Enhanced Cytokine Responsiveness Counteracts Age-Induced Decline in Hematopoietic Stem Cell Function

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2342-2342
Author(s):  
Gudmundur L Norddahl ◽  
Martin Wahlestedt ◽  
Santiago Gisler ◽  
Mikael Sigvardsson ◽  
David Bryder
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Adaptor Protein ◽  
Physiological Age ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Age Related ◽  
Lineage Distribution

Abstract Abstract 2342 In recent years, it has become increasingly clear that physiologic aging has several profound cell intrinsic effects on hematopoietic stem cell (HSC) aging. This includes an altered output of mature progeny and an expansion of the HSC pool, although the latter is accompanied with several functional shortcomings. Because of the hierarchical structure of hematopoiesis, improving HSC function should present a promising avenue to restore aberrant hematopoietic function. The signal adaptor protein Lnk, a relay of cytokine signaling, has been shown to negatively regulate hematopoiesis at several cellular stages. As a consequence of Lnk deficiency, mice display increased B lymphopoiesis and increased HSC numbers. The increase in HSC numbers has been suggested to arise by an increased ability of HSCs to self-renew. However, it has remained unclear how the enhanced sensitivity to cytokine signaling would affect hematopoiesis upon physiological age. In the present study, we investigated the effects of physiological aging on hematopoiesis in the Lnk−/− mice. Aged wild-type mice showed a number of age-related alterations in the hematopoietic system including; increased HSC numbers, decreased lymphopoiesis and decreased reconstitution potential upon competitive transplantation. Most of these parameters were dramatically altered as a consequence of Lnk deficiency. When competitively transplanted, aged Lnk−/− HSCs displayed a dramatically increased overall reconstitution potential compared to WT mice. Increases in reconstitution potential were observed in both primary and serially reconstituted recipient animals. Upon physiologic age, the lineage distribution of transplanted aged wild-type HSCs is skewed towards the myeloid lineage. By contrast, transplanted aged Lnk−/− HSCs gave rise to a hematopoietic system with a balanced lineage distribution resembling that generated by young wild-type HSCs. The enhanced HSC potential of aged Lnk−/− mice was uncoupled from effects on telomere length maintenance but displayed a distinct molecular profile in whole genome expression analysis. In summary, we demonstrate that deficiency of the signal adaptor molecule Lnk appears to functionally protect HSCs from many of the consequences of physiologic age, which at a broader level perhaps suggest a potential of cytokines to counteract age-related HSC decline. Disclosures: No relevant conflicts of interest to declare.

scholarly journals LNK (SH2B3) Deficiency Ameliorates Hematopoietic Stem Cell Defects in Fanconi Anemi

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2670-2670
Author(s):  
Joanna Balcerek ◽  
Jing Jiang ◽  
Qinqin Jiang ◽  
Kaosheng Lyu ◽  
Roger Greenberg ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Genome Instability ◽  
Bone Marrow Failure ◽  
Adaptor Protein ◽  
Replication Stress ◽  
Cytokine Signaling ◽  
Hematopoietic Stem

Abstract Fanconi Anemia (FA) is one of the most common inherited bone marrow failure (iBMF) syndromes. Although initially identified over 85 years ago, FA remains a fatal genetic disease. Nineteen FA genes cooperate in a genome stability pathway that is essential for repair of DNA damage and tolerance of replication stress. Cells derived from FA patients are hypersensitive to DNA interstrand crosslink (ICL)-inducing agents, such as Mitomycin C (MMC), and exhibit DNA damage checkpoint and mitosis defects. Mutations in FA genes result in hematopoietic stem cell (HSC) defects, bone marrow failure and cancer predisposition. Importantly, interventions to mitigate HSC defects do not exist, aside from allogeneic bone marrow transplantation (BMT). HSCs deficient for FancD2, a central component of the FA signaling pathway, are markedly compromised in reconstituting the hematopoietic system in murine BMT models. Remarkably, we found that loss of the adaptor protein Lnk (also called Sh2b3) restores HSC function in FancD2 knockout mice without accelerating neoplastic transformation. LNK negatively regulates HSC self-renewal, in part by attenuating cytokine signaling-activated JAK2 signaling in HSCs (J Clin Invest. 2008;118(8):2832-2844). Fancd2-/-;Lnk-/- (DKO) mice exhibit increased phenotypic HSCs in comparison to wildtype (WT) animals, and DKOHSC function is also largely restored to WT levels in serial transplantation assays. Primary DKOHSC and progenitors (HSPCs) are still sensitive to MMC, indicating that LNK does not play an overt role in ICL repair. Instead, Lnk deficiency notably reduces spontaneous DNA damage and genome instability. This is in agreement with recent studies that reveal a requirement for FA proteins in replication stress, which is a separable function from their role in DNA repair (Cell. 2011;145(4):529-542; Cancer Cell. 2012;22(1):106-116). Strikingly, we demonstrated that Lnk deficiency mitigates replication stress by stabilizing stalled replication forks, and that this effect is dependent upon cytokine signaling. Together, our data demonstrate that Lnk deficiency ameliorates FA HSPC defects by alleviating replication stress associated DNA damage and genome instability. To our knowledge, this is one of the first examples of in vivo genetic suppression of FA-associated HSPC defects. Our work sheds light on mechanisms underlying the origin of bone marrow failure in FA patients and has implications for new therapeutic strategies to treat FA associated bone marrow failure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Engraftment of rare, pathogenic donor hematopoietic mutations in unrelated hematopoietic stem cell transplantation

