity environment onboard the International Space Station. Each team is pursuing a different approach to creating vascularized tissues, and each has their own unique strategies and hurdles ahead. Here is a quick snapshot of what some of the teams have been doing and what they are planning for their next steps toward winning the Challenge before the sunset of the award at the end of 2019.

iTEAMS, Stanford University

Over the past year, iTEAMS has proposed and proved an integrated multi-scale, multi-modular system approach to overcome the challenges and tradeoff in functional vasculature requirements between major vascular lasting perfusion and capillary rapid sprouting and extensive coverage for diffusion. The former requires a slowly degradable biomaterial for sustained perfusion and the latter requires a fast biodegradable biomaterial for rapid sprouting and diffusion. The next steps being pursued are an optimization of perusable channel pathways, biomaterial candidates, and fabrication parameters. A critical upcoming milestone is to demonstrate functional microvasculature at a large scale for a long term in vitro. Team iTEAMS is working towards conducting their Vascular Tissue Challenge trials in 2018.

BioPrinter, Florida Institute of Technology

The team have developed a self-contained bioprinting system that is being used to generate tissue samples with high resolution and cell viability. They plan to use this printer to develop a sacrificial technique of bioprinting channels within a tissue sample. These channels will be used for the exchange of nutrients to cells needed to maintain viable tissue for an extended period of time. Currently, research is being conducted with various concentrations of bioink to obtain values that will result in high quality bioprinted tissue samples. In parallel, research on sacrificial techniques to create channels for nutrient flow is being conducted. The team anticipates that an official trial for the Vascular Tissue Challenge to be initiated in 2018.

Flow, Maize, and Blue, University of Michigan

The team has built a perfusion bioreactor that it is currently optimizing for customized tissue engineered vascular networks. The team hope to accomplish long-term perfusion of these vascular networks in the next 6 months with an official Vascular Tissue Challenge trial occurring sometime after that research is completed.

Techshot

Last summer, Techshot began formal efforts toward winning the Vascular Tissue Challenge by 3D printing biological materials and adult stem cells into vascular and cardiac structures on board a Zero Gravity Corporation aircraft. Test structures were printed during cycles of both zero G and high G forces, permitting evaluation of low viscosity, biological material printing in multiple gravity environments. As expected, the cycles of microgravity facilitated layer-by-layer printing of 3D structures with very low viscosities (these materials become puddles if printed on the ground). The team's next large step forward is a "Tissue Cassette" experiment that will be conducted this summer. Building upon last summer's work, Techshot will bioprint larger cardiac and vascular structures within a specialized container, a bioreactor they refer to as a "Tissue Cassette". This Tissue Cassette will not only provide an appropriate environment for culturing the 3D printed structure, it will impart physical and electrical cues to accelerate cell growth and tissue development. The bioreactor will also permit perfusion of the 3D bioprinted structure to further support cell growth in the larger printed volume.

The planned experiments will start by bioprinting identical sets of cardiac and vascular structures with an initial print size of 20mm x 30mm x 10mm. One set will stay on the ground. The second set will be loaded into a Techshot ADSEP system and launched to the International Space Station aboard SpaceX Cargo Dragon (CRS-12) on August 1, 2017. These experiments will provide insight into bioprinted cell behavior in microgravity and the associated differences in tissue development. This will provide a preliminary test of the technology Techshot plans to use for their Vascular Tissue Challenge trials that they expect to conduct after getting these results back.

Team Penn State, Pennsylvania State University

The team has made substantial progress with their research on micro-vascularization in engineered islets. In addition, the team has scaled up tissue constructs to a sub-cm^3 level and are working on expanding to the cm^3 level for the VTC trial. They have demonstrated viable vascularization with mouse cells and are currently conducting research to overcome technical issues with the co-culture of stem cell-derived human beta cells and microvascular endothelial cells. Finalizing the research to reach vascularization with these cells at the cm^3 level is the next critical step for this team, which they expect to take them into 2018 before conducting their final trials for the VTC.

Team WFIRM Bioprinting, Wake Forest University

During the past year, the WFIRM Bioprinting Team was focusing on the development of tissue-specific bioink systems that could mimic the microenvironments of each target tissues. The team assumes that these tissue-specific bioink systems can enhance the cell-cell and cell-matrix interactions that can accelerate tissue maturation/formation and functions. Up next in the team's research is to combine microvasculature created by endothelial cells with tissue-specific printed constructs. They plan to investigate the effects of endothelialized microvasculature on cell viability and tissue-specific functions of the tissue-specific printed constructs. It is not yet clear when the team's VTC trials will start, more will be known after their next research projects are completed.

