Hematopoietic Stem Cell Aging Research Articles

Tetrahydroxy stilbene glucoside rejuvenates aging hematopoietic stem cells with predilection for lymphoid differentiation via AMPK and Tet2

IntroductionAging of hematopoietic stem cells (HSCs) has emerged as an important challenge to human health. Recent advances have raised the prospect of rejuvenating aging HSCs via specific medical interventions, including pharmacological treatments. Nonetheless, efforts to develop such drugs are still in infancy until now. ObjectivesWe aimed to screen the prospective agents that can rejuvenate aging HSCs and explore the potential mechanisms. MethodsWe screened a set of natural anti-aging compounds through oral administration to sub-lethally irradiated mice, and identified 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) as a potent rejuvenating agent for aging HSCs. Then naturally aged mice were used for the follow-up assessment to determine the HSC rejuvenating potential of TSG. Finally, based on the transcriptome and DNA methylation analysis, we validated the role of the AMP-activated protein kinase (AMPK)-ten-eleven-translocation 2 (Tet2) axis (the AMPK-Tet2 axis) as the underlying mechanisms of TSG for ameliorating HSCs aging. ResultsTSG treatment not only significantly increased the absolute number of common lymphoid progenitors (CLPs) along with B lymphocytes, but also boosted the HSCs/CLPs repopulation potential of aging mice. Further elaborated mechanism research demonstrated that TSG supplementation restored the stemness of aging HSCs, as well as promoted an epigenetic reprograming that was associated with an improved regenerative capacity and an increased rate of lymphopoiesis. Such effects were diminished when the mice were co-treated with an AMPK inhibitor, or when it was performed in Tet2 knockout mice as well as senescent cells assay. ConclusionTSG is effective in rejuvenating aging HSCs by modulating the AMPK- Tet2 axis and thus represents a potential candidate for developing effective HSC rejuvenating therapies.

Epigenetics of hematopoietic stem cell aging.

The development of new antiaging medicines is of great interest to the current elderly and aging population. Aging of the hematopoietic system is attributed to the aging of hematopoietic stem cells (HSCs), and epigenetic alterations are the key effectors driving HSC aging. Understanding the epigenetics of HSC aging holds promise of providing new insights for combating HSC aging and age-related hematological malignancies. Aging is characterized by the progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death. During aging, the HSCs undergo both quantitative and qualitative changes. These functional changes in HSCs cause dysregulated hematopoiesis, resulting in anemia, immune dysfunction, and an increased risk of hematological malignancies. Various cell-intrinsic and cell-extrinsic effectors influencing HSC aging have also been identified. Epigenetic alterations are one such mechanism. Cumulative epigenetic alterations in aged HSCs affect their fate, leading to aberrant self-renewal, differentiation, and function of aged HSCs. In turn, these factors provide an opportunity for aged HSCs to expand by modulating their self-renewal and differentiation balance, thereby contributing to the development of hematological malignancies.

Variation in mesenchymal KITL/SCF and IGF1 expression in middle age underlies steady-state hematopoietic stem cell aging

AbstractIntrinsic molecular programs and extrinsic factors including proinflammatory molecules are understood to regulate hematopoietic aging. This is based on foundational studies using genetic perturbation to evaluate causality. However, individual organisms exhibit natural variation in the hematopoietic aging phenotypes and the molecular basis of this heterogeneity is poorly understood. Here, we generated individual single-cell transcriptomic profiles of hematopoietic and nonhematopoietic cell types in 5 young adult and 9 middle-aged C57BL/6J female mice, providing a web-accessible transcriptomic resource for the field. Among all assessed cell types, hematopoietic stem cells (HSCs) exhibited the greatest phenotypic variation in expansion among individual middle-aged mice. We computationally pooled samples to define modules representing the molecular signatures of middle-aged HSCs and interrogated, which extrinsic regulatory cell types and factors would predict the variance in these signatures between individual middle-aged mice. Decline in signaling mediated by ADIPOQ, KITL and IGF1 from mesenchymal stromal cells (MSCs) was predicted to have the greatest transcriptional impact on middle-aged HSCs, as opposed to signaling mediated by endothelial cells or mature hematopoietic cell types. In individual middle-aged mice, lower expression of Kitl and Igf1 in MSCs was highly correlated with reduced lymphoid lineage commitment of HSCs and increased signatures of differentiation-inactive HSCs. These signatures were independent of expression of aging-associated proinflammatory cytokines including interleukin-1β (IL-1β), IL-6, tumor necrosis factor α and RANTES. In sum, we find that Kitl and Igf1 expression are coregulated and variable between individual mice at the middle age and expression of these factors is predictive of HSC activation and lymphoid commitment independently of inflammation.

Aging-induced MCPH1 translocation activates necroptosis and impairs hematopoietic stem cell function.

