Overarching Goals and Structure
The Rejuvenome is designed to uncover the effects of aging-interventions (treatments targeting the biological processes of aging) throughout the entire body, spanning molecular and cellular changes to functional differences of individual organs and the body as a whole. For robustness, the study will use genetically diverse mice that more closely resemble the human population, and interventions will be tested successively over the next ~7 years. Ultimately, dozens of individual interventions and their combinations will be assessed using measurements including cross-sectional multiomics (multiple types of molecular measurements requiring euthanasia conducted at pre-designated time points) in over 20 organ types, and longitudinal measures (those that can be conducted repeatedly in the same mouse over its lifetime). New technologies will be incorporated into the Rejuvenome as they are invented, and early results will guide the selection of new interventions and the design of intervention combinations.
A variety of interventions will be included spanning the spectrum of well established (e.g. rapamycin), to promising (e.g. senolytics), to emerging (e.g. partial reprogramming), and speculative (e.g. combinations), so long as the evidence and rationale warrants inclusion. These interventions need not be limited to small molecules (drugs). Any method of improving lifespan or healthspan will be considered (e.g. dietary, surgical, gene therapy). Considerations for inclusion are being solicited from the community: ideas can be submitted here.
An in-house computational team will lead the central bioinformatics analyses and the development of new methods, including those incorporating machine learning. This team will also make all data and results freely available as soon as possible, create a user-friendly web-based platform for basic data visualization and analysis, and analyze and interpret the results together with the Rejuvenome Consortium, a group of scientists with expertise in each organ and intervention. Areas of high interest may warrant ancillary studies, either internally, or with collaborative third parties with special expertise or facilities.
The study is meant to be designed by the community, for the community, and analyzed with the community, with the initial project steering the selection of future interventions (or combinations of interventions) for similar profiling. The idea of Rejuvenome emerged from discussions with dozens of scientists and has been refined over time with continuous input from experts. It was also inspired by the Interventions Testing Program (ITP), the Study of Longitudinal Aging in Mice (SLAM), Tabula Muris Senis, and the growing number of published and ongoing studies in genetically diverse mice. It is meant to combine the best aspects of each at a scale never before attempted.
- Astera-Buck Partnership: Co-designers and scientific directors of Rejuvenome. Astera provides funding and houses the Rejuvenome computational team. The Buck, with vivarium and wet-lab infrastructure amenable to large-scale endeavors (thousands of mice), hosts all internal mouse and wet-lab experiments.
- The Rejuvenome Consortium: Together with Astera and Buck, a collaboration of labs with expertise in critical areas, including specific organs, specific interventions, or specific methodologies. The Consortium assists with experimental design and is essential to ensuring optimal sample preparation, analysis, and interpretation of results.
- Genetically diverse mice: Outbred cohorts (populations derived from combining multiple inbred strains) such as HET3s or diversity outbred mice (DOs) better represent human genetic diversity and will allow Rejuvenome to test for robustness across genetic backgrounds. Pilot studies comparing the two options are underway. One will be selected by late 2022 for use in the remainder of the project.
- High-throughput multi-omics: Spatial transcriptomics, single-nucleus transcriptomics, plasma proteomics, and DNA-methylation are included for initial cohorts. Additional methods such as metabolomics, ATAC-seq, and histology are under consideration. Such profiling will capture unprecedented details of the underlying mechanisms of aging and rejuvenation, allowing for the future design of synergistic multi-intervention paradigms. Technical pilots are underway comparing different platforms and establishing protocols in organs of interest, and the list may narrow once it is determined which are capturing the most useful information.
- Organism-wide inclusion: Early cohorts will include all major organs, but the list will likely narrow as it becomes clear which are most informative for determining how to combine interventions. Organs under consideration include: Adipose, adrenal gland, aorta, bladder, bone, bone marrow, brain, diaphragm, ear, eye, heart, kidney, large intestine, limb muscle, liver, lung, lymph node, mammary gland, ovary, pancreas, PBMCs, pituitary gland, prostate, skin, small intestine, spleen, stomach, testis, thymus.