Team Vital Organs, Rice University

At Rice University, Team Vital Organs is continuing to build out their 3D printing technology, characterizing the precision, cell viability and activity, designing assays for tissue assessment, and designing proper vascular architectures for complete tissue integration. Perfusion systems are complicated, but the team has a new large incubator that can now accommodate their proposed perfusion systems for the VTC. They are now working on validating long-term sterility and measurements from longitudinal assays. The team is looking forward to finishing these feasibility studies and putting together an official trial to win the Vascular Tissue Challenge within the next year.

For older groups, being overweight [body mass index (BMI): 25 to 30] is reportedly associated with a lower or similar risk of mortality than being normal weight (BMI: 18.5 to 25). However, this "risk paradox" is partly explained by smoking and disease-associated weight loss. This paradox may also arise from BMI failing to measure fat redistribution to a centralized position in later life. This study aimed to estimate associations between combined measurements of BMI and waist-to-hip ratio (WHR) with mortality and incident coronary artery disease (CAD). This study followed 130,473 UK Biobank participants aged 60-69 years (baseline 2006-2010) for 8.3 years (n = 2974 deaths). Current smokers and individuals with recent or disease-associated (e.g., from dementia, heart failure, or cancer) weight loss were excluded, yielding a "healthier agers" group.

Ignoring WHR, the risk of mortality for overweight subjects was similar to that for normal-weight subjects. However, among normal-weight subjects, mortality increased for those with a higher WHR (hazard ratio: 1.33) compared with a lower WHR. Being overweight with a higher WHR was associated with substantial excess mortality (hazard ratio: 1.41) and greatly increased CAD incidence compared with being normal weight with a lower WHR. Thus for healthier agers (i.e., nonsmokers without disease-associated weight loss), having central adiposity and a BMI corresponding to normal weight or overweight is associated with substantial excess mortality. The claimed BMI-defined overweight risk paradox may result in part from failing to account for central adiposity, rather than reflecting a protective physiologic effect of higher body-fat content in later life.

Findings from prospective cohort studies based on populations from Australia, Sweden, Denmark, the US, and the UK indicate that higher cognitive ability (intelligence) measured with standard tests in childhood or early adulthood is related to a lower risk of total mortality by mid to late adulthood. The association is evident in men and women; is incremental across the full range of ability scores; and does not seem to be confounded by socioeconomic status of origin or perinatal factors.

Several hypotheses have been proposed to explain associations between intelligence and later risk of mortality. The suggested causal mechanisms put forward, in which cognitive ability is the exposure and disease or death the outcome, include mediation by adverse or protective health behaviours in adulthood (such as smoking, physical activity), disease management and health literacy, and adult socioeconomic status (which could, for example, indicate occupational hazards). Recent evidence of a genetic contribution to the association between general cognitive ability and longevity, however, might support a system integrity theory that posits a "latent trait of optimal bodily functioning" proximally indicated by both cognitive test performance and disease biomarkers. None of these possibilities are mutually exclusive.

We investigated the magnitudes of the association between childhood intelligence and all major causes of death, using a whole year of birth population followed up to older age, therefore capturing sufficient numbers of cases for each outcome. All individuals born in Scotland in 1936 and registered at school in Scotland in 1947 were targeted for tracing and subsequent data linkage to death certificates. For most endpoints, higher childhood intelligence was associated with a lower risk of cause specific death. Risk of death related to lifetime respiratory disease was two thirds lower in the top performing 10th for childhood intelligence versus the bottom 10th. Furthermore for deaths from coronary heart disease, stroke, smoking related cancers, digestive diseases, and external causes, risk of mortality was halved for those in the highest versus lowest 10th of intelligence. The risk of dementia related mortality and deaths by suicide were reduced by at least a third in the highest performing quarter of intelligence test score versus the lowest quarter.

In the literature, the familial component of human longevity has been investigated using survival to extreme age and age at death as phenotypes of survival. The former actually refers to longevity whereas the latter refers to individual or population based lifespan. Both definitions are often used in the context of longevity research which is confusing and incorrect. Another complication is that most studies exclude infant and child mortality by applying a lower limit age threshold when considering the lifespan of a population or group of individuals. Unfortunately, there is no consensus on the age threshold for longevity studies. As a result of both the inconsistent use of terminology and different lower and upper limit age thresholds, the comparison of longevity studies is generally problematic. We will refer to longevity as survival into extreme old ages whereas lifespan refers to age at death related measures.

