Intestinal stem cells (ISCs) drive the rapid regeneration of the gut epithelium to maintain organismal homeostasis. Aging, however, significantly reduces intestinal regenerative capacity. While cellular senescence is a key feature of the aging process, little is known about the in vivo effects of senescent cells on intestinal fitness. Here, we identify the accumulation of senescent cells in the aging gut and, by harnessing senolytic CAR T cells to eliminate them, we uncover their detrimental impact on epithelial integrity and overall intestinal homeostasis in natural aging, injury and colitis. Ablation of intestinal senescent cells with senolytic CAR T cells in vivo or in vitro is sufficient to promote the regenerative potential of aged ISCs. This intervention improves epithelial integrity and mucosal immune function. Overall, these results highlight the ability of senolytic CAR T cells to rejuvenate the intestinal niche and demonstrate the potential of targeted cell therapies to promote tissue regeneration in aging organisms.

Competing Interest Statement
S.B. received research funding from Caper Labs, Elstar Therapeutics and Revitope Oncology for research unrelated to this study. S.B. is an advisor for Caper Labs. S.B and C.A. are listed as inventors on a patent application related to the regenerative effects of senolytic CAR T cells (63/510,997). M.S and C.A. are listed as the inventor of several patent applications (62/800,188; 63/174,277; 63/209,941; 63/209,940; 63/209,915; 63/209,924; 17/426,728; 3,128,368; 20748891.7; 2020216486) related to senolytic CAR T cells. M.S. is also listed on other unrelated patents concerning CAR T technology. M.S. and C.A. are advisors for Fate Therapeutics.

Senolytic CAR T cells reverse aging-associated defects in intestinal regeneration and fitness

It is believed that senescent cells are unable to grow; however, we have treated non-growing senescent cells with low frequency ultrasound (LFU) and they are rejuvenated for growth. Further, the senescent cells are characterized by low levels of motility but LFU treatment significantly increases the level of whole cell and organelle motility. At a mechanistic level, LFU causes Ca2+ entry that precedes dramatic increases in autophagy with the inhibition of mTORC1 and the movement of Sirtuin 1 from the nucleus to cytoplasm. Other characteristics of senescent cells are also reversed including blocking SASP secretion, restoring telomere length, plus nuclear 5mc levels, H3K9me3 levels, γH2AX levels, p53 nuclear staining, ROS and mitoROS levels are all restored to normal levels. Repeated LFU treatments enables expansion of primary cells and stem cells beyond normal replicative limits without altering cell phenotype. Rejuvenation is enhanced by rapamycin and Rho kinase inhibition but inhibited by blocking Sirtuin 1, Piezo1 or TRPV1. Thus, we suggest that mechanical pressure waves similar to those generated by exercise can reverse senescence at the cell level, which provides a way to rejuvenate senescent cells in vivo.

One-Sentence Summary
Low frequency ultrasound alone rejuvenates senescent cells by activating autophagy.

Competing Interest Statement
The authors have declared no competing interest.

Rejuvenation of Senescent Cells by Low Frequency Ultrasound without Senolysis

Reproductive aging is a major cause of fertility decline, attributed to decreased oocyte quantity and competence. Follicular somatic cells play crucial roles in the growth and development of the oocyte by providing nutrients and regulatory factors. Here we investigated how oocyte quality is affected by its somatic cell environment by creating chimeric follicles, whereby an oocyte from one follicle was transplanted into and cultured within another follicle whose native oocyte was removed. Somatic cells within the chimeric follicle re-establish connections with the oocyte and support oocyte growth and maturation in a three-dimensional (3D) culture system. We show that young oocytes transplanted into aged follicles exhibited reduced meiotic maturation and developmental potential, whereas the young follicular environment significantly improved the rates of maturation, blastocyst formation and live birth of aged oocytes. Aged oocytes cultured within young follicles exhibited enhanced interaction with somatic cells, more youth-like transcriptome, remodelled metabolome, improved mitochondrial function, and enhanced fidelity of meiotic chromosome segregation. These findings provide the basis for a future follicular somatic cell-based therapy to treat age-associated female infertility.

