Young Hematopoietic Stem Cells Research Articles

Age-Associated Decrease of the Histone Methyltransferase SUV39H1 in HSC Perturbs Heterochromatin and B Lymphoid Differentiation.

SummaryThe capacity of hematopoietic stem cells (HSC) to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA miR-125b, a known regulator of HSC function, and that expression of miR-125b increases with age in human HSC. Overexpression of miR-125b and inhibition of SUV39H1 in young HSC induced loss of B cell potential. Conversely, both inhibition of miR-125 and enforced expression of SUV39H1 improved the capacity of HSC from elderly individuals to generate B cells. Our findings highlight the importance of heterochromatin regulation in HSC aging and B lymphopoiesis.

FEATURES OF SUBPOPULATION COMPOSITION OF BLOOD LYMPHOCYTES IN RECIPIENTS WITHIN THE FIRST MONTH AFTER LIVER TRANSPLANTATION

Ratios of subpopulations of mononuclear blood cells in 68 patients were registered by method of flow cytometry during one month before or after transplantation of a cadaveric liver with the aim of a comparative assessment of the contribution of cellular factors into graft acceptance. Results. The obtained data allow considering the changes of separate Treg subpopulation after transplantation as a partial outcome of generalized shift of the whole process of lymphocytes differentiation, involving hematopoietic stem cells and prelymphocytes. This shift is followed by an increase of young hematopoietic stem cells, precursors of lymphocytes, angiogenic cells, and by a concomitant reduction of the majority of more matured subpopulations of lymphocytes. Conclusion . Diminishment of «tolerogenic» liver activity of before transplantation and its restoration after the organ’s replacement is explained by morphogenic/trophic mechanism. The basis of this mechanism is an increase in blood of hematopoietic stem cells and other cells transferring angiogenic and regenerative information to the graft.

Mitigation of a Newly Discovered Phenomenon, Extra Physiologic Oxygen Shock/Stress (EPHOSS), Mediated By the Mitochondria Permeability Transition Pore, Greatly Improves Stem Cell Collection and Transplantation

Hematopoietic stem cells (HSCs) reside in hypoxic niches within the bone marrow (BM). Yet, all HSC studies have been performed to date with cells immediately isolated in non-physiologic ambient air, whether or not they are subsequently processed in low oxygen tension. By collecting/manipulating BM in physiologically native conditions of hypoxia where all procedures are performed inside a hypoxic chamber, we demonstrate that brief exposure of mouse BM or human cord blood (CB) to ambient oxygen decreases recovery of phenotypically-defined and functional self-renewing long-term repopulating HSC and concomitantly increases numbers of progenitor cells, a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS). This new phenomenon is exquisitely sensitive to oxygen and great care must be taken to ensure all reagents, solutions, plastics, and anything that will come into contact with the cell suspension, is extensively pre-equilibrated in hypoxia. Up to 5-fold greater numbers of long-term (LT)-HSCs (CD34-CD150+Lin-Sca1+c-kit+CD41-CD48- or CD34-CD135-Lin-Sca1+c-kit+) could be recovered from mouse BM harvested in 3% O2 compared to BM harvested in air, or even BM harvested in 3% O2 and then exposed to air for as little as 30 min before analysis, even if subsequently returned to hypoxia. There was a concomitant decrease in short term-HSCs and multipotent progenitors when BM was harvested in hypoxia, an effect associated with decreased functional cytokine-stimulated colony formation of hematopoietic progenitor cells (HPC: CFU-GM, BFU-E, and CFU-GEMM). Similarly, if human CB was harvested under similar low oxygen conditions, a 3-fold increase in recovered HSCs (Lin-CD34+CD38-CD45RA+CD90+CD49f+) could be achieved compared to CB harvested in air. Using a custom mouse respirator to conduct competitive repopulating transplant experiments completely in a 3% O2 environment revealed an increase in competitive repopulating units (CRUs) up to more than 42 fold was recovered when BM is harvested in hypoxic conditions compared to air harvested BM in primary recipients, thus demonstrating the beneficial effects of hypoxic harvest on functional/transplantable HSCs with secondary transplant capability. These data strongly support the surprising conclusion that, until now, the true numbers of HSCs and the transplantation potency of BM and CB has been routinely and consistently underestimated because of rapid initiation of differentiation of LT-HSCs in ambient air. We present evidence linking mitochondrial function and cyclophilin D to EPHOSS. Genetically or pharmacologically suppressing cyclophilin D function, or p53 gene deletion link production of reactive oxygen species (ROS) to induction of the mitochondrial permeability transition pore (MPTP) as a molecular mechanism of EPHOSS, where rapid ROS generation in HSCs after exposure to “hyperoxic” room air initiates an irreversible cascade of differentiation signals (see illustration). We present additional evidence from gene knock-out model studies implicating roles for miR210 and Hif-1a in EPHOSS. The MPTP inhibitor, cyclosporine A, protects phenotypically-defined as well as functional and transplantable HSCs from EPHOSS during collection in air resulting in at least a 3-fold increase in HSC recovery as well as increased transplantation potency. Thus, pharmacological mitigation of EPHOSS during HSC collections for use in patient transplantation procedures may be clinically advantageous. Because cyclosporine A is already in use clinically, this EPHOSS-reducing strategy may be readily and easily tested for efficacy in a hospital setting. Because many different adult stem cells exist naturally in hypoxic niches, EPHOSS is likely relevant to other stem cells routinely harvested in air. Evidence suggests that aged HSCs may be more sensitive to the deleterious effects of EPHOSS than young HSCs. We propose that metabolic profiling of stem cells, including cancer stem cells, may not accurately represent the metabolism, behavior, and responses of these cells as they exist in their native hypoxic environments because they are harvested and studied in air. Thus, experimental designs that include a consideration of EPHOSS effects may be required to obtain a more complete understanding of stem cell metabolism and biology especially as it relates to stem cell aging or responses of cancer stem cells to chemotherapy. [Display omitted] DisclosuresBroxmeyer:CordUse: Membership on an entity’s Board of Directors or advisory committees.