Progress in longevity research is also hampered by the fact that longevity is likely dependent on an interplay between combinations of multiple genes and environmental factors which makes it difficult to separate environmental from genetic influences. In fact, environmental influences likely moderate genetic effects on longevity. Several genealogical studies have attempted to estimate the heritability of lifespan and longevity. These studies can be divided into two categories based on the type of data they used; (1) twin data and (2) pedigree data. Unlike animal studies in a lab setting, the effects of the environment on longevity in human studies cannot be controlled. In twins at least the variation in early environment is minimized as compared to other family based studies. In all cases, heritability estimates and the effect of specific gene variants on lifespan and longevity depends on the populations studied and their past and present environmental conditions.

It can be concluded from study results that the heritability of lifespan is between 0.01 and 0.27 in the population at large. The large variation in the heritability estimates indicates a prominent role for differential environmental influences on the estimates. Studies showing that siblings of centenarians and longevous sib-pairs have a high probability to also become a centenarian or longevous, respectively, and studies, which show that longevous parents have a high probability to bear longevous offspring, provide indications that the heritability of longevity may be higher than that of lifespan.

However, the heritability of longevity has only been investigated once in a twin study design, though of limited sample size. In addition, the heritability of longevity has been investigated more often in pedigree studies but the studies raise several questions about their design, sample size, and generalizability. Establishing the heritability of longevity is necessary for case definitions in genetic studies focused on gene mapping. Hence, researchers' attention should shift from lifespan to longevity and the heritability of longevity should be estimated in an appropriate design with a sufficiently large sample.

The main purpose of cellular senescence is to prevent the proliferation of damaged or stressed cells and to trigger tissue repair. However, upon persistent damage or during aging, the dynamic process of tissue repair becomes inefficient and senescent cells tend to accumulate. This accumulation in tissues is believed to impair tissue functions and accelerate aging. It has been demonstrated that genetic ablation of senescent cells ameliorates a variety of aging-associated diseases, reverts long-term degenerative processes, and extends longevity. Inspired by these findings, strategies to prevent, replace, or remove senescent cells have become of interest. For instance, there is an increasing interest in the development of senolytic molecules able to induce apoptosis preferentially in senescent cells.

A related key issue in this field is the design of probes to accurately detect senescent cells in aged or damaged tissues. However, one of the major obstacles limiting progress in this research area is the lack of real-time methods to selectively track senescence in in vivo systems. Detection of senescent cells usually relies on the detection of senescence-associated βGal (SAβGal), and several fluorescent or chromogenic probes have been reported for the visualization of this enzymatic activity. However, these first-generation probes are usually unsuitable for in vivo imaging as they rely on chromogenic changes or on the use of classical one-photon fluorescence excitation. As an alternative, recent stimulating studies developing two-photon fluorescent probes for the visualization of βGal activity have been described. However, some of the reported probes are synthesized by using tedious multistep protocols. Another common drawback is the fact that probes are tested in cultured cells or in animal models that were not directly related to senescence.

In view of the aspects mentioned above, we report herein a novel molecular probe for the two-photon fluorogenic in vivo detection of senescence. The probe (AHGa) is based on a naphthalimide fluorophore as a signaling unit containing an L-histidine methyl ester linker and an acetylated galactose attached to one of the aromatic nitrogen atoms of the L-histidine through a hydrolyzable N-glycosidic bond. Probe AHGa is transformed into AH in senescent cells resulting in an enhanced fluorescent emission intensity. Targeting of senescent cells in vitro with AHGa was validated with the SKMEL-103 cancer cell line treated with the chemotherapeutic palbociclib to induce senescence. A remarkable fluorescence emission enhancement (ca. 10-fold) in the presence of AHGa for palbociclib-treated SK-MEL-103 (senescent) cells was observed when compared with control SK-MEL-103 cells, due to the formation of AH.

The ability of tracking senescence of probe AHGa was also studied in vivo by employing mice bearing subcutaneous tumor xenografts generated with SK-MEL-103 melanoma cells and treated with palbociclib. Tumors in palbociclib-untreated mice showed negligible fluorescence emission both in the absence or in the presence of AHGa, whereas tumors in mice treated with palbociclib and intravenously injected with AHGa showed a clear fluorescent signal. A marked emission enhancement (ca. 15-fold) in tumors treated with palbociclib compared to nontreated tumors was observed. AH fluorescence was only found in senescent tumors but not in other organs. The combination of selectivity, sensitivity, and straightforward synthesis make AHGa an efficient OFF-ON two-photon probe for the in vivo signaling of senescence.

Life extensionism is a global movement with long-term traditions. The idea, that aging is similar to a disease and should be treated as such, was first suggested in the early 1900s. Since then, the study of aging biology has revealed the underlying processes of aging, such as DNA damage, toxic proteins aggregation and cross-links, cellular senescence, nutrient sensing deregulation and others, and proven the plausibility to address these processes to modify the dynamics of aging.