DNA damage contributes to the aging of hematopoietic stem cells (HSCs), yet the underlying molecular mechanisms are not fully understood. In this study, we identified a heterogeneous functional role of microcephalin (MCPH1) in the nucleus and cytoplasm of mouse HSCs. In the nucleus, MCPH1 maintains genomic stability, whereas in the cytoplasm, it prevents necroptosis by binding with p-RIPK3. Aging triggers MCPH1 translocation from cytosol to nucleus, reducing its cytoplasmic retention and leading to the activation of necroptosis and deterioration of HSC function. Mechanistically, we found that KAT7-mediated lysine acetylation within the NLS motif of MCPH1 in response to DNA damage facilitates its nuclear translocation. Targeted mutation of these lysines inhibits MCPH1 translocation and, consequently, compromises necroptosis. The dysfunction of necroptosis signaling, in turn, improves the function of aged HSCs. In summary, our findings demonstrate that DNA damage-induced redistribution of MCPH1 promotes HSC aging and could have broader implications for aging and aging-related diseases.

Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing

Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid–liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.

Abstract 4398: TET2 accelerates clonal hematopoiesis in an age-dependent manner

Abstract Aging is a process of systemic deterioration and the most significant risk factor for cancers. Clonal hematopoiesis (CH) commonly occurs with aging and links to higher mortality, leukemia risk, and cardiovascular diseases. Age-related CH involves the abnormal clonal expansion of hematopoietic stem cells (HSCs) bearing somatic mutations in genes frequently mutated in leukemia, including genes encoding epigenetic regulators, such as the DNA demethylase TET2. While such mutations are known to alter the HSC epigenome, the mechanisms through which these mutations drive HSC self-renewal, myeloid transformation, and inflammatory response remain elusive. In this study, we hypothesize that aging and CH mutations cooperatively reshape the HSC transcriptomic landscape and enhancing HSC competitive advantage that facilitates clonal expansion. Recently, TET2-mutant CH in humans has been strongly associated with epigenetic age acceleration. Using single-cell multi-omic analyses and flow-cytometric phenotyping, we demonstrated, for the first time, that HSC aging processes at transcriptomic, epigenomic, and cellular levels are accelerated by Tet2 deficiency in mice, in an age-dependent manner. Tet2-deficient HSCs increase the HSC aging scores at an older age of greater than or equal to 16 months, but not at a young or mid-age, based on gene expression and chromatin accessibility at a single-cell resolution. This age acceleration was further validated by epigenetic clock analysis of DNA methylation mapping. Moreover, we found that Tet2 deficiency drives a significant expansion of HSCs, a cellular hallmark of HSC aging, in an age-dependent manner. Specifically, we observed increased expression of S100a6, Cdk6, Egr1 in Tet2-deficient HSCs from older mice, suggesting that Tet2 deficiency hijacks the HSC expansion strategy during HSC aging by enhanced self-renewal. We also observed that the differentially expressed genes between Tet2-deficient older HSCs and control older HSCs are associated with open chromatin accessibility. Many of these genes are enriched in the targets of specific transcription factors (TFs), such as Egr1, Creb1, Stat family and Hoxb8. These target genes play critical roles in inflammatory pathways and HSC functions. In conclusion, our findings reveal that Tet2 deficiency significantly contributes to the acceleration of HSC aging, and hijacks the HSC expansion strategy (e.g., self-renewal) during HSC aging, which exacerbates age-related clonal hematopoiesis. By elucidating the transcriptomic and epigenomic alterations in Tet2-deficient HSCs, our study provides novel insights into how age and somatic mutations collaborate to promote the pathogenesis of age-related hematological diseases. These discoveries not only enhance our understanding of HSC aging mechanisms but also offer potential biomarkers for early detection and targets for therapeutic intervention in age-associated clonal disorders. Citation Format: Shilpita Karmakar, Yang Liu, Efthymios Motakis, Kaustav Sengupta, Travis Roeder, Kristina Mujica, Jennifer Trowbridge, Hideyuki Oguro, Sheng Li. TET2 accelerates clonal hematopoiesis in an age-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4398.

The Effect of Circulating Exosomes Obtained from Young and Old Individuals on the Aging Related hTERT and P16 Expression in Hematopoietic Stem Cells

Introduction: Functional reduction of telomeres can induce DNA damage response through cell cycle checkpoints and contribute to the senescence of stem cells. The effect of exosomes on the aging and rejuvenation of hematopoietic stem cells (HSCs) is not well known. Therefore, the present study is designed to examine the impact of plasma exosomes derived from young and old individuals on hTERT and P16 expression involved in the cellular aging process. background: Functional reduction of telomeres can induce DNA damage response through cell cycle checkpoints and contribute to the senescence of stem cells. The effect of exosomes on the aging and rejuvenating in hematopoietic stem cells (HSCs) is not well known. Therefore, the present study designed to examine the impact of plasma exosomes derived from young and old individuals on hTERT and P16 expression involved in cellular aging process. Methods: Exosomes isolated from four young (Y-Exo) and four old (O-Exo) men were evaluated for CD63 protein expression, morphology, size and zeta potential. HSCs were treated with exosomes, and then, the cell viability and the mRNA expression (hTERT and P16) were evaluated using MTT and qRT-PCR methods, respectively. To measure the hTERT protein level, a western blot technique was performed. Results: The gene expression of hTERT was significantly decreased in HSCs treated with 5 μg/ml (O5-Exo) and 10 μg/ml (O10-Exo) doses of exosomes obtained from elderly individuals compared to the cells treated with young exosomes and the untreated HSCs (p < 0.05). In addition, there was a profound elevation of hTERT protein in the HSCs treated with both doses of young exosomes in comparison with the cells treated with both doses of old exosomes (p < 0.05). Moreover, P16 expression was markedly upregulated in the O5-Exo and O10-Exo groups compared to the untreated group (p < 0.05). Conclusion: Our findings reinforce the concept that depending on the age of individuals, circulating exosomes may acquire properties that affect the pathways involved in the aging process in HSCs.