Microrna Mediated Regulation of Hematopoietic Stem Cell Aging

Aging hematopoietic stem cells (HSCs) exhibit numerous functional alterations including reduced capacity for self-renewal, myeloid-biased differentiation, and reduced production of mature lymphocytes and red blood cells. Interventions such as calorie restriction (CR) and rapamycin (Rapa) treatment have been shown to increase lifespan and to delay the onset of age-related diseases, and some studies have demonstrated that they may improve HSC function through poorly defined mechanisms. We and others have shown that microRNAs (miRNAs) are potent cell-intrinsic regulators of HSC self-renewal and lineage specification and also contribute to age-related disorders such as acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS). We hypothesized that miRNAs may underlie the recovery of HSC function observed in anti-aging mouse models, and thus we characterized miRNA expression profiles from HSCs (Lin-c-Kit+Sca-1+CD34-CD150+) from young mice (12-16 weeks old), old mice (20-22 months), and old mice that had been treated with anti-aging interventions. Evaluation of HSCs from CR and Rapa treated old mice revealed numerous changes consistent with inhibition/reversal of age-related HSC changes including a 5-fold reduction in HSC frequency (p=0.04), 2-fold increase in erythroid progenitors (pro-erythroblasts, p=0.04), 2.5 fold increase in common lymphoid progenitors (CLP; Lin-c-Kit+Sca-1+CD127+FLK2+, p=0.05), as well as 3.5-fold increase in peripheral blood B cells (p=0.002), 2.2 fold decrease in platelets (p=0.01), and increased red blood cells (p=0.04). These changes were associated with statistically significant increases in the percentage of HSCs in S/M/G2 (p=0.045), and undergoing apoptosis (p=0.05). Using a TaqMan-based qPCR expression profiling method evaluating 750 miRNAs, we found that old HSCs exhibited altered expression of 91 miRNAs compared to young (FDR <0.1, P <0.05). Moreover, HSCs from both CR and Rapa treated old mice exhibited expression of 60 miRNAs at levels similar to young, normal HSCs. miR-125b, a miRNA we and others previously showed to positively regulate HSC self-renewal, was reduced 2.2-fold in old mice, and its expression was restored in CR and Rapa treated HSCs. Lentivirally mediated expression of miR-125b in old HSCs increased their long-term reconstitution capacity 8.1-fold compared to control old HSCs based on donor chimerism levels at 16 weeks post-transplantation, resulting in chimerism levels similar to mice transplanted with young HSCs expressing miR-125b. The improved HSC engraftment capacity of old HSCs transduced with miR-125b was accompanied by statistically significant increases in the frequencies of lymphoid biased HSCs (Lin-c-Kit+Sca-1+CD34-CD150neg-low), megakaryocyte-erythroid progenitors (MEPs), CLPs, and peripheral blood B- and T-cells, compared to old HSCs transduced with control lentivirus (p<0.05 for all indicated cell types). While enforced expression of high levels of miR-125b in mouse HSPCs has been reported to induce myeloid leukemias, there was no evidence of a hematologic malignancy in mice transplanted with miR-125b transduced old HSCs up to 6 months post-transplantation. Overall, these results demonstrate that functional HSC aging phenotypes can be that inhibited/reversed by anti-aging interventions, that miR-125b regulates HSC aging, and that anti-aging interventions may exert their positive effects on HSC function by regulating miR-125b expression. DisclosuresNo relevant conflicts of interest to declare.