Even though aging itself is not described as a disease in the International Classification of Diseases (ICD), there is no doubt that aging is the major cause of many severe diseases, and the global population could benefit from bringing aging under medical control. Many existing drugs have been found to be geroprotective (protecting the body against the the aging process). However, what would happen if scientists applied geroprotective technologies to humans, remains a subject of numerous misconceptions.

This is the human life course as it was before the development of modern medicine: somewhere around their 50s people started to develop different age-related diseases, then died from them some 15-20 years later. As a result of the past century of development, however, now people reach their 50s, age-related diseases start to manifest, but modern medicine allows us to slow down their progression, so people live longer - but this is the period of illness that is extended, because this medicine fails to address the causes of aging. This is exactly why Brian Kennedy from the Buck Institute calls our healthcare system a "sickcare" system: we are keeping people alive for longer, but we are keeping them sick, generating a burden for our system of healthcare and social support: we have many people living longer in disability.

We can do better by developing interventions to address the aging processes. These interventions are meant to be applied in middle age, before the manifestation of age-related diseases, in order to extend the healthy period of life, or healthspan, while the period of illness is postponed and will remain relatively short. This could allow people in their 50s to look like they are 30, and in their 70s also look younger, be stronger, and feel as good as in their 50s. So what we mean by life extension is actually the extension of the healthy and productive period of life, free of disease and disability. In this "extended" society the majority of people could enjoy their lives for much longer and actively contribute to the development of the economy regardless of their chronological age.

Platelets (PLTs) fulfill essential functions in primary hemostasis and wound healing and maintain immunological properties, but also play a role in inflammation and cancer. In vivo, PLTs are formed by demarcation and cytoplasmatic shedding from one large precursor cell known as a megakaryocyte (MK). MKs reside within the bone marrow where they differentiate from hematopoietic stem cells. During their maturation they migrate to the sinusoids vessels, and extend protusions (proplatelets; proPLTs) through the vessel pores. The shear stress within the sinusoidal lumen supports the release of PLTs into the blood stream. Understanding the basic biology of thrombopoiesis and its physiological mechanisms is fundamental to efficiently mimic PLT production in vitro, an approach that is gaining importance for future transfusion and regenerative medicine. The demand for PLT transfusion is constantly rising. While the majority of PLT transfusions is provided to patients with reduced PLT counts after chemotherapy or hematopoietic progenitor cell transplantation, other clinical causes may require urgent PLT transfusion.

To achieve clinical numbers of in vitro generated MKs and PLTs, current next-generation strategies employ fluidic biomimetic reactors recapitulating the natural bone marrow environment. In 2006 researchers demonstrated PLT differentiation from human cord blood-derived CD34+ progenitor cells in a three-phase culture system. The first two differentiation phases were based on static cultures using hTERT human stroma cell as feeders. However, the final maturation of MKs and PLTs occurred in a suspension culture system. Another new feature introduced by this study was the co-culture of MKs in combination with human umbilical vein endothelial cells (HUVEC), since endothelial cells are known to fulfil stimulatory functions on proPLT formation.

In 2009, researchers published the first 3D PLT bioreactor built from a modular perfusion system. The device contained a central producer cell disc covered by a layer of pre-expanded CD34+ progenitor cells, while medium and gas flow occurred in separate spaces above and below this cell layer. This setup allowed the harvest of PLTs from the lower medium space over 30 days. Later, they further improved this bioreactor prototype. They increased PLT production by regulating the oxygen supply and inducing controlled shear stress with help of a continuous medium flow though the cell scaffold. These first approaches demonstrated the feasibility to produce PLTs not only in suspension cultures but also in continuous perfusion systems which can significantly facilitate the upscaling of PLT production.

In 2011 researchers established a 3D model one step ahead to a close technical analogue of the bone marrow microenvironment by the application of silk protein biomaterial. To simulate the natural niche, growth factor-coated silk microtubes (mimicking sinusoidal vessels) were embedded in modules filled with type I collagen gel. MKs were differentiated from CD34+ cells and seeded between the collagen gel and each microtube. They migrated towards the microtube and released proPLTs into the constitutive flow of media within the microtubes. However, only 7% of MKs exhibited proPLT production. In 2015 researchers presented a follow-up of this prototype, equipped with an additional silk sponge encompassing the microtube to better mimic the stiffness of the sinusoidal vessel surrounding. Moreover, they improved the entrapment of growth factors and extracellular matrix components, and seeded HUVEC into the lumen of the silk microtubes. These new features led to a threefold increase in numbers of released PLTs.

