Hematopoietic Stem Cell Aging Research Articles

3193 – EXPRESSION OF CD61 MARKS FUNCTIONALLY DISTINCT POPULATIONS OF AGED HEMATOPOIETIC STEM CELLS

Hematopoietic stem cells (HSCs) give rise to all hematopoietic cells throughout the lifespan of an organism. Upon ageing, murine HSCs gradually lose their self-renewal and regenerative potential, skew towards the myeloid lineage and expand in numbers. Also, the aged HSC pool becomes increasingly heterogeneous where some cells have remained ‘young-like'. Here we aimed to identify and isolate these ‘young-like' HSCs from aged mice to better understand the mechanisms that contribute to HSC ageing. We focused on CD61 (Integrin beta 3), a membrane-associated gene that we previously identified as one of the most consistently differentially expressed gene between young and old murine HSCs. CD61 plays a role in a variety of cellular processes, including cell adhesion and migration. We hypothesized that the level of CD61 expression on HSCs marks distinct populations of stem cells. We competitively transplanted 500 CD61High and 500 CD61Low young and aged long-term hematopoietic stem cells (LT-HSCs) alongside 2x106 competitor bone marrow cells into lethally irradiated recipients. Chimerism levels were analyzed by FACS every 4 weeks for 20 weeks. LT-HSCs from primary recipients were then competitively transplanted into secondary recipients to further investigate their functionality. In primary transplantation, young CD61High and CD61Low HSCs provided comparable engraftment. However, CD61High HSCs from aged donors resulted in, on average, 80% chimerism whereas the CD61Low HSCs provided only 20% chimerism. In contrast to primary transplantations, secondary transplantations with young CD61Low HSCs resulted in 20% lower chimerism compared to CD61High HSCs. Our data demonstrate that expression of CD61 becomes increasingly important as HSCs age. In addition, our data from secondary transplantation suggest that CD61 might mark intrinsically superior cells in the HSCs pool.

Exploring new molecules that regulate hematopoietic stem cells and early stages of lymphoid hematopoiesis: the functional significance of ESAM and SATB1

Hematopoietic stem cells (HSCs) possess multilineage differentiation capability, which sustains the production of blood and immune cells throughout life. However, the precise mechanisms by which HSCs initiate differentiation toward a particular lineage and the factors that attenuate their lymphopoietic potential with aging are yet to be elucidated. Our group has investigated this issue for over two decades. We initially developed a method for segregating early lymphoid progenitors from HSCs and identified two molecules: endothelial cell-selective adhesion molecule (ESAM), highly expressed in HSCs, and special AT-rich sequence binding protein 1 (SATB1), expressed in early lymphoid progenitors. ESAM marks HSCs across species, including humans. In addition to its significance in stress-induced hematopoiesis, ESAM is also useful in identifying features of human acute myeloid leukemia stem cells. Further, we determined the role of SATB1 in the early HSC differentiation processes toward the lymphoid lineage. Remarkably, SATB1 expression in HSCs significantly decreased with aging, whereas its exogenous induction in aged HSCs rejuvenated their lymphopoietic potential. Furthermore, SATB1-expressing HSCs demonstrated robust lymphopoietic and long-term reconstituting capability, whereas HSCs without SATB1 skewed toward the myeloid lineage. Thus, our continuing research has revealed the significance of ESAM and SATB1 in the fundamental biology of HSCs.

Ginsenoside Rg1 as a Potential Regulator of Hematopoietic Stem/Progenitor Cells.

Ginsenoside Rg1 (Rg1), a purified, active component of the root or stem of ginseng, exerts positive effects on mesenchymal stem cells (MSCs). Many recent studies have found that hematopoietic stem cells (HSCs), which can develop into hematopoietic progenitor cells (HPCs) and mature blood cells, are another class of heterogeneous adult stem cells that can be regulated by Rg1. Rg1 can affect HSC proliferation and migration, regulate HSC/HPC differentiation, and alleviate HSC aging, and these findings potentially provide new strategies to improve the HSC homing rate in HSC transplantation and for the treatment of graft-versus-host disease (GVHD) or other HSC/HPC dysplasia-induced diseases. In this review, we used bioinformatics methods, molecular docking verification, and a literature review to systematically explore the possible molecular pharmacological activities of Rg1 through which it regulates HSCs/HPCs.

DNA damage and aging on hematopoietic stem cells: Impact of oxidative stress in ApoE-/- mice.

The effects of aging on ROS production and DNA damage were assessed in hematopoietic stem cells (HSCs) from apolipoprotein E-deficient (ApoE-/-) mice (2-, 12- and 24-month-old), a traditional experimental model of atherogenic dyslipidemia. HSCs from aged ApoE-/- mice were associated with increased ROS levels, leading to loss quiescence, DNA damage, apoptosis and telomere shortening. The concurrence of lack of ApoE and aging result in exhaustion and senescence of HSCs accompanied by increased oxidative stress and inflammation. Therefore, our data open avenues to a better understanding of age-related changes and genetic factors, which may synergistically compromise the efficacy of aged HSC recovery and/or transplantation.