Asymmetry in skeletal distribution of mouse hematopoietic stem cell clones and their equilibration by mobilizing cytokines

Hematopoietic stem cells (HSCs) are able to migrate through the blood stream and engraft bone marrow (BM) niches. These features are key factors for successful stem cell transplantations that are used in cancer patients and in gene therapy protocols. It is unknown to what extent transplanted HSCs distribute throughout different anatomical niches in the BM and whether this changes with age. Here we determine the degree of hematopoietic migration at a clonal level by transplanting individual young and aged mouse HSCs labeled with barcoded viral vector, followed by assessing the skeletal distribution of hundreds of HSC clones. We detected highly skewed representation of individual clones in different bones at least 11 mo after transplantation. Importantly, a single challenge with the clinically relevant mobilizing agent granulocyte colony-stimulating factor (G-CSF) caused rapid redistribution of HSCs across the skeletal compartments. Old and young HSC clones showed a similar level of migratory behavior. Clonal make-up of blood of secondary recipients recapitulates the barcode composition of HSCs in the bone of origin. These data demonstrate a previously unanticipated high skeletal disequilibrium of the clonal composition of HSC pool long-term after transplantation. Our findings have important implications for experimental and clinical and stem cell transplantation protocols.

Understanding epigenetic changes in aging stem cells – a computational model approach

During aging, a decline in stem cell function is observed in many tissues. This decline is accompanied by complex changes of the chromatin structure among them changes in histone modifications and DNA methylation which both affect transcription of a tissue-specific subset of genes. A mechanistic understanding of these age-associated processes, their interrelations and environmental dependence is currently lacking. Here, we discuss related questions on the molecular, cellular, and population level. We combine an individual cell-based model of stem cell populations with a model of epigenetic regulation of transcription. The novel model enables to simulate age-related changes of trimethylation of lysine 4 at histone H3 and of DNA methylation. These changes entail expression changes of genes that induce age-related phenotypes (ARPs) of cells. We compare age-related changes of regulatory states in quiescent stem cells occupying a niche with those observed in proliferating cells. Moreover, we analyze the impact of the activity of the involved epigenetic modifiers on these changes. We find that epigenetic aging strongly affects stem cell heterogeneity and that homing at stem cell niches retards epigenetic aging. Our model provides a mechanistic explanation how increased stem cell proliferation can lead to progeroid phenotypes. Adapting our model to properties observed for aged hematopoietic stem cell (HSC) clones, we predict that the hematopoietic ARP activates young HSCs and thereby retards aging of the entire HSC population. In addition, our model suggests that the experimentally observed high interindividual variance in HSC numbers originates in a variance of histone methyltransferase activity.

Heterogeneity of young and aged murine hematopoietic stem cells revealed by quantitative clonal analysis using cellular barcoding

The number of hematopoietic stem cells (HSCs) that contributes to blood formation and the dynamics of their clonal contribution is a matter of ongoing discussion. Here, we use cellular barcoding combined with multiplex high-throughput sequencing to provide a quantitative and sensitive analysis of clonal behavior of hundreds of young and old HSCs. The majority of transplanted clones steadily contributes to hematopoiesis in the long-term, although clonal output in granulocytes, T cells, and B cells is substantially different. Contributions of individual clones to blood are dynamically changing; most of the clones either expand or decline with time. Finally, we demonstrate that the pool of old HSCs is composed of multiple small clones, whereas the young HSC pool is dominated by fewer, but larger, clones.

Heterogeneity of young and aged murine hematopoietic stem cells revealed by quantitative clonal analysis using cellular barcoding

AbstractThe number of hematopoietic stem cells (HSCs) that contributes to blood formation and the dynamics of their clonal contribution is a matter of ongoing discussion. Here, we use cellular barcoding combined with multiplex high-throughput sequencing to provide a quantitative and sensitive analysis of clonal behavior of hundreds of young and old HSCs. The majority of transplanted clones steadily contributes to hematopoiesis in the long-term, although clonal output in granulocytes, T cells, and B cells is substantially different. Contributions of individual clones to blood are dynamically changing; most of the clones either expand or decline with time. Finally, we demonstrate that the pool of old HSCs is composed of multiple small clones, whereas the young HSC pool is dominated by fewer, but larger, clones.

Proliferation-Dependent Alterations of the DNA Methylation Landscape Underlie Hematopoietic Stem Cell Aging

The functional potential of hematopoietic stem cells (HSCs) declines during aging, and in doing so, significantly contributes to hematopoietic pathophysiology in the elderly. To explore the relationship between age-associated HSC decline and the epigenome, we examined global DNA methylation of HSCs during ontogeny in combination with functional analysis. Although the DNA methylome is generally stable during aging, site-specific alterations of DNA methylation occur at genomic regions associated with hematopoietic lineage potential and selectively target genes expressed in downstream progenitor and effector cells. We found that age-associated HSC decline, replicative limits, and DNA methylation are largely dependent on the proliferative history of HSCs, yet appear to be telomere-length independent. Physiological aging and experimentally enforced proliferation of HSCs both led to DNA hypermethylation of genes regulated by Polycomb Repressive Complex 2. Our results provide evidence that epigenomic alterations of the DNA methylation landscape contribute to the functional decline of HSCs during aging.