Step by step, bioreactor bioengineering for efficient PLT production became increasingly complex. In 2014, researchers presented the first PLT bioreactor-on-a-chip that, despite its small size, considered a broad spectrum of parameters to recapitulate the bone marrow microenvironment. To mimic the stiffness of the natural bone marrow, MKs were seeded in hydrogels such as alginate. To improve MK trapping, extracellular matrix proteins were added into the surrounding media, or used to coat the membrane separating the MK chamber from the lower flow chamber. ProPLT formation was stimulated with help of endothelial cell contacts, and PLT release was optimized using controlled hemodynamic vascular shear stress. In 2016 researchers developed a 'microfluidic model of the PLT generating organ', constituted by a single-flow chip, in which MKs derived from human cord blood (hCB) CD34+ cells were constitutively perfused and captured by thousands of vWF (von Willebrand factor)-coated micropillars to release PLTs into the media flow. This setup enabled a high throughput of millions of MKs.

In summary, it is currently possible to efficiently differentiate MKs from induced pluripotent stem cells (iPSCs), but they show a restricted capacity to produce PLTs in vitro. Physiologically, one MK produces thousands of PLTs into the circulation. In contrast, the protocols available only allow the production of up to hundreds PLTs per MK. This delays the possibility for clinical application of in vitro produced PLTs. Yields of PLT production may profit in the future from the harmonization of MK expansion and differentiation culture systems towards a synchronized PLT formation and release. The application of shear stress in the designed bioreactor aimed to provide a physical cue to induce a synchronized proPLT formation, extension, and PLT release. However, it remains highly desirable to identify biological or chemical signals that might support this process.

A number of hypotheses have been postulated for cardiac aging including oxidative stress, mitochondrial injury, autophagy dysregulation, and intracellular Ca2+ mishandling. Nonetheless, the precise machineries behind cardiac aging still remain somewhat elusive. Recent evidence from our laboratory and others has depicted a unique role for phosphoinositide 3-kinase (PI3K) and its downstream-signaling target protein kinase B (Akt) in aging-induced pathological changes in the heart. It was shown that the on-and-off switching of the PI3K/Akt pathway, particularly by insulin and insulin-like growth factor-1 (IGF-1), serves as a powerful physiological integrator rudimentary to life span and aging.

Our data have revealed an essential role for diminished autophagy, an evolutionarily conserved lysosome-dependent process for turnover of proteins and organelles, in Akt overactivation-induced accentuation of cardiac aging process. Autophagy plays a key role for biological aging process and cardiac homeostasis. Diminished autophagy has been shown to accelerate mammalian aging, in association with accumulation of damaged intracellular components including protein aggregate. Moreover, defective autophagy facilitates ventricular remodeling, contractile defects, and heart failure. Given the critical role for Akt in the regulation of cardiac survival and life span, this study was designed to examine the role of Akt2 ablation on aging-induced geometric, functional, and intracellular Ca2+ homeostatic changes in the heart, with a focus on autophagy and mitochondrial integrity.

Our findings indicated that Akt2 ablation prolongs life span and improves myocardial contractile function with a possible adaptive cardiac remodeling through the Sirt1-mediated autophagy regulation. In addition, Akt2 ablation alleviated aging-associated mitochondrial injury. Cardiac aging is characterized by unfavorable cardiac remodeling and function including cardiac hypertrophy, interstitial fibrosis, compromised contractility, and prolonged diastolic duration. To our surprise, Akt2 ablation negated aging-induced cardiac contractile dysfunction with a more pronounced remodeling. More prominent changes in heart mass/size, and cardiomyocyte cross-sectional area (but not fibrosis) were noted in aged Akt2-/- mice, favoring an important role for Akt2 in aging as opposed to young hearts. With the improved cardiac function in aging, the more pronounced cardiac hypertrophy in the face of Akt2 ablation seemed to suggest a state of adaptive cardiac hypertrophy in aged Akt2-/- hearts. Akt2 knockout did not elicit any notable cardiac effect at young age, suggesting that ablation of Akt2 may take time to impose cardiac remodeling and contractile effects.