JAK2V617F Mutant Megakaryocytes Contribute to Hematopoietic Aging in a Murine Model of Myeloproliferative Neoplasm

IntroductionRecent studies implicated megakaryocytes (MKs) in regulating hematopoietic stem cell (HSC) function. Abnormal MK hyperplasia is a hallmark feature of myeloproliferative neoplasms (MPNs). In the present study, we investigated the effects of JAK2V617F-bearing MKs on HSC aging in a murine model of MPN during a 2-yr follow up.MethodsJAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) and Pf4-Cre mice (which express Cre under the promoter of platelet factor 4, a MK-specific gene) were crossed to generate MK lineage-specific human JAK2V617F transgenic mouse line (Pf4 +FF1 +).ResultsDuring aging, the Pf4 +FF1 +mice maintained an essential thrombocythemia phenotype with no evidence of transformation to leukemia or myelofibrosis (Figure 1).Myeloid-biased HSCs (Lin -cKit +Sca1 +CD150 +CD48 -CD41 +) were significantly expanded in 1yr old and 2yr old Pf4 +FF1 +mice compared to age-matched control mice, while no difference in myeloid-biased HSC numbers was detected between young (6mo) Pf4 +FF1 +mice and control mice. Competitive repopulation assays and serial transplantation assays showed that HSCs from old Pf4 +FF1 +mice had a reduced engraftment and self-renewal capacity with a skewed differentiation towards the myeloid lineage. Results from the serial transplantation experiments also indicated that the functional decline of HSCs in aged Pf4 +FF1 +mice were HSC-intrinsic and was not reversible. Both cell cycle analysis by Hoechst33342 and Pyronin Y staining and cell proliferation analysis by in vivo BrdU labeling revealed that the JAK2V617F mutant MK niche can promote hematopoietic aging in old Pf4 +FF1 +mice by increasing HSC proliferation/cycling. Taken together, the Pf4 +FF1 +mice demonstrated several hallmarks of accelerated HSC aging.(Figure 2)Next, we examined the hematopoietic microenvironment in the old Pf4 +FF1 + mice. Using whole-mount immunofluorescent staining and confocal imaging, we found that the cKit +HSPCs were located further from MKs in old Pf4 +FF1 +mice compared to old control mice. We also found that CD45 -CD31 +Sca1 - sinusoidal marrow endothelial cell (EC) number (by flow cytometry analysis) and marrow vascular area (by in vivo VE-cadherin staining) were significantly decreased in aged Pf4 +FF1 +mice compared to control mice. Tube formation assays confirmed that conditioned medium from old Pf4 +FF1 +MK culture significantly inhibited EC angiogenesis in vitro. These findings suggest that the JAK2V617F mutant MK niche not only altered its own interaction with HSCs during aging, but also suppressed the vascular niche function to promote HSC aging. (Figure 3)To further understand the mechanisms by which JAK2V617F mutant MKs promote HSC aging, we performed both RNA sequencing and targeted cytokine arrays of wild-type and JAK2V617F mutant MKs from both young (6mo) and old (2yr) Pf4-cre control and Pf4 +FF1 +mice. We found that HSC aging had a profound effect on MK transcriptomic profiles and dysregulated pathways in cell adhesion molecules, MAPK signaling, NF-kappa B signaling, hematopoietic cell lineage, and cytokine-cytokine receptor interaction were highly upregulated in old JAK2V617F mutant MKs compared to young JAK2V617F mutant MKs. FAS ligand, IL-12, tissue inhibitor of metalloproteinases-1, IL-10, IL-6, MIG, macrophage inflammatory protein 1a, GCSF, IL-5, MIP-1g were produced in increased amounts in old mutant MKs compared to old wild-type MKs. Many of these factors are involved in inflammation, angiogenesis, extracellular matrix remodeling, and hematopoiesis regulation.Sensitive PCR assays confirmed that human JAK2 gene was expressed in MKs but not in HSCs in 2yr old Pf4 +FF1 +mice. In vitro co-culture and in vivo co-transplantation assays provided further evidence that the JAK2V617F mutant MKs affected wild-type HSC function directly. Therefore, the hematopoietic aging phenotype we have observed was not caused by any direct effect of the JAK2V617F mutation on HSC function because the Pf4 promoter was ‘leaky’.ConclusionsResults from this study support that, as a hematopoietic niche cell, MKs represent an important connection between the extrinsic and intrinsic mechanisms for HSC aging in MPNs – the JAK2V617F-bearing MKs can alter the hematopoietic niche to accelerate HSC aging, and HSC aging in turn can profoundly remodel the niche e.g. by affecting MK transcriptomics. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Age-Related Changes in Human HSC Methylomes Affect Gene Regulatory Elements Critical to HSC Function and Reveal Novel HSC Aging Candidate Genes

Age-Related Changes in Human HSC Methylomes Affect Gene Regulatory Elements Critical to HSC Function and Reveal Novel HSC Aging Candidate Genes

Unraveling Heterogeneity of Aged Hematopoietic Stem Cells By Single-Cell RNA Sequence Analysis