- Longitudinal blood profiling: Multi-omics (especially proteomics and DNA methylation) will help generate biomarkers capable of measuring health during aging or in response to intervention.
- Longitudinal functional measures: Metabolic cages with activity monitoring, bone density, muscle strength, echocardiography, arterial stiffness, retinal scans, and a frailty index will estimate the success of putative interventions and measure aspects of quality of life, providing a link from molecular data and biomarkers to functional outcome.
- Lifespan: With ~100 mice per intervention (equally split between male and female), this study is not powered to detect small lifespan differences. However, the age of death will be linked to data from longitudinal assays, with the hope of discovering if these measurements can act as surrogate markers of mortality.
- Aging Interventions: Nine mouse cohorts are planned, with a new cohort (of 1-month-old mice) enrolled every 6 months beginning in mid-2022. Six to eight interventions or intervention combinations will be included per cohort, each tested in 100 18-month-old mice. Interventions started at a younger age will be considered with sufficient rationale.
- Open data and transparency: All datasets will be made freely available to the community. Raw data from all assays will become available as soon as possible after quality control, and preliminary results and interpretations will be available prior to release on BioRxiv. Experimental design (e.g. which interventions will be tested) for future cohorts will be released ahead of time, as well, and the experimental design may change over time with feedback from the community and from internal results.
- Open-ended design: If successful, this project could establish a new paradigm for conducting mouse studies. If additional funding is secured, the project will continue into the future, beyond the first 9 cohorts.
Project Structure and Timeline
The Rejuvenome is composed of 4 major components: Pilots, the Baseline, the Rejuvenome Cohorts, and Peromyscus. The Pilots are small-scale technical experiments designed to inform key components of the Rejuvenome, such as the choice of single-cell RNA-seq technology. The Baseline is designed to put these methods into practice at moderate scale prior to beginning the Rejuvenome Cohorts, while generating novel data quickly. As the core of the project, the Rejuvenome Cohorts will be the gold-standard, high-throughput platform for characterizing interventions in genetically diverse mice with multiomics and multiple longitudinal measurements. Finally, aging of Peromyscus leucopus—a common North American deer mouse reported to live up to 8 years in captivity, over twice that of the common laboratory mouse Mus musculus—will be characterized both longitudinally and with multiomics across its lifespan.
Pilot 1: Mouse strain comparison
The Diversity Outbred (DO) mouse stock is a population of genetically distinct individuals derived from 8 inbred strains, making it the most diverse stock readily available for scientific research. However, early reports of male aggression led to some doubt on its reliability for a large-scale study, and several active studies use only female DOs for this reason. HET3s are also genetically distinct, but less diverse than DOs because they are derived from only 4 inbred strains. HET3s are all siblings with identical mitochondria, as they are all born from mothers of the same CB6F1 strain (in both mice and humans, mitochondrial DNA is inherited from the mother). In contrast, DOs are bred to have mitochondria derived from all 8 of the founding strains. We are comparing HET3s and DOs in terms of aggression and variability in the longitudinal assays. All else being equal, if aggression is not a major factor in our hands, DOs will be chosen for the remainder of the project due to their higher diversity.
Pilot 2: Single-nucleus RNA-seq comparison, and establishment of standard protocols
Single-nucleus RNA-seq, rather than single-cell RNA-seq, was selected for the Rejuvenome because of its immense practical advantages for a project of this scale. We are comparing two commercial platforms for high throughput single-nucleus RNA-seq: those offered by 10x Genomics and Parse Biosciences. Methods will be compared in terms of data quality, reliability, scalability of workflow, and cost. For many organ types, single-nucleus RNA-seq has not been attempted. We will therefore practice and establish protocols for all organs under consideration.
Pilot 3: Spatial Transcriptomics, establishment of standard protocols
Spatial technologies are rapidly advancing, but these methods have not yet been leveraged to study aging biology. After reviewing multiple platforms, we have chosen NanoString Technologies’ GeoMx instrument for the Rejuvenome Baseline. For many, or indeed most, of our chosen mouse organs, standard procedures do not exist. We will perform experiments to test, troubleshoot, and optimize such protocols, including in samples from old animals. NanoString’s upcoming CosMx based on in-situ hybridization is also under close consideration.