Perhaps the most intriguing finding from our study is that Akt2 ablation prolonged life span and rescued against aging-induced cardiac dysfunction despite more pronounced cardiac hypertrophy. Several theories may be proposed for Akt2 ablation-elicited responses in aging. Earlier findings from our group depicted dampened phosphorylation of the Akt-negative regulator PTEN with aging, consistent with present observation of Akt activation in aging and the rationale of beneficial Akt2 ablation. Second, restored autophagy and mitophagy seem to play an important role for Akt2 ablation-induced cardioprotection. Both Akt activation, a key molecule governing cardiac survival, autophagy, and mitochondrial function, and aging have been shown to suppress autophagy. Our results revealed that Akt2 ablation restored autophagy and mitophagy in aging hearts. Our in vitro findings further revealed that autophagy induction with rapamycin improved mitophagy and contractile function. It is likely that restored autophagy and mitophagy may be responsible for prolonged survival in Akt2 knockout mice, in line with the prolonged life span with autophagy induction. Improved autophagy may improve diastolic function in senescent myocardium via preserved intracellular Ca2+ handling.

In summary, our findings suggest that Akt2 seems play an essential role in the regulation of longevity, cardiac geometry, and function in aging. Our data favor the notion that increased Akt signaling and downregulated Sirt1 with advanced aging may underscore reduced autophagy and mitophagy in aging, indicating the therapeutic potentials for Akt and autophagy/mitophagy in the management of cardiac aging. Although our study sheds some light on the interaction of Akt-Sirt1 signaling cascades on autophagy and cardiac homeostasis, the pathogenesis of cardiac dysfunction in aging, particularly in association with autophagy and mitochondria still deserves further investigation.

"Companies have put fisetin into various health products but there hasn't been enough serious testing of the compound. Based on our ongoing work, we think fisetin might be helpful as a preventative for many age-associated neurodegenerative diseases, not just Alzheimer's disease (AD), and we'd like to encourage more rigorous study of it." Previous research found that fisetin reduced memory loss related to Alzheimer's in mice genetically modified to develop the disease. But that study focused on genetic (familial) AD, which accounts for only 1 to 3 percent of cases. By far the bigger risk factor for developing what is termed sporadic AD, as well as other neurodegenerative disorders, is simply age. For the current inquiry, researchers turned to a strain of laboratory mice that age prematurely to better study sporadic AD. By 10 months of age, these mice typically show signs of physical and cognitive decline not seen in normal mice until two years of age.

The team fed the 3-month-old prematurely aging mice a daily dose of fisetin with their food for 7 months. Another group of the prematurely aging mice was fed the same food without fisetin. During the study period, mice took various activity and memory tests. The team also examined levels of specific proteins in the mice related to brain function, responses to stress and inflammation. At 10 months, mice not treated with fisetin had difficulties with all the cognitive tests as well as elevated markers of stress and inflammation. Brain cells called astrocytes and microglia, which are normally anti-inflammatory, were now driving rampant inflammation. Mice treated with fisetin, on the other hand, were not noticeably different in behavior, cognitive ability or inflammatory markers at 10 months than a group of untreated 3-month-old mice with the same condition. Additionally, the team found no evidence of acute toxicity in the fisetin-treated mice, even at high doses of the compound. "Mice are not people, of course. But there are enough similarities that we think fisetin warrants a closer look, not only for potentially treating sporadic AD but also for reducing some of the cognitive effects associated with aging, generally."

Zymo Research Corp. announced today the release of a new service that can quantify biological aging in a precise manner using the myDNAge test. Based on Horvath's Clock, Zymo Research's proprietary technology is used to analyze DNA methylation patterns of over 500 loci. The new test will be sold to consumers via a newly formed company, Epimorphy, LLC, an associated company of Zymo Research, created to provide epigenetic-based tests to the consumer marketplace.

The detailed data report that the consumer will receive compares the customer's biological age to their chronological age, which could provide insight on how lifestyle and disease may have influenced their aging process, and could also be used to develop new anti-aging therapies. This test is not intended to diagnose, treat, cure or prevent any disease. Consumers can order the test online at a cost of 299 per test. Once the sample is submitted, it is prepared and analyzed using DNAge technology. A report including data analysis and biological age determination is made available for consumers.

The research community in academia and biopharma markets can also order the service for mice and human samples. "Epigenetics may hold important keys to unlocking a myriad of diseases and disorders, such as cancer, autism, Alzheimer's, and diabetes, among many others. This groundbreaking tool could provide profound insight on how biological aging is assessed. We are pleased to be able to provide this technology not only to the researcher but also to the consumer marketplace."

Research into parabiosis can be technically challenging, and had more or less died out by the late 1970s. These days, though, it is back in the news - for a string of recent discoveries have suggested that previous generations of researchers were on to something. The blood of young animals, it seems, may be able to ameliorate at least some of the effects of ageing. In 2005, research joined the circulatory systems of mice aged between two and three months with members of the same strain that were 19-26 months old. That is roughly equivalent to hooking a 20-year-old human up to a septuagenarian. After five weeks, the researchers deliberately injured the older mice's muscles. Usually, old animals heal far less effectively from such injuries than young ones do. But these mice healed almost as well as a set of young control animals. The young blood had a similar effect on liver cells, too, doubling or tripling their proliferation rate in older animals.