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes deregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old) and aged (20-month-old) mice to gain insight into the dynamics of HSC aging.scRNA-seq analysis revealed three major clusters (A, B, C) and three minor clusters (D, E, F) in young HSCs. Of interest, aged HSCs also showed similar cluster formation. One of the minor clusters was characterized by gene signature associated with inflammatory response and significantly increased with age, while the remaining two minor clusters, which showed cell cycle gene signature, did not change in proportion. One of the major clusters, Cluster C, which showed the strongest expression of HSC-specific gene sets compared with other clusters, moderately increased with age.Our scRNA-seq analysis confirmed upregulation of age-related genes previously reported, such as Selp, Mt1, and Vwf, in a considerable portion of aged HSCs. von Willebrand factor (Vwf) encodes a blood glycoprotein produced by megakaryocytes and endothelial cells. Several groups have reported that Vwf-expressing HSCs show myeloid/platelet-biased differentiation (Joana C et al., Nature 2013; Sandra P et al., Developmental Cell 2018). Importantly, our scRNA-seq data identified that Clusterin (Clu) is rarely expressed in young HSCs and is dramatically upregulated in aged HSCs. Clu expression was predominantly increased in one of the major clusters, Cluster C. Clusterin encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis. We hypothesized that Clu would be useful as a marker of a unique subpopulation of aged HSCs, and thus conducted further analysis by using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory sequences of the BAC gene.Clu/GFP was preferentially expressed in HSCs and at lower frequencies in MPP1 than HSCs in Lineage -Sca-1 +c-Kit + (LSK) cell fraction. Clu-positive HSCs expressed high levels of CD150 and were detected in 10% and 60% of HSCs in 10-week-old young mice and 8-month-old middle-aged mice, respectively, indicating that Clu-positive HSCs increase with aging. We next assessed the function of Clu-positive HSCs in young mice. Clu-positive young HSCs established significantly lower chimerism than Clu-negative young HSCs and preferentially differentiated into myeloid cells in competitive transplantation assays. RNA-seq analysis of Clu-positive and Clu-negative HSCs from young mice confirmed that Clu-positive HSCs show the gene signature of myeloid-biased HSCs. Characterization of Clu-positive and negative subpopulations in aged HSCs is currently underway. These results suggest that Clu-positive HSCs represent myeloid-biased HSCs which expand with aging, thus Clu expression serves as a novel marker to monitor the alterations in HSC heterogeneity with aging. DisclosuresIwama: Nissan Chemical Corporation: Research Funding.

Microbiota Signals Suppress the Lymphoid Specification of Hematopoietic Stem Cells

Aging is associated with an expansion of phenotypic hematopoietic stem cells (HSCs) with reduced self-renewal capacity and myeloid-skewed lineage differentiation. Signals from commensal flora support basal myelopoiesis in young mice; however, their contribution to hematopoietic aging is largely unknown. Here, we characterize hematopoiesis in young and middle-aged mice housed under specific pathogen free (SPF) and germ-free (GF) conditions. We did not analyze older mice due to the difficulty in maintaining mice in a gnotobiotic facility for more than one year. Consistent with prior studies, there is a shift in hematopoiesis in aged SPF mice towards granulopoiesis, with a significant increase in the percentage of granulocytic cells and a decrease in B lineage cells in the bone marrow. The marked shift from lymphopoiesis to myelopoiesis that develops during aging of SPF mice is mostly abrogated in GF mice. Compared with aged SFP mice, there is a marked expansion of B lymphopoiesis in aged GF mice, which is evident at the earliest stages of B cell development.To investigate the impact of microbiota signals on multipotent HSPCs, we first quantified HSPCs by flow cytometry (Figure 1A-B). In aged SPF mice, the number of lineage - Sca1 + cKit + CD150 + CD48 - (LSK-SLAM) cells and CD34 - LSK-SLAM cells is increased 6.4 ± 1.7-fold and 3.4 ± 1.2-fold, respectively. Similar increases were observed in aged GF mice, with LSK-SLAM increasing 5.3 ± 1.6-fold (p=NS compared to SPF mice) and CD34 - LSK-SLAM cells increasing 2.8 ± 0.31-fold (p=NS). To quantify functional HSCs, limiting dilution transplantation experiments using unsorted bone marrow cells was performed. Although on a per cell basis the repopulating activity of aged HSCs is reduced, due to the large increase in phenotypic HSCs, the number of functional HSCs actually increases with aging, with similar increases in functional HSCs in aged SPF and GF mice (Figure 1C). Finally, to assess lineage-bias, we transplanted a limiting number of sorted HSCs and assessed lineage output. As expected, in young SPF mice, the majority of HSCs displayed a balanced myeloid/lymphoid lineage output, with a significant increase in myeloid-biased HSCs observed with aging (Figure 1D). In young GF mice, the majority of HSCs are lymphoid-biased. Moreover, although the myeloid output increased modestly with aging, the majority of HSCs in aged GF remained lymphoid-biased or balanced. Consistent with these data, RNA expression profiling of phenotypic HSCs from aged GF mice show enrichment for non-myeloid biased HSCs. Surprisingly, the RNA expression profiling data also suggest that inflammatory signaling is increased in aged GF HSCs compared with aged SPF HSCs. Collectively, these data suggest that microbiota-related signals suppress the lymphoid potential of HSCs, contributing to the expansion of myeloid-biased HSCs that occurs with aging. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cytoplasmic Labile Iron Accumulates in Aging Stem Cells Perturbing a Key Rheostat for Identity Control