Rejuvenome Baseline: Aging and rapamycin in C57BL/6
The genetically diverse mice used in the Rejuvenome Cohorts are not available pre-aged. With the first Cohort of 1-month-olds slated to begin aging in-house in May, the first interventions will not start until October, 2023. To establish methods at scale in preparation for these immense Cohorts while generating novel data as soon as possible, we have designed the Rejuvenome Baseline. Here we will perform longitudinal measures with metabolic cages, and collect cross sectional samples across the lifespan and at several time points after rapamycin treatment (126ppm) in pre-aged C57BL/6 mice. Multi-omic measures will feature spatial transcriptomics, single-nucleus RNA-seq, and DNA-methylation.
Rejuvenome Cohorts: Intervention characterization in HET3s or DOs
As the core of the Rejuvenome Project, genetically diverse cohorts (either HET3s or DOs, to be determined based on Pilot 1 [see above] and Cohort 1 [see below]) will be enrolled every six months beginning in May, 2022. The first interventions will start in October, 2023, when Cohort 1 reaches 18 months of age. In general, each of the planned 9 Cohorts will contain 6-8 interventions, each tested in 50 males and 50 females (the max capacity at any given time is 5,000 mice). In addition to a battery of longitudinal measures collected across the lifespan, three cross sectional time points of 10 mice per sex are included at 1, 3, and 6 months post intervention start, corresponding to ages of 19, 21, and 24 months.
Cohort 1: Aging and rapamycin in C57BL/6, HET3, and DO
The first Rejuvenome Cohort is an exception to the high-throughput intervention testing of later Cohorts, and instead focuses on establishing an aging and rapamycin-treated reference dataset comparing C57BL/6, HET3, and DO. Over the last several decades there has been a push to use genetically diverse mice that better mimic the diverse human population. This led to the development of such mouse stocks such as the UM-HET3 and DO, but their use by the research community has been, overall, limited. Now, the push to replace genetically identical strains like C57BL/6 with diverse stocks is stronger than ever, with more and more researchers using or considering using them. However, the choice of which stock to use is not clear, and in fact if they are truly “better” than C57BL/6 (e.g. in terms of phenotypic variability) is debated. The purpose of Cohort 1 is to help resolve this debate, or at least provide a large, centralized study to which researchers can refer when considering these options.
Cohort 2: Multiple interventions in HET3 or DO
Interventions under serious consideration for this cohort include senolysis, partial reprogramming, and exercise. Specific suggestions for these categories, or other interventions or combinations, are being accepted here.
Cohorts 3-9: Multiple interventions and combinations in HET3 or DO
Interventions for these Cohorts have not yet been selected. By this time, new interventions may exist that warrant consideration, and advances in technology may warrant the inclusion of new assays.
Several species of the genus Peromyscus, commonly called deer mice, are long-lived in captivity. Reportedly, species like Peromyscus leucopus show a maximum lifespan of over 8 years, more than twice that of the distantly related house mouse, Mus musculus, used almost ubiquitously in laboratory research. In this arm of Rejuvenome, P. leucopus will be characterized across its lifespan with longitudinal measurements cross sectional multiomics assays. However, these mice will undergo no interventions, as one might consider this species difference to be an intervention in and of itself.
Transparency and Community Interaction
We hope to maintain transparency in all phases of the Rejuvenome, starting with this release of experimental plans, as our efforts are not meant to be competitive or duplicative. Rather, we hope to work with the research community to advance our knowledge about aging and aging interventions as quickly as possible. It is our hope that such transparency encourages researchers working on similar avenues to reach out to coordinate efforts or collaborate. Similarly, we hope to continue discussions with the research community regarding experimental design, both of planned and future experiments.
A caveat to such transparency prior to conducting experiments is that plans can, and will, change. Our goal is to continuously update our experimental designs, and to explain why those decisions were made, with future blog posts.