HSC Aging Epigenome: Widespread Alterations in DNA Methylation and Transcription.

AbstractAbstract 2329Age is the most important risk factor for myelodysplastic syndrome (MDS), a premalignant state that transforms into acute myelogenous leukemia in one third of cases. Indeed with normal aging, hematopoietic stem cell (HSC) regenerative potential diminishes and differentiation skews from lymphopoiesis toward myelopoiesis. The expansion in the HSC pool with aging provides sufficient but abnormal blood production, and animals experience a decline in immune function. Previous studies from our lab established that the DNA methyltransferase 3a (Dnmt3a) enables efficient differentiation by critically regulating epigenetic silencing of HSC genes (Challen et al. 2012) Interestingly, Dnmt3a expression is decreased in old HSCs, leading us to hypothesize that epigenetic changes in old HSCs may partially mimic the changes seen in Dnmt3a mutant HSCs. We propose that revealing the genome-wide DNA methylation and transcriptome signatures will lead to a greater understanding of HSC aging and MDS, which is characterized by frequent epigenetic abnormalities.In this study, we investigated genome-wide DNA methylation and transcripts by whole genome bisulfite sequencing (WGBS) and transcriptome sequencing (mRNA-seq)in young and old HSCs. For WGBS, we generated ∼600M raw reads resulting in ∼ 60 raw Gb of paired-end sequence data and aligned them to either strand of the reference genome (mm9), providing an average 40-fold sequencing depth. Globally, there was a 1.1% difference in the DNA methylation between young and old HSCs. Of these differences, 38% (172,609) of the CpG dinucleotides were hypo-methylated, and 62% (275,557) were hyper-methylated in old HSCs. To understand where the methylation changes predominantly occurred, the genome was subdivided into 77 features. Among these features, SINEs, especially Alu elements, exhibited the highest level of DNA methylation (90.94% in young HSCs, and 91.87% in old HSCs). CpG islands (CGIs) adjacent to the transcription start sites (TSS) exhibited the lowest level of DNA methylation (2.02% in young HSCs, and 2.11% in old HSCs). Interestingly strong hypo-methylation was observed in ribosomal RNA regions (68.04% in young HSCs, 59.04% in old HSCs), and hyper-methylation was observed in LINEL1 repetitive elements (88.62% in young HSCs, 90.12% in old HSCs). Moreover, the examination of differentially methylated promoters identified enrichment of developmentally important transcription factors such as Gata2, Runx1, Gfi1b, Erg, Tal1 Eto2, Cebpa and Pu.1. Additionally, we compare our ∼10,000 differentially methylation regions (DMRs, regions with clustered DNA methylation changes) with a chip-seq data set containing binding of 160 ChIP-seq analyses of hematopoietic transcription factors in different hematopoietic cells. We found significant overlaps between DMRs and transcription factor binding regions. We found DMRs which were hypermethylated showed association with differentiation-promoting Ets factors, in particular Pu.1 from a range of different blood cell types. In contrast, hypomethylated DMRs showed associations with HSC-associated transcription factors such as Scl and Gata2. Further examination of the differentially methylated gene bodies, intragenic and intergenic DMRs identified some previously noted targets for epigenetic silencing or alteration in AML and also novel transcripts including long non-coding RNAs (lincRNA) and upstream regulatory elements (URE). We found significant correlation between RNA-seq expression and DMRs within +1kb upstream of TSS. RNA-sequencing provided complementary and distinct information about HSC aging. We identified differentially expressed genes, novel RNA transcripts, differential promoter, coding sequence, and splice variant usage with age. Gene set enrichment analysis of up- and down- regulated genes, revealed ribosomal protein and RNA metabolism as critical contributors to HSC aging.In conclusion, our study marks a milestone in the mouse HSC epigenome, reporting the first complete methylome and transcriptome of pure HSC using whole-genome bisulfite sequencing and RNA-seq. These provide novel information about the magnitude and specificity of age-related epigenetic changes in a well-defined HSC population. Understanding the roles of DNA methylation and transcription in normal HSC function will allow for greater therapeutic exploitation of HSCs in the clinic. Disclosures:No relevant conflicts of interest to declare.