Bone marrow resident and rarely dividing hematopoietic stem cells (HSC) harbor an extensive self-renewal capacity to sustain life-long blood formation, albeit their function declines during ageing. Various molecular mechanisms confer stem cell identity, ensure long-term maintenance and are known to be deregulated in aged stem cells. How these programs are coordinated, particularly during cell division, and what triggers their ageing-associated dysfunction has been unknown.We have previously uncovered that iron chelator exposure increases the number of functional HSC ex vivo and in vivo (Kao et al., Science Transl Med 2018). While ensuring a sufficient amount of redox active, readily available iron which is required in numerous electron transfer reactions governing fundamental cellular processes, cells tightly regulate the size of the intracellular labile iron pool (LIP) to limit adverse ROS generation. Perturbations in the ability to limit intracellular iron is detrimental for cells and known to compromise HSC maintenance and function via altered redox signaling and increased macromolecule oxidation and damage. The HSC stimulatory effects of iron chelator (IC) treatment and the well characterized central roles of redox active intracellular iron in sustaining basic cell function prompted us to examine a potential regulatory role of the LIP in controlling somatic stem cell function.In this study, we quantified LIP in young and aged HSC and monitored iron homoeostasis pathway activation, hallmarked by the stabilization of transferrin receptor (Tfrc) mRNA, in stem cells for which we developed a single molecule RNA fluorescence in situ hybridization (smRNA FISH) assay enabling the quantification of Tfrc dynamics with unparalleled resolution and sensitivity. We have further used experimental LIP modulation in primary hematopoietic stem cell models to characterize the consequences of iron homeostasis pathway activation in young and aged stem cells; and employed integrated comparative quantitative transcriptomics (single cell RNA-seq) and proteomics along with genetic and pharmacological rescue models to identify the consequences and mechanisms of LIP size alterations. Our findings demonstrate that HSC, containing the lowest amount of cytoplasmic chelatable iron hematopoietic cells, activate a limited iron response during mitosis. Engagement of this iron homeostasis pathway elicits mobilization and β-oxidation of arachidonic acid and enhances stem cell-defining transcriptional programs governed by histone acetyl transferase Tip60/KAT5. We further find an age-associated expansion of the labile iron pool, along with loss of Tip60/KAT5-dependent gene regulation to contribute to the functional decline of ageing HSC, which can be mitigated by iron chelation.Together, our work reveals cytoplasmic redox active iron as a novel rheostat in adult stem cells; it demonstrates a role for the intracellular labile iron pool in coordinating a cascade of molecular events which reinforces HSC identity during cell division and to drive stem cell ageing when perturbed. As loss of iron homeostasis is commonly observed in the elderly, we anticipate these findings to trigger further studies into understanding and therapeutic mitigation of labile iron pool-dependent hematopoietic stem cell dysfunction in a wide range of degenerative and malignant hematologic pathologies. DisclosuresD'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

The Transcriptional and Epigenetic Reprogramming of Aged Hematopoietic Stem Cells Drives Myeloid Rewiring in Clonal Hematopoiesis-Associated Cytopenias