Deep learning-based predictive classification of functional subpopulations of hematopoietic stem cells and multipotent progenitors.

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) play a pivotal role in maintaining lifelong hematopoiesis. The distinction between stem cells and other progenitors, as well as the assessment of their functions, has long been a central focus in stem cell research. In recent years, deep learning has emerged as a powerful tool for cell image analysis and classification/prediction. In this study, we explored the feasibility of employing deep learning techniques to differentiate murine HSCs and MPPs based solely on their morphology, as observed through light microscopy (DIC) images. After rigorous training and validation using extensive image datasets, we successfully developed a three-class classifier, referred to as the LSM model, capable of reliably distinguishing long-term HSCs, short-term HSCs, and MPPs. The LSM model extracts intrinsic morphological features unique to different cell types, irrespective of the methods used for cell identification and isolation, such as surface markers or intracellular GFP markers. Furthermore, employing the same deep learning framework, we created a two-class classifier that effectively discriminates between aged HSCs and young HSCs. This discovery is particularly significant as both cell types share identical surface markers yet serve distinct functions. This classifier holds the potential to offer a novel, rapid, and efficient means of assessing the functional states of HSCs, thus obviating the need for time-consuming transplantation experiments. Our study represents the pioneering use of deep learning to differentiate HSCs and MPPs under steady-state conditions. This novel and robust deep learning-based platform will provide a basis for the future development of a new generation stem cell identification and separation system. It may also provide new insight into the molecular mechanisms underlying stem cell self-renewal.

Time-series analysis of hematopoietic stem cells.

This study aimed to investigate the molecular mechanisms underlying the aging of hematopoietic stem cells (HSCs). Gene expression profile GSE32719 was downloaded from the Gene Expression Omnibus database, including 14 young, 5 middle, and 8 old HSCs. Differential expression analysis, short time-series expression miner analysis, and weighted co-expression network analysis were conducted to screen for hub genes whose expression changed over time during HSC aging. Subsequently, functional enrichment and multiple regulatory network analyses of the hub genes were performed. A total of 124 intersecting time-dependent differentially expressed and module genes were obtained, which were considered hub genes whose expression changed over time during HSC aging. Hub genes were significantly enriched in pathways such as the Hippo and AMP-activated protein kinase (AMPK) signaling pathways. Moreover, AP-1 Transcription Factor Subunit (FOS) and sirtuin 1 (SIRT1) had higher degrees in the protein-protein interaction network, were regulated by more transcription factors (TFs), such as Sp1 transcription factor (SP1) and BRCA1 DNA repair-associated (BRCA1), in the TF-mRNA-miRNA network, were associated with more diseases in the disease-gene network, and could be targeted by more drugs in the drug-gene network. Furthermore, SIRT1 was targeted by miR-9-5p in the TF-mRNA-miRNA network. Hub genes such as FOS and SIRT1 and key pathways such as the Hippo and AMPK signaling pathways may play crucial roles in HSC aging. Moreover, FOS and SIRT1 were regulated by SP1 and BRCA1, respectively, during HSC aging. Furthermore, miR-9-5p may modulate HSC aging by targeting SIRT1. Thus, FOS and SIRT1 may be potential therapeutic targets for age-related hematopoietic dysfunction.

Age-related noncanonical TRMT6-TRMT61A signaling impairs hematopoietic stem cells.

Aged hematopoietic stem cells (HSCs) exhibit compromised reconstitution capacity and differentiation bias toward myeloid lineages. However, the molecular mechanism behind HSC aging remains largely unknown. In this study, we observed that RNA N1-methyladenosine-generating methyltransferase TRMT6-TRMT61A complex is increased in aged murine HSCs due to aging-declined CRL4DCAF1-mediated ubiquitination degradation signaling. Unexpectedly, no difference of tRNA N1-methyladenosine methylome is observed between young and aged hematopoietic stem and progenitor cells, suggesting a noncanonical role of the TRMT6-TRMT61A complex in the HSC aging process. Further investigation revealed that enforced TRMT6-TRMT61A impairs HSCs through 3'-tiRNA-Leu-CAG and subsequent RIPK1-RIPK3-MLKL-mediated necroptosis cascade. Deficiency of necroptosis ameliorates the self-renewal capacity of HSCs and counters the physiologically deleterious effect of enforced TRMT6-TRMT61A on HSCs. Together, our work uncovers a nonclassical role for the TRMT6-TRMT61A complex in HSC aging and highlights a therapeutic target.

Implications of stress-induced gene expression for hematopoietic stem cell aging studies.