Histone Alterations Are Associated with Hematopoietic Stem Cell (HSC) Differentiation and Aging

AbstractAbstract 1188Chromatin regulators, including both Trithorax-group (trxG) and Polycomb-group (PcG) families, have been reported to maintain hematopoietic stem cells (HSC) self-renewal and differentiation. However, their primary targets in HSC remain to be fully identified. Moreover, those histone modifiers have been recently found to control longevity in C.elegans, which leads us to further investigate their roles in HSC aging. HSCs increase in number, decrease in regeneration capacity, and exhibit a myeloid biased differentiation with age. In this study, we profiled global mRNA (RNA-seq) and chromatin changes (ChIP-seq) in highly purified young (4 month) and old (24 month) murine bone marrow-derived HSCs (SP-KSL-CD150+). One key challenge in this study is that mouse HSCs represent less than 0.01% of all bone marrow cells. Thus, we first developed and optimized a method for successful application of ChIP-seq to a limited number of cells (<20,000 cells). This method allowed us to generate the binding profiles for H3K4me3, H3K27me3 and H3K36me3 in young and old HSCs, differentiated granulocytes (Gr1+) and B (B220+) cells.In young HSCs, we determined ∼18,000 peaks for the active gene mark H3K4me3. For the repressive mark H3K27me3, we identified ∼6000 peaks across the genome, 2591 of which were present in the promoter region defined as the transcriptional start site (TSS) ± 100 bp. Strikingly, 70.2% (1820 out of 2591) of H3K27me3-enriched genes were also bound by H3K4me3. These so-called “Bivalent genes”, with both H3K4me3 and H3K27me3, are also found in embryonic stem cell (ESC) where they represent master regulators for lineage development. Here we found that these 1820 genes in HSC are prevalent in development, transcriptional regulation and RNA metabolism. They include many lineage-specific transcription factors, such as Cebpa, Ebf-1, Pax5, Gata3, Tbx21, Runx3 and Eomes. During HSC differentiation to granulocytes or B cells, HSC pluripotency regulators acquired the H3K27me3 repressive mark, while lineage regulators lost it in differentiated lineages. In addition, we observed with HSC differentiation alternative promoter usage on many epigenetic modifiers or transcription factors, such as Dnmt3a, Dnmt3b and Kdm6b (Jmjd3) and Runx3. The different isoforms may have different functions in hematopoiesis.Compared to HSC differentiation, HSC aging showed less extensive chromatin changes. Increased H3K4me3 binding at the TSS was identified for ∼300 genes, including Selp, Nupr1, Sdpr, Plscr2, Slamf1 (CD150) and Mt1. Interestingly, several genes in the Hoxb cluster showed increased H3K4me3 binding and higher expression with age. For H3K27me3, although PcG family member EZh2 expression decreases with HSC aging, we did not detect a global H3K27me3 decrease. On the contrary, H3K27me3 binding increased at ∼500 genes and decreased at ∼100 genes. Among them, the lymphoid transcription factors Pax5 and Ebf1 exhibited increased H3K27me3 binding. The enhanced repression of these lymphoid regulators may explain the myeloid-skewed differentiation that occurs with age. We also observed increased H3K27me3 on several Wnt ligands, including Wnt2a, Wnt8a and Wnt8b. As Wnt signaling is required for active HSC cycling, the increased H3K27me3 binding on these ligands may reinforce HSC quiescence. One well known target of the PcG family in aging is Cdkn2a (p16), which showed progressive loss of H3K27me3 repression with aging in neural stem cell (NSC). In contrast, our results revealed that p16 is repressed by H3K27me3 in both 4M and 24M HSC, and we did not observe its expression increase with aging. However, another cell cycle regulator p21 expression increases with aging, accompanied by a decrease of H3K27me3.In summary, we mapped chromatin state alterations with HSC differentiation and aging in this study. These findings will advance our fundamental understanding of HSC biology. Furthermore, chromatin regulators, such as Bmi1 and EZh2, have been implicated in leukemia transformation. This study provides a mechanistic link into how deregulation of these factors correlates with cancer progression. Disclosures:No relevant conflicts of interest to declare.

Cdc42 Activity Regulates Hematopoietic Stem Cell Aging and Rejuvenation

The decline in hematopoietic function seen during aging involves a progressive reduction in the immune response and an increased incidence of myeloid malignancy, and has been linked to aging of hematopoietic stem cells (HSCs). The molecular mechanisms underlying HSC aging remain unclear. Here we demonstrate that elevated activity of the small RhoGTPase Cdc42 in aged HSCs is causally linked to HSC aging and correlates with a loss of polarity in aged HSCs. Pharmacological inhibition of Cdc42 activity functionally rejuvenates aged HSCs, increases the percentage of polarized cells in an aged HSC population, and restores the level and spatial distribution of histone H4 lysine 16 acetylation toa status similar to that seen in young HSCs. Ourdata therefore suggest a mechanistic role for Cdc42 activity in HSC biology and epigenetic regulation, and identify Cdc42 activity as a pharmacological target for ameliorating stem cell aging.