Patients (pts) with myelodysplastic syndromes (MDS) have few therapy options. Interventions to improve outcomes should consider strategies that arrest MDS in its early phases, when symptoms are minimal and prolonged survival is expected.To develop prevention strategies that arrest MDS before the disease outcomes become irreversibly dismal, we dissected the molecular and biological mechanisms that maintain MDS in one of its premalignant phases, clonal cytopenia of undetermined significance (CCUS). Recognizing that CCUS is an aging-related disease, we first sought to determine, at the single-cell level, how CCUS affects the transcriptional and epigenetic profile of the aging hematopoietic stem and progenitor cell (HSPC) compartment and overcomes aging-induced degenerative phenotypes. We performed single-cell RNA sequencing (scRNA-seq) analysis of Lin -CD34 + HSPCs isolated from the bone marrow (BM) of 3 young healthy donors (yHDs), 4 elderly HDs (eHDs), and 3 elderly pts with CCUS carrying mutations in common MDS driver genes.We found that the frequencies of hematopoietic stem cells (HSCs) and megakaryocytic (Mk)/erythroid (Er) progenitors were increased at the expense of myeloid (My) progenitors in eHDs as compared with yHDs (Fig. a). In contrast, CCUS pts had a predominant My-biased HSPC distribution (Fig. a). However, immunophenotypic quantification in large cohorts of eHDs and CCUS pts revealed that CCUS pts' BM had significantly fewer CD34 +CD38 - HSC populations and CD34 +CD38 + My progenitors, suggesting that My bias in CCUS results from the aberrant My differentiation of HSCs rather than My cell expansion. Further differential expression analysis among the scRNA-seq clusters showed that, compared with yHD HSCs, eHD HSCs were characterized by a significant upregulation of genes involved in the TNFα-induced activation of NF-κB (e.g., MCL1; Fig. b), which is consistent with previous findings that aged HSCs undergo transcriptional reprogramming to maintain their survival in response to changes in the systemic environment (He et al. Blood 2020). In contrast, CCUS HSCs, compared with eHD HSCs, overexpressed regulators of translation, respiratory electron transport, and mitochondrial translation initiation (Fig. c), which underscores these cells' state of proliferation and metabolic activation and their ability to overcome aging-induced phenotypic alterations.To evaluate whether the aberrant lineage differentiation in eHD and CCUS HSPCs arose from the altered fate determination of HSCs, we performed single-cell assays for transposase-accessible chromatin sequencing to profile chromatin accessibility in sorted HSCs or Lin -CD34 + HSPCs from yHDs, eHDs, and CCUS pts. Consistent with our transcriptomic data, compared with yHD HSCs, eHD HSCs were mostly poised for Mk differentiation, whereas CCUS HSPCs were poised for lymphoid/My differentiation. Indeed, eHD HSCs had an increased activity of transcriptional factors belonging to the NF-κB family and open peaks at the distal elements of genes involved in hemostasis (Fig. d). In contrast, CCUS HSCs were poised to downregulate the expression of genes involved in NF-κB signaling and Mk/Er differentiation (Fig. e).These results suggested that CCUS HSCs are highly metabolically active to maintain My differentiation. Indeed, metabolomic analyses confirmed that intermediates of oxidative phosphorylation were significantly upregulated in CCUS CD34 + cells as compared with eHD CD34 + cells (Fig. f). Further, scRNA-seq analysis of mononuclear cells isolated from the BM of 3 CCUS and 3 eHD samples revealed the widespread upregulation of genes involved in protein processing and mitochondrial metabolism. This analysis also revealed impaired terminal My differentiation despite the HSPC My bias, with decreased frequencies of monocytic cells, and an intriguing expansion of cytotoxic cell subsets in the BM of CCUS pts.In conclusion, our results demonstrate that CCUS HSCs carrying MDS driver mutations evade aging-induced phenotypic degeneration, become metabolically active, and have aberrant My skewing. Our study clarifies the molecular mechanisms underlying MDS initiation and offers an opportunity for early therapeutic intervention. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A FLI1-KLF6 Axis Regulates Aging in Human Hematopoietic Stem and Progenitor Cells and Normalization of KLF6 Levels in Aged Cells Leads to Their Rejuvenation

Aging causes a gradual decline in hematopoietic stem cell (HSC) function, which increases the risk for hematological malignancies. While much has been done in murine models, human HSC aging impairment is less understood. We recently showed that Krüppel-like transcription factor 6 (KLF6) is among the top downregulated genes during human HSC aging, which correlates with H3K27ac loss at several upstream putative enhancers. Moreover, loss of KLF6 in human CD34 + cells resulted in impaired in vitro differentiation, increased colony-forming potential and a transcriptional profile similar to that of aged CD34 + CD38 - cells. We hypothesized that age-acquired deregulation of KLF6 may be a key player in age-related HSC dysfunction and sought to fully characterize this. Thus, we isolated CD34 + cells from young (<32 y.o) and aged (>65 y.o.) healthy donors and performed CRISPR-Cas9 genome editing and transcriptional activation of KLF6, respectively, followed by epigenetic and transcriptional reprogramming, in vivo hematopoietic reconstitution, and analysis of DNA damage, apoptosis, and reactive oxygen species (ROS) levels.KLF6 knock-out (KO) and non-targeting control (NTC) cells from young healthy donors were engrafted into immunodeficient NSGS mice. Hematopoietic reconstitution analysis showed that KLF6 KO cells led to increased myeloid and reduced lymphoid reconstitution in peripheral blood (PB; p<1.62 -7) and an increase in immunophenotypically defined HSC and CD34 + CD38 - progenitor fractions in the bone marrow (BM; p=0.02, and p=0.04, respectively). H3K27ac analysis of KLF6 KO cells revealed a loss of 3,390 ChIP-seq peaks (FDR < 0.05) and 285 peaks gained. Functional annotation using ChIP-Enrich showed that H3K27ac loss associates with myeloid homeostasis, erythroid differentiation and oxidative stress (FDR < 0.05). Three putative enhancer (E) regions upstream of the KLF6 locus showed loss of H3K27ac with aging. Depletion of the E1 but not E2 or E3 regions phenocopied in vitro and in vivo findings of KLF6 KO. Transcription factor (TF) ChIP-seq data analysis revealed FLI1, ERG, and RUNX1 binding overlapping the E1 region. Knockdown of FLI1 but not ERG or RUNX1 led to an increase in KLF6. Notably, FLI1 mRNA levels, but not ERG or RUNX1, are increased during normal aging.We next performed in vitro KLF6 activation in aged CD34 + (KLF6a) cells using a dCas9-VP64 system to test if we could rejuvenate these cells. KLF6a cells exhibited a decrease in their in vitro myeloid differentiation potential, compared to aged NTC CD34 + cells (p<0.0041), and behaved instead similar to young controls. ChIP-seq analysis of KLF6a showed marked decrease of H3K4me1 (n=3,273 peaks) with relatively few regions with increased H3K4me1 (n=602) (FDR < 0.05). In contrast, we observed an increase in H3K27ac (n=3,361 peaks) with only 71 peaks lost compared to aged NTC (FDR < 0.05). Regions that gained H3K27ac in KLF6a were associated with platelet activation, cell junction and adhesion. In vivo analysis of KLF6a cells injected into NSGS mice revealed a significant reduction in the PB myeloid fraction compared to NTC (p<1.2-8), with a concomitant expansion in the lymphoid compartment (p<4.4 -11). BM composition analysis at week 16 showed a decrease in the HSC fraction in KLF6a cells (p=0.0029) as well as a reduction in CD34 +CD38 -, CD34 +CD38 + and MEPs (p=0.036, p<0.0001 and p=0.041, respectively). We next examined the impact of KLF6 modulation on DNA damage and observed that young human KLF6 KO cells had a significant increase in gH2AX and 53BP1 (p<0.0001, for both) whereas KLF6a in aged CD34 + cells exhibited reduced gH2AX and 53BP1 foci in comparison to aged NTC (p<0.0001, for both). In addition, apoptotic levels in KLF6 KO cells were higher than in NTC cells (p=0.006) whereas aged KLF6a cells showed a reduction in the incidence of apoptotic cells compared to NTC (p=0.019). Finally, ROS analysis in young KLF6 KO showed increased levels of total and mitochondrial ROS compared to NTC (p=0.0008 and p<0.0001, respectively) whereas both ROS fractions were reduced in KLF6a cells (p=0.0002 and p<0.0001, respectively).In summary, these results show that the FLI1-KLF6 axis plays a key role in regulating HSPC aging and that KLF6 is required for normal HSPC function and differentiation. In addition, normalization of KLF6 levels in aged HSPCs resulted in reprogramming and rejuvenation HSPCs, confirming the central role of this TF in aging HSPC biology. DisclosuresNo relevant conflicts of interest to declare.