A decline in hematopoietic stem cell (HSC) function is believed to underlie hematological shortcomings with age; however, a comprehensive molecular understanding of these changes is currently lacking. Here we provide evidence that a transcriptional signature reported in several previous studies on HSC aging is linked to stress-induced changes in gene expression rather than aging. Our findings have strong implications for the design and interpretation of HSC aging studies.

Differential Effects of Endurance Exercise on Musculoskeletal and Hematopoietic Modulation in Old Mice.

One of the most important strategies for successful aging is exercise. However, the effect of exercise can differ among individuals, even with exercise of the same type and intensity. Therefore, this study aims to confirm whether endurance training (ETR) has the same health-promoting effects on the musculoskeletal and hematopoietic systems regardless of age. Ten weeks of ETR improved endurance exercise capacity, with increased skeletal muscle mitochondrial enzymes in both young and old mice. In addition, age-related deterioration of muscle fiber size and bone microstructure was improved. The expression levels of myostatin, muscle RING-finger protein-1, and muscle atrophy F-box in skeletal muscle and peroxisome proliferator-activated receptor-γ in the femur increased with age but decreased after ETR. ETR differentially modulated hematopoietic stem cells (HSCs) depending on age; ETR induced HSC quiescence in young mice but caused HSC senescence in old mice. ETR has differential effects on modulation of the musculoskeletal and hematopoietic systems in old mice. In other words, endurance exercise is a double-edged sword for successful aging, and great effort is required to establish exercise strategies for healthy aging.

Methods for Estimating Personal Disease Risk and Phylogenetic Diversity of Hematopoietic Stem Cells.

An individual's chronological age does not always correspond to the health of different tissues in their body, especially in cases of disease. Therefore, estimating and contrasting the physiological age of tissues with an individual's chronological age may be a useful tool to diagnose disease and its progression. In this study, we present novel metrics to quantify the loss of phylogenetic diversity in hematopoietic stem cells (HSCs), which are precursors to most blood cell types and are associated with many blood-related diseases. These metrics showed an excellent correspondence with an age-related increase in blood cancer incidence, enabling a model to estimate the phylogeny-derived age (phyloAge) of HSCs present in an individual. The HSC phyloAge was generally older than the chronological age of patients suffering from myeloproliferative neoplasms (MPNs). We present a model that relates excess HSC aging with increased MPN risk. It predicted an over 200 times greater risk based on the HSC phylogenies of the youngest MPN patients analyzed. Our new metrics are designed to be robust to sampling biases and do not rely on prior knowledge of driver mutations or physiological assessments. Consequently, they complement conventional biomarker-based methods to estimate physiological age and disease risk.

Zinc chloride may regulate hematopoietic stem cell aging and pro-inflammatory cytokines in systemic lupus erythematosus

Background: The immune cells of patients with systemic lupus erythematosus (SLE) age earlier than those of normal subjects. However, the senescence of circulating hematopoietic stem cells (HSCs) in patients with SLE is not well understood, and it is unclear whether zinc treatment can regulate the senescence and proinflammatory cytokine production of HSCs in these patients. Methods: Clinical data were collected on 38 patients with SLE and 35 healthy controls (HCs), and the complete blood count, circulating HSC number, and p16 (a senescence marker) expression in the peripheral blood of these participants were analyzed via flow cytometry. Pooled circulating HSCs were isolated using leukapheresis. The effects of zinc chloride exposure on the pooled HSCs of each group were determined in vitro. Levels of the proinflammatory cytokines IL-6 and IL17, regulatory cytokine TGF-β, p16, and regulator T-cells (Tregs) were evaluated 72 h after incubation with 50 or 100 µM zinc chloride. Results: The number of circulating HSCs did not differ between the two groups (p=0.1685). The expression of p16 in HSCs was higher in the SLE group than in the HC group (p = 0.0043), and patients with SLE exhibited higher levels of IL-6, IL-17, and p16 in pooled HSCs (p =0.0025, p<0.0001, and p = 0.0003, respectively), although TGF-β levels did not differ between the groups (p=0.9816). Zinc chloride reduced IL-6, TGF-β, IL-17, and p16 expression in patients with SLE toward HC levels. Treg frequency in pooled HSCs was comparable between the groups (p=0.3997), although a 100 µM zinc chloride treatment significantly depleted the Treg population of patients with SLE (p=0.0001). Conclusions: Circulating HSCs in SLE are more aged and produce more proinflammatory cytokines. Zinc chloride treatment might prevent immunoaging and inhibit proinflammatory cytokine–producing cells in patients with SLE.