Accelerated Aging versus Rejuvenation of the Immune System in Heterochronic Parabiosis

The emergence of immune disorders in aging is explained by many factors, including thymus dysfunction, decrease in the proportion and function of naïve T cells, and so forth. There are several approaches to preventing these changes, such as thymus rejuvenation, stem cells recovery, modulation of hormone production, and others. Our investigations of heterochronic parabiosis have shown that benefits of a young immune system, e.g., actively working thymus and regular migration of young hematopoietic stem cells between parabiotic partners, appeared unable to restore the immune system of the old partner. At the same time, we have established a progressive immune impairment in the young heterochronic partners. The mechanism of age changes in the immune system in this model, which may lead to reduced life expectancy, has not been fully understood. The first age-related manifestation in the young partners observed 3 weeks after the surgery was a dramatic increase of CD8(+)44(+) cells population in the spleen. A detailed analysis of further changes revealed a progressive decline of most immunological functions observable for up to 3 months after the surgery. This article reviews possible mechanisms of induction of age-related changes in the immune system of young heterochronic partners. The data obtained suggest the existence of certain factors in the old organisms that trigger aging, thus preventing the rejuvenation process.

Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age

In the human hematopoietic system, aging is associated with decreased bone marrow cellularity, decreased adaptive immune system function, and increased incidence of anemia and other hematological disorders and malignancies. Recent studies in mice suggest that changes within the hematopoietic stem cell (HSC) population during aging contribute significantly to the manifestation of these age-associated hematopoietic pathologies. Though the mouse HSC population has been shown to change both quantitatively and functionally with age, changes in the human HSC and progenitor cell populations during aging have been incompletely characterized. To elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated immunophenotypic HSC and other hematopoietic progenitor populations from healthy, hematologically normal young and elderly human bone marrow samples. We found that aged immunophenotypic human HSC increase in frequency, are less quiescent, and exhibit myeloid-biased differentiation potential compared with young HSC. Gene expression profiling revealed that aged immunophenotypic human HSC transcriptionally up-regulate genes associated with cell cycle, myeloid lineage specification, and myeloid malignancies. These age-associated alterations in the frequency, developmental potential, and gene expression profile of human HSC are similar to those changes observed in mouse HSC, suggesting that hematopoietic aging is an evolutionarily conserved process.

Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells

Hematopoietic stem cell (HSC) populations change with aging, but the extent to which this is caused by qualitative versus quantitative alterations in HSC subtypes is unclear. Using clonal assays, in this study we show that the aging HSC compartment undergoes both quantitative and qualitative changes. We observed a variable increase of HSC pool size with age, accompanied by the accumulation of predominantly myeloid-biased HSCs that regenerate substantially fewer mature progeny than young myeloid-biased HSCs and exhibit reduced self-renewal activity as measured by long-term secondary transplantation. Old HSCs had a twofold reduction in marrow-homing efficiency and a similar decrease in functional frequency as measured using long-term transplantation assays. Similarly, old HSCs had a twofold reduced seeding efficiency and a significantly delayed proliferative response compared with young HSCs in long-term stromal cell co-cultures but were indistinguishable in suspension cultures. We show that these functional defects are characteristics of most or all old HSCs and are not indicative of a nonfunctional subset of cells that express HSC markers. Furthermore, we demonstrate that cells with functional properties of old HSCs can be generated directly from young HSCs by extended serial transplantation, which is consistent with the possibility that they arise through a process of cellular aging.

Effects of Age and Environment on Short-Term Homing and Function of Mouse Hematopoietic Stem Cells.