Competing Interest Statement
We would like to disclose that we have filed a patent for this study. The applicants and inventors for this patent are Rong Li and HaiYang Wang. This patent application, titled 'Somatic Cell-Based Therapy to Treat Female Infertility', was filed under the following numbers: PCT/SG2023/050339 and has been published with the Publication Number: WO 2023/224556 A1. The remaining authors declare no competing interests.

Rejuvenation of aged oocyte through exposure to young follicular microenvironment

Recent studies proposing induced glia-to-neuron conversion raised the potential for generating new neurons to replace those lost due to injury, aging or neurodegenerative diseases. Here, single-cell spatial transcriptomics [Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH)] is used to construct a spatial cell atlas of the subventricular and dentate gyrus neurogenic niches of young and aged adult murine brain. RNAs that encode the RNA binding protein Polypyrimidine Tract-Binding Protein (PTBP1) in the aged murine brain are determined to be highest in glia that line previously active neurogenic niches. A glial cell population with ependymal character within an initially quiescent subventricular neurogenic niche in the aged murine brain is identified that upon transient suppression of PTBP1 reenters the cell cycle, replicates DNA, and converts into neurons through a canonical adult neurogenesis pathway. Glia-derived neurons migrate from this niche, with some neurons transiting to the striatum and acquiring a transcriptome characteristic of GABAergic inhibitory neurons. Similar PTBP1 expressing quiescent glia are identified in the corresponding neurogenic niche of aged human brain. Thus, transient reduction of PTBP1 holds potential for inducing the generation of new neurons in quiescent neurogenic niches of the aged nervous system, thereby offering promising therapeutic applications.

Re-activation of neurogenic niches in aging brain

Aging across most species, including mice and humans, is characterized by an exponential acceleration of mortality rates. In search for the molecular basis of this phenomenon, we analyzed DNA methylation (DNAm) changes in aging mice. Utilizing principal component analysis (PCA) on DNAm profiles, we identified a primary aging signature with an exponential age dependency, closely reflecting the Gompertz law’s description of mortality acceleration. This signature is the manifestation of the dynamic instability in the organism’s state that drives the aging process in mice. It aligns closely with regression-based aging clocks and responds to interventions such as caloric restriction and parabiosis. Additionally, we identified a linear DNAm signature, indicative of a global demethylation level. Through single-cell DNAm (scDNAm) data from aging animals, we demonstrate that this signature captures the exponential expansion of the state space volume spanned by individual cells within an aging organism, and thus quantifying linearly increasing configuration entropy, likely an irreversible process. Consistent with this interpretation, we found that neither caloric restriction (CR) nor parabiosis significantly impacts the entropic feature, reinforcing its link to irreversible damage.

Differential Responses of Dynamic and Entropic Aging Factors to Longevity Interventions

Aging and neurodegeneration entail diverse cellular and molecular hallmarks. Here, we studied the effects of aging on the transcriptome, translatome, and multiple layers of the proteome in the brain of a short-lived killifish. We reveal that aging causes widespread reduction of proteins enriched in basic amino acids that is independent of mRNA regulation, and it is not due to impaired proteasome activity. Instead, we identify a cascade of events where aberrant translation pausing leads to reduced ribosome availability resulting in proteome remodeling independently of transcriptional regulation. Our research uncovers a vulnerable point in the aging brain’s biology – the biogenesis of basic DNA/RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity and the biosynthesis of macromolecules.

Impaired biogenesis of basic proteins impacts multiple hallmarks of the aging brain

Although most drugs currently approved are meant to treat specific diseases or symptoms, it has been hypothesized that some might bear a beneficial effect on lifespan in healthy older individuals, outside of their specific disease indication. Such drugs include, among others, metformin, SGLT2 inhibitors and rapamycin. The UK biobank has recorded prescription medication and mortality data for over 500’000 patients during more than 40 years. In this work we examined the impact of the top 406 prescribed medications on overall mortality rates within the general population of the UK. As expected, most drugs harbor a negative effect on lifespan, probably due to the underlying negative effect of the disease the drug is intended for. Importantly, a few drugs seem to have a beneficial effect on lifespan, including notably Sildenafil, Atorvastatin, Naproxen and Estradiol. These retrospective results warrant further investigation in randomized controlled trials.