The Aging Bone Marrow Niche Disrupts Glutamine Metabolism By Upregulating HSC Derived Glutamine Synthetase

As our aging population grows it is becoming increasingly important to better understand the physiological changes that drive that drive the aging process. Aging has long been known to drive haematopoietic stem cell (HSCs) dysfunction and therefore has been implicated in promoting the development of age-related haematopoietic diseases, such as leukaemia and myeloma. It is likely that a combination of intrinsic changes within the HSCs and extrinsic changes within the bone marrow (BM) microenvironment contribute to HSC aging. We have previously shown that the bone marrow niche accumulates senescent cells which contribute to impaired HSC response to stress 1. Here we investigate the intrinsic changes that can be observed in aged HSCs and how these are influenced by the bone marrow niche. In order to investigate the intrinsic changes that drive metabolic HSC aging we BM was isolated from young (8-12 weeks) and aged (18-24 months). Single HSCs were then FACS purified and sequenced using the SmartSeq2 protocol. Three transcriptionally distinct populations were identified, HSCs from young mice (Y), HSCs from aged mice with an aging transcriptional signature (O) and HSCs from aged mice that are transcriptionally similar to HSCs from young mice (OY). Assessment of differentially expressed genes between these three populations revealed that glutamine synthetase ( glul) is significantly upregulated in the O population compared to both the Y and OY population. Glul is responsible for the conversion of glutamate to glutamine and therefore levels of both were next measured in FACS purified HSCs from aged and young mice. Results show that whilst there was no difference in glutamate levels, the glutamine levels were significantly reduced in HSCs from aged mice. These data suggest that glutamine metabolism is impaired in aged HSC. Next, to determine the impact of the aged BM niche on glul expression in aged HSCs, HSCs from young and aged PepCboy (CD45.1+) mice were FACS purified and adoptively transferred into young C57Bl/6 mice. After 12 weeks the mice were sacrificed and engraftment of CD45.1+ cells was confirmed by flow cytometry. In addition CD45.1+ HSCs were FACS purified to assess glul expression and measure glutamine and glutamate levels. Results confirm that the mechanism for changes in glul expression and glutamine levels is driven by the aging BM niche and transplantation into a young niche reverses this change. To understand the importance of dysregulated glutamine metabolism in aged humans we collected BM from older patients undergoing elective hip replacement surgeries. All samples were collected following informed consent and under approval of the UK NHS Health Research Authority (ref07/H0310/146). CD34+ cells were isolated and RNA extracted to measure glul expression. Similarly, to data from aged mice glul expression was elevated in aged CD34+ cells compared to young CD34+ controls. Furthermore, glutamine levels were also low in aged CD34+ cells. This suggests that the changes in HSCs from aged mice correlate with finding in human stem cells and mice are therefore a suitable species to study dysregulated glutamine metabolism in aged HSCs. Together this work demonstrates that aging causes a disruption of glutamine and glutamate metabolism in HSCs. The cause for this disruption and its direct consequences on overall HSC metabolism remains to be explored. However, it is likely that these metabolic changes will have a knock-on effect on HSC function and may elucidate some of the drivers of metabolic HSC aging.

Low CD117 Expression Identifies Hematopoietic Stem Cell Subsets with Enhanced Lymphoid Potential in Mice and Humans

Background: Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective de novo T-cell generation and thymic recovery, restoring the adaptive immune system after Hematopoietic Cell Transplantation (HCT). Recent studies have brought new insights into functional heterogeneity within HSCs, revealing an organized and predictable framework governing the adoption of lineage-restricted fates. However, the gene-regulatory networks underlying lymphoid determination and their conservation with aging in HSCs need to be elucidated. This study aims to comprehensively investigate the molecular profile of young and old HSCs and identify HSC subsets with multilineage transcriptional programs. Results: We conducted single-cell multiomic RNA and ATAC sequencing of young and old HSCs defined by Lineage-CD34-CD48-CD150+Sca-1+Kit+. We identified two CD117 (Kit) HSC subsets, Kit lo and Kit hi, with unique transcriptional profiles. Kit hi HSCs were characterized by quiescence, platelet bias, and low-output gene signatures, while Kit lo HSCs enriched for multi-lineage and high-output gene signatures. We observed a decreased frequency of Kit lo HSCs in aged mice. Although Kit lo HSCs have been previously described to exhibit increased self-renewal (Shin et al. JEM, 2014), their T-cell potential and the molecular underpinnings governing lymphoid differentiation programs are unknown. We performed in vitro studies, S17 stromal assay, and artificial thymic organoids (ATOs), which revealed an enhanced output of lymphoid progenitors and T-cells from Kit lo HSCs, consistent across all age cohorts. We next compared Kit lo vs. Kit hi HSCs in a competitive allogeneic HCT model. Kit lo HSCs demonstrated improved thymic recovery and post-HCT T-cell reconstitution, independent of age. To orthogonally evaluate post-HCT thymic function, we assessed Recent Thymic Emigrants (RTEs) output, which further substantiated superior recovery in mice receiving Kit lo HSCs. We further observed that Kit lo HSC-derived T-cells exhibited better proliferation in response to an acute Listeria monocytogenes infection. Next, we transplanted middle-aged mice to evaluate the rejuvenation potential of different Kit HSC subsets. We observed that Kit lo HSCs partly mitigated age-related changes in the thymic microenvironment, specifically by enhancing the regeneration of thymic epithelial cells (TECs) in middle-aged mice, thereby reversing the decline in T-cell production. We found differential expression and activity of key transcription factors (TFs), including Runx3, Zbtb7a, and Ezh1, associated with lymphoid differentiation through integrative transcriptional and chromatin accessibility analyses of Kit lo HSCs. These TFs notably showed distinct patterns in Kit lo HSCs independent of age, indicating their potential role in driving their enhanced T-cell potential. To establish the existence of a comparable human subset, we interrogated a human BM CITE-Seq dataset (Sommarin et al. Biorxiv 2021) for the mouse Kit lo gene signature. In concordance with our findings in mice, we observed an enrichment of the Kit lo gene program in young HSCs and decreased frequency of Kit lo HSCs in old BM. Next, we used ATOs to validate differential lymphoid potential. We found increased T-cell output originating from Kit lo HSCs, thus underscoring their enhanced lymphoid capacity and potential clinical relevance. Conclusion: Collectively, we demonstrate a distinct HSC subset, Kit lo HSC, with an age-conserved lymphoid gene program that enhances T-cell production and facilitates thymic regeneration. Importantly, we demonstrate the presence of an analogous HSC subset in humans, revealing the relevance of these insights to human health and disease. Thus, Kit lo HSCs have therapeutic potential to counteract age-associated immune senescence and treatment-related immune suppression.