AbstractAbstract 1616The C57BL/6 mouse is one of the best studied models of hematopoietic stem cell (HSC) aging. Characteristic age-related changes include an increase in HSC frequency, a corresponding decrease in functional activity, myeloid skewing, and decreased in vivo homing ability. Studies of age-dependent differences in homing have primarily relied upon the injection of young or old bone marrow into irradiated recipients, with subsequent quantification of functional homed HSCs using secondary transplantation assays (e.g. Liang et al, Blood 2005). While this is a relevant and informative approach, it is limited because it measures homed cells indirectly, using an assay that itself is dependent on homing. To study this phenomenon more directly, we compared the short-term homing efficiency of purified HSCs from old and young mice by co-injecting them into old or young recipients. This approach enabled us to ascertain whether the previously reported decrease in homing efficiency is due to differences in physical homing to the bone marrow, or a reduced function per homed cell, or some combination of both. Second, it allowed us to determine the relationship between the decreased in vivo homing efficiency of old HSCs and their functional properties when assayed in a homing-independent stem cell assay. Third, by varying the age and irradiation status of the homing recipients, we could test directly the influence of these factors on HSC homing and function. To accomplish these objectives, we purified HSCs (CD150+CD48-LSK bone marrow cells) from old (23-26 months) GFP+ and young (3-4 months) CFP+ mice, combined them at known ratios, and injected them into non-irradiated young, non-irradiated old, or lethally irradiated young recipients. 22 hours later, these recipients were sacrificed and the ratio of GFP+ and CFP+ cells found homed to the bone marrow were compared to the ratio in which they were injected. In this way, we determined that relative to their young counterparts, the 22-hour homing efficiency of old CD150+CD48-LSK cells was consistently 2-fold lower when injected into young recipients. However, when the same cells were injected into non-irradiated old recipients, the relative homing efficiency varied from 2-fold lower to 1.1-fold higher. This suggests that the factors affecting homing are similar between individual young recipients, while the microenvironments of individual old recipients are variable in their relative ability to support homing of young and old HSCs. We recently demonstrated that the proportion of purified old HSCs that were functional when assayed clonally in vitro (cobblestone area forming cell [CAFC] assay) declined approximately 2-fold compared to young HSCs. To determine whether the non-homed HSCs were the same cells that were non-functional in the CAFC assay, we compared the CAFC seeding efficiency of old and young cells before and after homing. There was no functional improvement in homed old LSK150+48- cells, suggesting that the homing defect and reduced CAFC efficiency are non-overlapping and may be characteristics of all old HSCs. To determine if the homing microenvironment also had an effect on the function of the homed cells, CAFC assays were initiated with old and young HSCs before and after homing into each of the three recipient types. This analysis revealed that the function of the homed cells was significantly decreased for age-mismatched or irradiated recipients compared to pre-homed cells. In contrast, CAFC activity of young or old cells homed to non-irradiated age-matched recipients did not change. This suggests that the environment of an age mismatched and/or irradiated recipient can have a negative effect on the function of homed cells within a matter of hours. In conclusion, these co-homing studies reveal that compared to young HSCs, old HSCs are physically less able to home and migrate into the bone marrow within a 22-hour period post-transplantation. However, this relative homing defect is most pronounced and consistent when injected into young recipients. Second, the homing defect and reduced CAFC efficiency are non-overlapping and may be characteristics of all old HSCs. Third, old or young HSCs that do successfully home to irradiated or age-mismatched recipients become functionally compromised compared to pre-homed HSCs, suggesting that the homing defects of old HSCs can be ameliorated or exacerbated depending on the microenvironment in which they are transplanted. Disclosures:No relevant conflicts of interest to declare.

Human cytomegalovirus-specific CD4+ and CD8+ T-cell response determination: Comparison of short-term (24h) assays vs long-term (7-day) infected dendritic cell assay in the immunocompetent and the immunocompromised host

Human cytomegalovirus (HCMV)-specific CD4(+) and CD8(+) T-cells were measured in the immunocompetent host as well as in 13 solid-organ transplant recipients (SOTR), and 12 young hematopoietic stem cell transplant recipients (HSCTR) by using a long-term (7-day) assay based on PBMC stimulation by HCMV-infected dendritic cells (iDC), and two short-term (24h) assays, one for CD4(+) stimulation by infected cell lysate (iCL), and the other for CD8(+) stimulation by a pool of 34 epitopic peptides (pep-pool). In the immunocompetent, the number of T-cells activated by either iCL or the pep-pool was significantly reduced with respect to iDC. In both SOTR and HSCTR, the number of T-cells activated by iDC was comparable to that activated by iCL or the pep-pool. A significant correlation between iDC-activated T-cells and T-cells activated by either iCL or the pep-pool was observed. In conclusion, whenever a rapid result is needed, short-term assays may efficiently replace the iDC assay.

Hematopoiesis in the Elderly: Age-Associated Effects in Frequency, Function, and Gene Expression of Human Hematopoietic Stem Cells.