Age-Acquired Downregulation of Lmna Leads to Epigenetic Deregulation and Altered HSPC Function

Hematopoietic stem cells (HSCs) exhibit epigenetic reprogramming and decline in function with aging, and these changes may be predisposing mechanisms for development of clonal hematopoiesis and myeloid malignancies. Our recent study characterizing the epigenetic and transcriptional landscape of young (18-30 years) and aged (65-75 years) human CD34 +CD38 - bone marrow (BM) cells identified Lamin-A/C (LMNA) as one of the most downregulated genes with aging (7.9-fold, p=1.9x10 -13). LMNA encodes the nuclear lamina protein Lamin-A/C and is mutated in the premature aging disorder Hutchinson-Gilford Progeria Syndrome.To determine whether downregulation of LMNA contributes to the phenotype of human HSC aging, we knocked down LMNA (LMNA KD) using shRNA in young, mobilized peripheral blood CD34 + cells. With an average knockdown of 60%, we observed ~33% increase in myeloid colony forming potential (p<0.05). LMNA KD also impaired differentiation in liquid culture as determined by persistence of CD34 expression in twice as many cells as controls (p<0.0001) while induction of myeloid CD11b expression was reduced by ~29% (p<0.05), as well as a trend to reduced erythroid (CD71 +GYPA +) differentiation. To investigate the effects of Lmna loss-of-function in vivo, we conditionally deleted Lmna in mice using Vav-Cre. Loss of Lmna (Lmna KO) had no effect on steady state hematopoiesis in young (10 weeks) or middle-aged (14 months) knockout mice. However, Lmna KO BM cells from middle-aged mice generated 2-fold more total colonies than wild-type, floxed controls (WT=126 vs KO=292, p<0.01).Given that nuclear architecture provides a layer of epigenetic regulation through chromatin-lamina interactions, we investigated how the epigenome changes in the context of LMNA deficiency. By super-resolution microscopy, we observed loss of LMNA localization from the nuclear periphery in LMNA KD human CD34 +cells (p<0.05). Since lamina-associated domains generally contain regions of repressed chromatin, we stained for histone H3K9me2 and observed that LMNA KD cells displayed increased scattering throughout the nucleus without a change in its density. Next, we performed ATAC-seq and identified 603 open chromatin sites showing changes in accessibility (FDR<0.1). Over 73% of these sites show reduced accessibility and were associated with genes showing reduced expression by RNA-seq. Gene ontology analysis of these sites identified genes associated with heme biosynthetic and cell differentiation processes (FDR<0.005). Moreover, gene set enrichment analysis of the RNA-seq confirmed that downregulated genes were enriched for genes downregulated in our previously reported CD34 + aging signature (NES=-1.46, FDR=0.001). To determine whether LMNA KD alters the epigenome, we performed ChIP-seq for active (H3K4me3, H3K27ac) and repressive (H3K9me2) histone marks. LMNA KD resulted in significant reduction of 36% of H3K27ac peaks, which mimics our previous observation of marked age-related losses of H3K27ac. Most differential peaks localized to gene promoters (50%) and introns (34%) and include genes involved in nuclear pore organization and gene silencing (FDR<0.001). Notably, for peaks located at intergenic regions, >35% overlapped with active enhancers we reported as lost with aging and were associated with genes involved in regulation of p38 MAPK cascade (enrichment=2.34E -5), an important signaling pathway regulating proliferation and differentiation of HSCs and leukemic cells.Taken together, LMNA deficiency recapitulates features of aging at the functional and epigenetic level. LMNA KD in young, human CD34 + cells impaired their differentiation while increasing their colony forming potential. Similarly in middle-aged mice, LMNA KO BM cells showed increased colony forming potential. Genomic changes induced by LMNA deficiency include reduced accessibility and gene transcription, accompanied by changes in localization and occupancy of histone H3K9me2 and H3K27ac, respectively. These epigenetic changes affect genes regulating differentiation and signaling pathways. Thus, we have demonstrated that in addition to its structural role, LMNA also contributes to chromatin regulation of hematopoietic pathways important for normal CD34 + function. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Stem Cell Regeneration through Paracrine Regulation of the Wnt5a-Prox1 Signaling Axis