Detection and Prevention of ADAR1p150-Induced Hematopoietic Stem and Progenitor Cell Aging

Introduction: Over the last decade, ADAR1p150 has been linked by our research team and others to malignant progenitor reprogramming and therapeutic resistance in a broad array of malignancies. However, ADAR1p150's role in accelerated hematopoietic stem and progenitor cell (HSPC) aging in response to inflammatory cytokines had not been clearly elucidated. Here, we investigate ADAR1 splice isoform switching and the ADAR1p150 inhibitory effects of Rebecsinib on human HSPC function in normal humanized aged normal bone marrow (aNBM) HSPC mouse models. Methods: Whole transcriptome RNA sequencing (RNA-seq) was utilized with splice isoform and editome analysis to detect ADAR1 splice isoform switching and adenosine to inosine RNA editing. Rebecsinib (17S-FD-895), a pre-IND drug candidate, has shown promising potential as an inhibitor of ADAR1p150-mediated RNA editing ( Crews, Ma...Jamieson. Cell Stem Cell 2023), which is an important therapeutic target in patients with myelofibrosis or acute myeloid leukemia that overexpress ADAR1p150 compared with ADAR1p110. Human CD34 + cells, isolated from aged normal bone marrow (aNBM) from patients undergoing elective hip replacement surgery, were transduced with a lentiviral ADAR1-nano-luciferase reporter and transplanted into NSG-SGM3 adult mice that secrete human SCF, IL-3, and GM-CSF cytokines that support normal hematopoiesis. After engraftment confirmation peripheral blood human CD45 flow cytometric assessment and IVIS 200 system imaging of ADAR1-nano-luc-GFP reporter expressing HSPC engraftment, the mice were randomly divided into vehicle-treated and Rebecsinib-treated groups (15mg/kg, twice a week for two weeks). Peripheral blood, bone marrow, and spleen were collected upon treatment completion for analysis. Results: By HSPC RNA-seq, we detected an imbalance between ADAR1p150 and ADAR1p110 expression together with increased RNA editing and splicing alterations using whole transcriptome RNA sequencing (RNA-seq) of aged compared with young bone marrow hematopoietic stem and progenitor cells (HSPCs). However, the functional relevance of ADAR1 splice isoform switching had not been evaluated in human-aged normal bone marrow HSPC xenograft (PDX) mouse models nor had this cytokine-induced HSPC aging been reversed. Flow cytometric analysis and immunohistochemistry staining revealed that Rebecsinib treatment at a dose of 10mg/kg, twice a week for two weeks, effectively spared normal human immune cells (CD3+, CD14+, and CD19+ cells). Remarkably, Rebecsinib treatment not only preserved Hematopoietic Stem (CD34+CD38-) cells and Progenitor (CD34+CD38+) cells in peripheral blood, bone marrow, and spleen but also promoted their expansion. These findings demonstrate the potential of Rebecsinib to enhance the retention of HSPC populations in the bone marrow niche. Conclusions: Our pre-clinical experiments utilizing RNA-seq, a novel ADAR1-nano-luciferase-GFP reporter, and aNBM PDX mouse models provide compelling evidence supporting the potential of Rebecsinib to restore the balance of ADAR1p150:p110 ratios and engraftment in humanized aNBM HSPC mouse models. These findings highlight Rebecsinib as a promising therapeutic candidate for targeting leukemia and myelofibrosis while preserving essential HSPC populations.