Abstract 1505Poster Board I-528Aging of the human hematopoietic system is associated with an increase in the development of anemia, myeloid malignancies, and decreased adaptive immune function. While the hematopoietic stem cell (HSC) population in mouse has been shown to change both quantitatively as well as functionally with age, age-associated alterations in the human HSC and progenitor cell populations have not been characterized. In order to elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated and compared HSC and other hematopoietic progenitor populations prospectively isolated via fluorescence activated cell sorting (FACS) from 10 healthy young (20-35 years of age) and 8 healthy elderly (65+ years of age) human bone marrow samples. Bone marrow was obtained from hematologically normal young and old volunteers, under a protocol approved by the Stanford Institutional Review Board. We determined by flow cytometry the distribution frequencies and cell cycle status of HSC and progenitor populations. We also analyzed the in vitro function and generated gene expression profiles of the sorted HSC and progenitor populations.We found that bone marrow samples obtained from normal elderly adults contain ∼2-3 times the frequency of immunophenotypic HSC (Lin-CD34+CD38-CD90+) compared to bone marrow obtained from normal young adults (p < 0.02). Furthermore, upon evaluation of cell cycle status using RNA (Pyronin-Y) and DNA (Hoechst 33342) dyes, we observed that a greater percentage of HSC from young bone marrow are in the quiescent G0- phase of the cell cycle compared to elderly HSC, of which there is a greater percentage in G1-, S-, G2-, or M-phases of the cell cycle (2.5-fold difference; p < 0.03). In contrast to the increase in HSC frequency, we did not detect any significant differences in the frequency of the earliest immunophenotypic common myeloid progenitors (CMP; Lin-CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP; Lin-CD34+CD38+CD123+CD45RA+), and megakaryocytic-erythroid progenitors (MEP; Lin-CD34+CD38+CD123-CD45RA-) from young and elderly bone marrow. We next analyzed the ability of young and elderly HSC to differentiate into myeloid and lymphoid lineages in vitro. We found that elderly HSC exhibit diminished capacity to differentiate into lymphoid B-lineage cells in the AC6.21 culture environment. We did not, however, observe significant differences in the ability of young and elderly HSC to form myeloid and erythroid colonies in methylcellulose culture, indicating that myelo-erythroid differentiation capacity is preserved in elderly HSC. Correspondingly, gene expression profiling of young and elderly human HSC indicate that elderly HSC have up-regulation of genes that specify myelo-erythroid fate and function and down-regulation of genes associated with lymphopoiesis. Additionally, elderly HSC exhibit increased levels of transcripts associated with transcription, active cell-cycle, cell growth and proliferation, and cell death. These data suggest that hematopoietic aging is associated with intrinsic changes in the gene expression of human HSC that reflect the quantitative and functional alterations of HSC seen in elderly bone marrow. In aged individuals, HSC are more numerous and, as a population, are more myeloid biased than young HSC, which are more balanced in lymphoid and myeloid potential. We are currently investigating the causes of and mechanisms behind these highly specific age-associated changes in human HSC. Disclosures:Weissman:Amgen: Equity Ownership; Cellerant Inc.: ; Stem Cells Inc.: ; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

Multiple prethymic defects underlie age-related loss of T progenitor competence

AbstractAging in mice and humans is characterized by declining T-lymphocyte production in the thymus, yet it is unclear whether aging impacts the T-lineage potential of hematopoietic progenitors. Although alterations in the lymphoid progenitor content of aged mouse bone marrow (BM) have been described, irradiation-reconstitution experiments have failed to reveal defects in T-lineage potential of BM hematopoietic progenitors or purified hematopoietic stem cells (HSCs) from aged mice. Here, we assessed T-progenitor potential in unmanipulated recipient mice without conditioning irradiation. T-progenitor potential was reduced in aged BM compared with young BM, and this reduction was apparent at the earliest stages of intrathymic differentiation. Further, enriched populations of aged HSCs or multipotent progenitors (MPPs) gave rise to fewer T-lineage cells than their young counterparts. Whereas the T-precursor frequency within the MPP pool was unchanged, there was a 4-fold decline in T-precursor frequency within the HSC pool. In addition, among the T-competent HSC clones, there were fewer highly proliferative clones in the aged HSC pool than in the young HSC pool. These results identify T-compromised aged HSCs and define the nature and cellular sites of prethymic, age-related defects in T-lineage differentiation potential.

Multiple prethymic defects underlie age-related loss of T progenitor competence (81.6)

Abstract Aging in mice and humans is characterized by declining T lymphocyte production in the thymus, yet it is unclear whether aging impacts the T lineage potential of hematopoietic progenitors. Although alterations in the lymphoid progenitor content of aged mouse bone marrow have recently been described, irradiation- reconstitution experiments have failed to reveal defects in T lineage potential of bone marrow (BM) progenitors or purified hematopoietic stem cells (HSC) from aged mice. Here, we assessed T progenitor potential in young recipient mice without conditioning irradiation. T potential was reduced in aged BM compared to young BM and this reduction was apparent at the earliest stages of intrathymic differentiation. Further, purified populations of aged HSC or multipotent progenitors (MPP) also gave rise to fewer T lineage cells than their young counterparts. Limiting dilution experiments in vitro showed that the T precursor frequency within the MPP pool was unchanged. However, there was a fourfold reduction in T precursor frequency within the aged HSC pool, indicating that there is an accumulation of T-incompetent cells in this population. Additionally, among the T-competent HSC clones, there were fewer highly proliferative clones in the aged HSC pool than in the young HSC pool. These results define the nature and cellular sites of prethymic age-related defects in T lineage differentiation potential.