Since then, a torrent of papers have shown matching improvements elsewhere in the body. No one has yet replicated the finding that young blood makes superannuated mice live longer. But it can help repair damaged spinal cords. It can encourage the formation of new neurons in mouse brains. It can help rejuvenate their pancreases. The walls of mouse hearts get thicker as the animals age; young blood can reverse that process as well. The effects work backwards, too. Old blood can impair neuron growth in young brains and decrepify youthful muscles. Finding out exactly what is happening is tricky. The working theory is that chemical signals in young blood are doing something to stem cells in older animals. Stem cells are special cells kept in reserve as means to repair and regrow damaged tissue. Like every other part of the body, they wear out as an animal ages. But something in the youngsters' blood seems to restore their ability to proliferate and encourages them to repair damage with the same vigour as those belonging to a younger animal would. In all probability, it is not one thing at all, but dozens or hundreds of hormones, signalling proteins and the like, working together. Researchers have been comparing the chemical composition of old and young blood, searching for those chemicals that show the biggest changes in level between the two.

Even with a list of targets, working out what is going on is hard. Blood is complicated stuff, and the tools available to analyse it are far from perfect. In 2014 a group suggested GDF-11 as a possible rejuvenating factor. The following year another team said that they were unable to replicate those results. They claimed the original test was sensitive to proteins besides GDF-11, messing up the results. The original team replied within months that, no, it was in fact the new test that was flawed, because it was itself picking up extra proteins. And there, at the moment, the matter stands. There are further possible explanations for parabiotic rejuvenation besides blood chemistry. One is that older animals may also benefit from having their blood scrubbed by young kidneys and livers, which mere blood transfusion would not offer. A 2016 paper described blood exchanges that were done in short bursts (thus eliminating the possibility of such scrubbing) and reported rejuvenating effects, but ones that were not as widespread as those obtained by full-on parabiosis. Another idea is that cells from the young animal, rather than chemicals in its blood, could be doing some of the work. The mechanisms by which parabiosis operates, then, are foggy.

Sure, you know how old you are, but what about your cells? Are they the same age? Are they older, younger? Why does it matter? A team of researchers is reporting progress in developing a method to accurately determine the functional age of cells, a step that could eventually help clinicians evaluate and recommend ways to delay some health effects of aging and potentially improve other treatments, including skin graft matching and predicting prospects for wound healing.

The researchers devised a system that can consider a wide array of cellular and molecular factors in one comprehensive aging study. These results show that the biophysical qualities of cells, such as cell movements and structural features, make better measures of functional age than other factors, including cell secretions and cell energy. The team examined dermal cells from just underneath the surface of the skin taken from both males and females between the ages of 2 to 96 years. The researchers hoped to build a system that through computational analysis could take the measure of various factors of cellular and molecular functions. From that information, they hoped to determine the biological age of individuals more accurately using their cells, in contrast to previous studies, which makes use of gross physiology, or examining cellular mechanisms such as DNA methylation.

Researchers trying to understand aging have up to now focused on factors such as tissue and organ function and on molecular-level studies of genetics and epigenetics, meaning heritable traits that are not traced to DNA. However, the level in between, the cells, have received relatively little attention. This research was meant to correct for that omission by considering also the biophysical attributes of cells, including such factors as the cells' ability to move, maintain flexibility and structure. This focus emerges from the understanding that changes associated with aging at the physiological level such as diminished lung capacity, grip strength and mean pressure in the arteries "tend to be secondary to changes in the cells themselves, thus advocating the value of cell-based technologies to assess biological age." For example, older cells are more rigid and do not move as well as younger cells, which most likely contributes to the slower wound healing commonly seen in older people.

From the analysis, researchers were able to stratify individuals' samples into three groups: those whose cells roughly reflected their chronological age, those whose cells were functionally older, and those whose cells were functionally younger. The results also showed that the so-called biophysical factors of cells determined a more accurate measure of age relative to biomolecular factors such as cell secretions, cell energy, and the organization of DNA. The more accurate system could eventually enable clinicians to see aging in the cells before the person experiences age-related health decline. This in turn could allow doctors to recommend treatments or changes in life habits, such as exercise or diet changes.