Reduction in Lymphoid-Supporting Factors By Mesenchymal Stromal Cells Drives Myeloid-Biased Hematopoiesis at Middle Age

The bone marrow “niche” for hematopoietic stem cells (HSCs) encompasses various cell types, extracellular matrices and secreted factors that maintain the specialized functions of HSCs such as quiescence, self-renewal, and production of multipotent progenitor cells. In old age, changes in this niche contribute to dysfunction of HSCs and the hematopoietic system including increased inflammation and myeloid-biased molecular programs. We have observed that HSC and hematopoietic aging phenotypes become apparent by mid-age (Young et al. Cell Stem Cell 2021, Young et al. J Exp Med 2016), leading to the hypothesis that evaluating alterations in the niche at this stage would reveal the earliest changes initiating HSC aging. To test this hypothesis, we examined both the hematopoietic and non-hematopoietic compartments in individual middle-aged (12-14 months of age, n = 9) and compared them to young C57BL/6 mice (2-4 months of age, n = 5). Immunostaining of femurs showed an increase in sinusoid volume, vessel dysmorphia, and an increase in megakaryocyte abundance in the bone marrow of middle-aged mice. No alterations were observed in arterioles nor adipocyte volume. This data provides supporting evidence that a subset of HSC niche remodeling phenotypes observed in old age are present by mid-age. We then performed single-cell RNA-seq to comprehensively assess changes in abundance and transcriptomes of hematopoietic and non-hematopoietic cells at mid-age. Analysis of this data revealed significant expansion of HSCs in middle-aged mice. In the expanded HSC population, a reduction in B lymphoid differentiation signatures was observed without alterations in inflammation or myeloid differentiation signatures. Computational predictions suggested that the reduced B lymphopoiesis program in middle-aged HSCs resulted from decreased interaction with soluble factors produced by subsets of mesenchymal stromal cells (MSCs), rather than endothelial cells or other hematopoietic cell compartments. Specifically, reduced MSC-to-HSC interactions were linked to a decline in signaling mediated by adiponectin (ADIPOQ; Adipoq), stem cell factor (SCF; Kitl), midkine (MDK; Mdk), and insulin-like growth factor 1 (IGF1; Igf1). Among these, reductions in Kitl and Igf1 in MSCs were correlated with the loss of B lymphopoiesis programs in HSCs comparing individual middle-aged mice. To test the functional impact of the decline in Igf1 in MSCs on lymphopoiesis, we created three MSC-selective Igf1 conditional knockout mouse models using distinct Cre recombinases (Lepr-Cre, nestin-CreER and Prx1-CreER). In all three of these models, MSC-selective knockout of Igf1 reduced B lymphopoiesis resulting in myeloid-biased hematopoiesis, replicating hematopoietic aging phenotypes observed at middle age. These findings indicate that the decline in B lymphopoiesis from HSCs during aging is initiated by a reduction in lymphoid-supporting factors produced by MSCs in the bone marrow microenvironment, and this occurs independently of inflammation or activation of myeloid-promoting transcriptional programs in HSCs.

Early Generation Telomerase Deficient Mice to Investigate Clonal Hematopoiesis in Aging

Clonal hematopoiesis is an age-associated condition characterized by the expansion of hematopoietic stem cell (HSC) pools with somatic mutations, observed in 10-30% of individuals older than 70 years. This phenomenon is primarily driven by mutations in transcriptional regulators such as DNMT3A, TET2, and ASXL1. While the clonal expansion of mutated HSCs is ubiquitous with age, it appears largely benign in the absence of strong selective pressures. These benign clones might compensate for the loss of regenerative HSC capacity during aging, and skew differentiation towards the myeloid lineage. Telomere erosion plays a significant role in this process. Telomeres normally prevent the activation of the DNA damage response (DDR) machinery. However, with each cell division, telomeres shorten until they reach a critical length, eventually leading to apoptosis or senescence. Previous studies have suggested a potential involvement of telomere attrition in the development of clonal hematopoiesis (Hinds et al. Blood 2016; Zink et al. Blood 2017). Late generation telomerase deficient (Terc-/- G5) mice develop premature bone marrow failure and mortality, however earlier generation Terc-/- G3 (3 homozygous crosses) have shortened telomeres but appear relatively normal. Importantly, inhibitors of telomerase have recently entered clinical trials for haematological malignancies and therefore understanding the long-term effects of telomerase inhibition is important. By studying the interplay between telomere attrition, HSC aging, and the development of CHIP, we aim to gain insights into the mechanisms underlying age-associated hematopoietic changes and their potential clinical implications. We examined young (age 8-10 week) and aged (10-12 month) Terc-/- G3 mice. Telomere lengths were significantly reduced in bone marrow cells from young Terc G3 (15 kb) when compared to wild-type mice (30 kb) with mild reduction in BM cellularity. Absolute long-term HSC numbers were reduced, as were megakaryocyte erythroid progenitors, but there was preservation of granulocyte macrophage progenitors and common myeloid progenitors. Spleen progenitor numbers were not affected. In comparison, aged (10-12 months old) Terc G3 mice showed enhanced differentiation skewing in the peripheral blood with significantly increased myeloid (n = 8 wt; n = 12 Terc ko; p = 0.0004) and concomitantly reduced B cell (p = 0.0002) and T cell (p = 0.0320) populations. There was marked mobilization of bone marrow progenitors to the spleen, most notably GMP (p = 0.0004) but also MEP and CMP in aged Terc G3. Furthermore, aged Terc G3 ko now exhibited marked reduction in bone marrow CMP (p < 0.0001) and MEP (p < 0.0001), but skewing towards GMP (p<0.001) resulting in normal total GMP numbers, but markedly reduced CMP and MEP numbers (p < 0.0001). Compared to young Terc G3 mice, the aged Terc G3 mice had relative expansion of the BM LT-HSC population, and therefore normal numbers of BM LT-HSC compared to aged matched controls. Aged Terc G3 BM LTHSC have been isolated for gene expression and clonality analysis. Together, these findings are consistent with accelerated aging in G3 Terc-/- mice, providing a model for examining clonal hematopoiesis. In order to assess the cell intrinsic function of Terc G3 LTHSC, we next performed non-competitive bone marrow transplantation experiments from Terc ko or wild-type donors into lethally irradiated young wild-type recipients (n = 9 per group), and analysed these at 4 months post-transplant. There was reduced, but stable reconstitution by Terc G3 cells, consistent with previous competitive bone marrow transplants in Terc G3 (Bruedigam et al. Cell Stem Cell 2014). We observed a similar reduction in bone marrow cellularity in Terc ko transplanted recipients (p = 0.003). Lineage skewing was less pronounced but CMP percentages were significantly reduced in Terc ko compared to wild-type conditions (p = 0.03). In contrast to primary mice, mobilization of progenitors to the spleen was not observed, suggesting that this finding was ameliorated by normal bone marrow and splenic supporting cells. The aged G3 Terc ko model faithfully recapitulates the haematopoietic phenotype of aging in humans and we propose to utilise this model to further our understanding of the mechanisms and physiological consequences of clonal hematopoiesis, to eventually help identify strategies to mitigate its clinical consequences.