Association between prescription drugs and all-cause mortality risk in the UK population

How cell replication ultimately results in aging and the Hayflick limit are not fully understood. Here we show that clock-like accumulation of DNA G-quadruplexes (G4s) throughout cell replication drives conserved aging mechanisms. G4 stimulates transcription-replication interactions to delay genome replication and impairs DNA re-methylation and histone modification recovery, leading to loss of heterochromatin. This creates a more permissive local environment for G4 formation in subsequent generations. As a result, G4s gradually accumulate on promoters throughout mitosis, driving clock-like DNA hypomethylation and chromatin opening. In patients and in vitro models, loss-of-function mutations in the G4-resolving enzymes WRN, BLM and ERCC8 accelerate the erosion of the epigenomic landscape around G4. G4-driven epigenomic aging is strongly correlated with biological age and is conserved in yeast, nematodes, insects, fish, rodents, and humans. Our results revealed a universal molecular mechanism of aging and provided mechanistic insight into how G-quadruplex processor mutations drive premature aging.

A universal molecular mechanism driving aging

Objective. Investigational cell therapies have been developed as disease-modifying agents for the treatment of osteoarthritis (OA), including those that inducibly respond to inflammatory factors driving OA progression. However, dysregulated inflammatory cascades do not specifically signify the presence of OA. Here, we deploy a synthetic receptor platform that regulates cell behaviors in an arthritis-specific fashion to confine transgene expression to sites characterized by cartilage degeneration.

Methods. An scFv specific for type II collagen (CII) was used to produce a synthetic Notch (synNotch) receptor that enables “CII-synNotch” mesenchymal stromal cells (MSCs) to recognize CII fibers exposed in damaged cartilage. Engineered cell activation by both CII-treated culture surfaces and on primary tissue samples was measured via inducible reporter transgene expression. TGFβ3-expressing cells were assessed for cartilage anabolic gene expression via qRT-PCR. In a co-culture with CII-synNotch MSCs engineered to express IL-1Ra, ATDC5 chondrocytes were stimulated with IL-1α, and inflammatory responses of ATDC5s were profiled via qRT-PCR and an NF-kB reporter assay.

Results. CII-synNotch MSCs are highly responsive to CII, displaying activation ranges over 40-fold in response to physiologic CII inputs. CII-synNotch cells exhibit the capacity to distinguish between healthy and damaged cartilage tissue and constrain transgene expression to regions of exposed CII fibers. Receptor-regulated TGFβ3 expression resulted in upregulation of Acan and Col2a1 in MSCs, and inducible IL-1Ra expression by engineered CII-synNotch MSCs reduced pro-inflammatory gene expression in chondrocytes.

Conclusion. This work demonstrates proof-of-concept that the synNotch platform guides MSCs for spatially regulated, disease-dependent delivery of OA-relevant biologic drugs.

Competing Interest Statement.
BLW, JMB, CLD, and KAH have filed a patent disclosure pertaining to the CII-synNotch receptor

A programmable arthritis-specific receptor for guided articular cartilage regenerative medicine

Centenarians provide a unique lens through which to study longevity, healthy aging, and resiliency. Moreover, models of human aging and resilience to disease that allow for the testing of potential interventions are virtually non-existent. We obtained and characterized over 50 centenarian and offspring peripheral blood samples including those connected to functional independence data highlighting resistance to disability and cognitive impairment. Targeted methylation arrays were used in molecular aging clocks to compare and contrast differences between biological and chronological age in these specialized subjects. Isolated peripheral blood mononuclear cells (PBMCs) were then successfully reprogrammed into high-quality induced pluripotent stem cell (iPSC) lines which were functionally characterized for pluripotency, genomic stability, and the ability to undergo directed differentiation. The result of this work is a one-of-a-kind resource for studies of human longevity and resilience that can fuel the discovery and validation of novel therapeutics for aging-related disease.