As insulin and Igf1 share nearly identical downstream signaling pathways in mammals, and act in part via hybrid insulin/Igf1 heterodimer receptors that can bind to either ligand, the relative functions of these two ligands have not been completely delineated. Many studies have focused on Igf1 as the primary ligand which mediates the lifespan-altering effects through this signaling cascade in mammals, but the impact of directly altering insulin levels on longevity had not been evaluated.

Insulin resistance is a common feature of mammalian aging and a risk factor for numerous age-related diseases. Although this suggests that reducing insulin signaling could be detrimental for mammalian healthspan, it is important to consider that conventionally defined insulin resistance (i.e., impaired insulin-stimulated glucose disposal) is not a generalized reduction of all insulin signaling. Instead, some insulin-regulated processes are maintained at normal capacity in the "insulin resistant" state, while others are downregulated. Moreover, circulating levels of the insulin ligand are elevated with insulin resistance. The commonly accepted paradigm posits that insulin levels rise as a compensatory response to prevent hyperglycemia when there is insufficient insulin-stimulated glucose uptake. However, causality between the closely associated conditions of systemic insulin resistance and insulin hypersecretion has remained controversial, and it has been suggested that hyperinsulinemia could be an early, primary cause of insulin resistance, obesity, and eventually type 2 diabetes. Genetic loss-of-function experiments targeting insulin itself present an ideal opportunity to disentangle hyperinsulinemia from insulin resistance and to evaluate the lifelong effects of limiting endogenous insulin production and secretion.

To determine how moderately lowering the insulin ligand would affect late-life glucose homeostasis and longevity in mammals, we compared mice with either full or partial expression of the ancestral insulin gene Ins2. Since altering insulin gene dosage does not affect circulating insulin levels in all contexts, we used a mouse model in which the rodent-specific insulin gene (Ins1) was fully inactivated to prevent compensatory Ins1 expression. We designed our experiment to evaluate these animals in the context of two distinct diets (diet A: moderate-energy diet; diet B: high-energy diet). Remarkably, we found that across both diets, mice with reduced circulating insulin levels had improved insulin sensitivity with advanced age and exhibited lifespan extension without changing Igf1 levels. These results suggest a causal contribution for hyperinsulinemia in age-dependent insulin resistance and point to the modest suppression of insulin as a safe and attainable strategy for extending lifespan.

"Older obese patients and sometimes women who suffer heart failure go to the cardiac catheterization lab and the cardiologist finds nothing that would explain their heart failure. They have normal large blood vessels in the heart still the heart failure has developed." What isn't readily seen with these routine exams is the thickened walls that can hinder dilation of the small capillaries fed by these bigger vessels, a condition called coronary microvascular dysfunction, or cardiac syndrome X.

In patients and animal models, who are both older and obese, researchers have found a key dynamic in the dysfunction is an enzyme called ADAM17, which is involved in a huge variety of functions like releasing growth factors as we develop, but also implicated in diseases from Alzheimer's to arthritis. ADAM17 levels increase in obesity while levels of its natural inhibitor, the protein caveolin-1, decrease with age, enabling the perfect storm. ADAM17 was discovered 20 years ago for its ability to cut and release previously inactive tumor necrosis factor, or TNF, from the cell membrane. TNF is a major promoter of inflammation that also directly impacts the function of the endothelial cells that line blood vessels. The scientist found that ADAM17 cleaves TNF from fat, releasing it into the bloodstream where it preferentially targets the heart. The bottom line: the walls of the hair-sized microvasculature become thicker, less elastic, less able to dilate and to properly sustain the heart.

The research team found ADAM17 highly expressed in fat and even higher in the blood vessels of aged human fat. The protein level was increased in younger mice on a high-fat diet, but the significant increase in its activity came with age and fat. In humans, they saw the ability of the tiny vessels to dilate significantly reduced in those ages 69 and older and further reduced in older individuals - males and females - who also were obese. They found ADAM17 present in the fat of young and old mice on high-fat diets compared to normals, but it was only significantly active in the older mice on a high-fat diet. When they looked at younger and older obese patients, again much like the mice, they found high levels of expression of ADAM17 in the lining of blood vessel walls. When they transplanted fat from aged obese mice to younger mice, it increased circulating levels of proinflammatory factors and impaired dilation of the coronary microvasculature. "It basically mimicked the old vascular phenotype in the young animals."

The researchers have started to look at antibodies that would directly target and ideally reduce levels of ADAM17 in the face of aged fat and at least delay development of small vessel disease. They further think a similar process may happen in the brains of older obese individuals, so have ongoing studies of how microvascular disease can lead to Alzheimer's in these individuals. The researchers note that young, obese individuals could help themselves avoid this and likely other diseases like diabetes, by losing weight while they are young.