Decline in Cardiolipin in Hematopoietic Stem Cell during Aging Alters Their Regenerative Potential

Hematopoietic Stem Cells (HSCs) are known for their regenerative potential. This property allowed the use of HSC in bone marrow transplantation to treat hematological disorders such as leukemia. However, aging influences these characteristics of HSCs. The major hallmarks of aging are limited self-renewal and reconstitution capacity resulting in HSC exhaustion in aged individuals. Mitochondrial metabolism and activity are active drivers of HSC fate decisions and data from our lab shows that mitochondria in aged HSCs have increased sphericity with a polarized mitochondrial network and a lower mitochondrial membrane potential. An important aspect of mitochondrial functions is the lipid composition of their membranes. A lipid trafficking assay showed an atypical pattern of lipid incorporation by mitochondria in aged HSCs suggesting that mitochondrial lipids become abnormal upon aging. The overall content of Cardiolipin (CL) which is a signature mitochondrial lipid, found exclusively in the inner mitochondrial membrane was also reduced during aging. This was done with flow cytometry using a CL marker NAO (Non-Acridine Orange). Tafazzin encoded by the gene TAZ is crucial for remodeling CL into its mature form which is required for maintaining mitochondrial functions. Mutations in the TAZ gene have been known to cause a reduction in the synthesis of mature CL which is often associated with HSCs functional defect. Western Blot analysis showed a considerable decrease in Taz protein expression in aged stem and progenitor cells (HSPC) compared to young HSPC. To understand the effect of reduced Taz expression on HSC functions, we used a doxycycline inducible, sh-RNA mediated TAZ knock-down (KD) mouse model. Bone marrow analysis indicates that the frequency of HSC and multipotent progenitors decreases significantly in 6-month-old TAZ KD mice compared to wild type (WT) mice. Our data shows that following 5FU induction TAZ KD mice have a significant reduction in the multipotent progenitor and HSC population on day 10 and day 21 post 5FU, indicating TAZ KD decrease the regenerative potential of HSCs under stress conditions. Further pointing towards the significance of cardiolipin in HSC regeneration. TAZ KD mice also have abnormal peripheral blood recovery post 5FU mainly affecting the platelets. We next examined the repopulation potential of HSCs isolated from TAZ KD mice using serial competitive transplantation assays. While primary transplanted mice of TAZ KD HSCs exhibited similar donor-cell chimerism compared to mice transplanted with WT HSCs, about 50-60%, secondary recipient of TAZ KD cells had significantly lower donor-cell chimerism than secondary recipients of WT cells. Further, donor-derived HSC frequency in the bone marrow of recipients of TAZ KD HSCs 24 weeks following engraftment was also significantly lower than recipients of WT HSCs. Hence, Taz-deficiency causes a defect in HSC self-renewal under regenerative conditions. We next evaluated whether restoring cardiolipin levels in mid-aged HSCs could rescue their functions. For this, mid-aged HSC were incubated for 3 days under conditions that maintain HSC functions in vitro with Alcar (Acetyl-L-Carnitine), which promotes CL production. In lipid incorporation assay, Alcar supplementation restored mitochondrial membrane potential, mitochondrial organization and lipid incorporation of mid-aged HSCs. In competitive transplant assay, while mid-aged WT HSC treated with vehicle gave rise to a myeloid-bias graft, Alcar supplemented mid-aged WT HSC gave rise to a more balanced graft with increased ratio lymphoid to myeloid cells in the peripheral blood 16 weeks following transplantation, albeit the rescue was partial. This study indicates that HSC mitochondrial membrane lipids become abnormal with aging which causes a decline in HSC function and highlights the critical role of mitochondrial cardiolipin in HSC functions. It implies that metabolite supplementation is a viable option for restoring age-related HSC functional defects.