The crosstalk between bone marrow (BM) microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration after injury. Here we show that deletion of the genes encoding the DNA repair-deficient syndrome Fanconi anemia (FA), Fanca and Fancc, in mice dampens HSC regeneration through both direct effects on HSCs and indirect effects on BM niche cells. Specifically, Fanca- or Fancc-deficiency compromises hematologic recovery and dampens HSC regeneration following irradiation. FA HSCs show persistent upregulation of the Wnt target Prox1, a homeobox transcription factor, in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improves long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in wild-type (WT) HSC mimics the defective repopulation phenotype of FA HSCs. By analyzing paracrine factors in Wnt signaling, we found that WT mice, but not FA mice, show significant induction by TBI of BM stromal Wnt5a protein, which is produced in LepR +CXCL12 + BM stromal cells. Wnt5a treatment of irradiated FA mice enhances hematopoietic recovery and HSC regeneration. Conversely, Wnt5a neutralization in co-cultured LepR + BM stromal cells inhibits HSC regeneration and hematopoietic recovery following TBI. Mechanistically, Wnt5a secreted by LepR +CXCL12 + BM stromal cells inhibits b-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSPCs. The detrimental effect of deregulated Wnt5a-Prox1 signaling on HSC regeneration and hematopoietic recovery is also observed in aged mice. Irradiation induces upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improves HSC regeneration and hematopoietic recovery after irradiation. Finally, treatment of aged mice with Wnt5a enhances hematopoietic repopulation. Collectively, these findings identify the novel paracrine Wnt5a-Prox1 signaling axis in regulating HSC regeneration under conditions of injury and aging. DisclosuresNo relevant conflicts of interest to declare.

Integrated Single-Cell Bioinformatics Analysis Reveals Intrinsic and Extrinsic Biological Characteristics of Hematopoietic Stem Cell Aging.

Hematopoietic stem cell (HSC) aging, which is accompanied by loss of self-renewal capacity, myeloid-biased differentiation and increased risks of hematopoietic malignancies, is an important focus in stem cell research. However, the mechanisms underlying HSC aging have not been fully elucidated. In the present study, we integrated 3 independent single-cell transcriptome datasets of HSCs together and identified Stat3 and Ifngr1 as two markers of apoptosis-biased and inflammatory aged HSCs. Besides, common differentially expressed genes (DEGs) between young and aged HSCs were identified and further validated by quantitative RT-PCR. Functional enrichment analysis revealed that these DEGs were predominantly involved in the cell cycle and the tumor necrosis factor (TNF) signaling pathway. We further found that the Skp2-induced signaling pathway (Skp2→Cip1→CycA/CDK2→DP-1) contributed to a rapid transition through G1 phase in aged HSCs. In addition, analysis of the extrinsic alterations on HSC aging revealed the increased expression levels of inflammatory genes in bone marrow microenvironment. Colony formation unit assays showed that inflammatory cytokines promoted cellular senescence and that blockade of inflammatory pathway markedly rejuvenated aged HSC functions and increased B cell output. Collectively, our study elucidated the biological characteristics of HSC aging, and the genes and pathways we identified could be potential biomarkers and targets for the identification and rejuvenation of aged HSCs.

Microbiota Signals Suppress B Lymphopoiesis With Aging in Mice.

Aging is associated with significant changes in hematopoiesis that include a shift from lymphopoiesis to myelopoiesis and an expansion of phenotypic hematopoietic stem cells (HSCs) with impaired self-renewal capacity and myeloid-skewed lineage differentiation. Signals from commensal flora support basal myelopoiesis in young mice; however, their contribution to hematopoietic aging is largely unknown. Here, we characterize hematopoiesis in young and middle-aged mice housed under specific pathogen free (SPF) and germ-free (GF) conditions. The marked shift from lymphopoiesis to myelopoiesis that develops during aging of SPF mice is mostly abrogated in GF mice. Compared with aged SPF mice, there is a marked expansion of B lymphopoiesis in aged GF mice, which is evident at the earliest stages of B cell development. The expansion of phenotypic and functional HSCs that occurs with aging is similar in SPF and GF mice. However, HSCs from young GF mice have increased lymphoid lineage output, and the aging-associated expansion of myeloid-biased HSCs is significantly attenuated in GF mice. Consistent with these data, RNA expression profiling of phenotypic HSCs from aged GF mice show enrichment for non-myeloid biased HSCs. Surprisingly, the RNA expression profiling data also suggest that inflammatory signaling is increased in aged GF HSCs compared with aged SPF HSCs. Collectively, these data suggest that microbiota-related signals suppress B lymphopoiesis at multiple stages of development and contribute to the expansion of myeloid-biased HSCs that occurs with aging.

Primary myelofibrosis in untreated sickle cell disease: Are adult patients at higher risk for developing hematological myeloid neoplasms?

Primary myelofibrosis in untreated sickle cell disease: Are adult patients at higher risk for developing hematological myeloid neoplasms?

Cdc42-Borg4-Septin7 axis regulates HSC polarity and function

Aging of hematopoietic stem cells (HSCs) is caused by the elevated activity of the small RhoGTPase Cdc42 and an apolar distribution of proteins. Mechanisms by which Cdc42 activity controls polarity of HSCs are not known. Binder of RhoGTPases proteins (Borgs) are known effector proteins of Cdc42 that are able to regulate the cytoskeletal Septin network. Here, we show that Cdc42 interacts with Borg4, which in turn interacts with Septin7 to regulate the polar distribution of Cdc42, Borg4, and Septin7 within HSCs. Genetic deletion of either Borg4 or Septin7 results in a reduced frequency of HSCs polar for Cdc42 or Borg4 or Septin7, a reduced engraftment potential and decreased lymphoid-primed multipotent progenitor (LMPP) frequency in the bone marrow. Taken together, our data identify a Cdc42-Borg4-Septin7 axis essential for the maintenance of polarity within HSCs and for HSC function and provide a rationale for further investigating the role of Borgs and Septins in the regulation of compartmentalization within stem cells.

Mitochondrial Contributions to Hematopoietic Stem Cell Aging.

Mitochondrial dysfunction and stem cell exhaustion are two hallmarks of aging. In the hematopoietic system, aging is linked to imbalanced immune response and reduced regenerative capacity in hematopoietic stem cells (HSCs), as well as an increased predisposition to a spectrum of diseases, including myelodysplastic syndrome and acute myeloid leukemia. Myeloid-biased differentiation and loss of polarity are distinct features of aged HSCs, which generally exhibit enhanced mitochondrial oxidative phosphorylation and increased production of reactive oxygen species (ROS), suggesting a direct role for mitochondria in the degenerative process. Here, we provide an overview of current knowledge of the mitochondrial mechanisms that contribute to age-related phenotypes in HSCs. These include mitochondrial ROS production, alteration/activation of mitochondrial metabolism, the quality control pathway of mitochondria, and inflammation. Greater understanding of the key machineries of HSC aging will allow us to identify new therapeutic targets for preventing, delaying, or even reversing aspects of this process.

Epigenetic memories inscribed in chromatin accessibility in aged hematopoietic stem cells

Epigenetic memories inscribed in chromatin accessibility in aged hematopoietic stem cells

Angelica Sinensis Polysaccharide Mediates Sirtl to Inhibit P53/P21 Pathway and Delay Hematopoietic Stem Cell Aging

To study the effect of Angelica sinensis polysaccharide (ASP) on the expression of Sirt1/p53/p21 in radiation-induced aging hematopoietic stem cells (HSCs) of mice, and to explore the possible mechanism of ASP regulating the aging of HSCs. Methods: C57BL/6J mice were randomly divided into control group, model group and ASP group. The model group was irradiated with X-ray 3.0Gy/8F to establish the aging model of HSCs in mice. ASP group was given ASP intragastric administration during irradiation; The control group and the model group were given equal volume of normal saline. HSCs were sorted by immunomagnetic beads, and cell cycle was detected by flow cytometry The aging cells were observed by α-galactosidase staining. The directional differentiation ability of HSCs was observed by mixed colony culture (CFU-Mix). The expression of Sirtl, p53 and p21 protein was detected by Western blot. Results: Compared with the control group. X-ray can significantly increase the proportion of HSCs Gi cells, the rate of SA-α-Gal positive cells and the expression of p53 and p21 protein in aging group (P&lt;0.05).The expression of Sirtl and mixed colony forming ability were decreased (P&lt;0.05). Compared with the model group, ASP inhibited the increase of aging HSCs Gicell ratio, SA-fJ-Gal positive cell rate and p53 and p21 protein expression (P&lt;0.05). At the same time, Sirtl expression and mixed colony forming ability were increased (P&lt;0.05). conclusion: ASP may inhibit p53/p21 pathway and delay the aging of mouse HSCs by regulating Sirtl.