2020 ◽  
Vol 12 (526) ◽  
pp. eaax6249 ◽  
Author(s):  
Wing Hing Wong ◽  
Sima Bhatt ◽  
Kathryn Trinkaus ◽  
Iskra Pusic ◽  
Kevin Elliott ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Cell Transplant ◽  
Younger Adults ◽  
Hematopoietic Stem ◽  
Clonal Hematopoiesis ◽  
Age Related ◽  
Over Time

Clonal hematopoiesis is associated with various age-related morbidities. Error-corrected sequencing (ECS) of human blood samples, with a limit of detection of ≥0.0001, has demonstrated that nearly every healthy individual >50 years old harbors rare hematopoietic clones below the detection limit of standard high-throughput sequencing. If these rare mutations confer survival or proliferation advantages, then the clone(s) could expand after a selective pressure such as chemotherapy, radiotherapy, or chronic immunosuppression. Given these observations and the lack of quantitative data regarding clonal hematopoiesis in adolescents and young adults, who are more likely to serve as unrelated hematopoietic stem cell donors, we completed this pilot study to determine whether younger adults harbored hematopoietic clones with pathogenic mutations, how often those clones were transferred to recipients, and what happened to these clones over time after transplantation. We performed ECS on 125 blood and marrow samples from 25 matched unrelated donors and recipients. Clonal mutations, with a median variant allele frequency of 0.00247, were found in 11 donors (44%; median, 36 years old). Of the mutated clones, 84.2% of mutations were predicted to be molecularly pathogenic and 100% engrafted in recipients. Recipients also demonstrated de novo clonal expansion within the first 100 days after hematopoietic stem cell transplant (HSCT). Given this pilot demonstration that rare, pathogenic clonal mutations are far more prevalent in younger adults than previously appreciated, and they engraft in recipients and persist over time, larger studies with longer follow-up are necessary to correlate clonal engraftment with post-HSCT morbidity.

scholarly journals Extreme disruption of heterochromatin is required for accelerated hematopoietic aging

Blood ◽  
2020 ◽  
Vol 135 (23) ◽  
pp. 2049-2058 ◽  
Author(s):  
Christine R. Keenan ◽  
Nadia Iannarella ◽  
Gaetano Naselli ◽  
Naiara G. Bediaga ◽  
Timothy M. Johanson ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Cell Types ◽  
Premature Aging ◽  
Thymic Involution ◽  
Hematopoietic Stem ◽  
Hematopoietic System

Abstract Loss of heterochromatin has been proposed as a universal mechanism of aging across different species and cell types. However, a comprehensive analysis of hematopoietic changes caused by heterochromatin loss is lacking. Moreover, there is conflict in the literature around the role of the major heterochromatic histone methyltransferase Suv39h1 in the aging process. Here, we use individual and dual deletion of Suv39h1 and Suv39h2 enzymes to examine the causal role of heterochromatin loss in hematopoietic cell development. Loss of neither Suv39h1 nor Suv39h2 individually had any effect on hematopoietic stem cell function or the development of mature lymphoid or myeloid lineages. However, deletion of both enzymes resulted in characteristic changes associated with aging such as reduced hematopoietic stem cell function, thymic involution and decreased lymphoid output with a skewing toward myeloid development, and increased memory T cells at the expense of naive T cells. These cellular changes were accompanied by molecular changes consistent with aging, including alterations in nuclear shape and increased nucleolar size. Together, our results indicate that the hematopoietic system has a remarkable tolerance for major disruptions in chromatin structure and reveal a role for Suv39h2 in depositing sufficient H3K9me3 to protect the entire hematopoietic system from changes associated with premature aging.

scholarly journals Albumin Modifies Responses to Hematopoietic Stem Cell Mobilizing Agents in Mice

Cells ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 4 ◽  
Author(s):  
Eva Danner ◽  
Halvard Bonig ◽  
Eliza Wiercinska
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Wild Type ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Split Dose ◽  
Healthy Donors ◽  
Hematopoietic Stem Cell Mobilization ◽  
Csf Levels ◽  
Cell Mobilization

Albumin, the most abundant plasma protein, not only controls osmotic blood pressure, but also serves as a carrier for various small molecules, including pharmaceuticals. Its impact on pharmacological properties of many drugs has been extensively studied over decades. Here, we focus on its interaction with the following mobilizing agents: Granulocyte-colony stimulating factor (G-CSF) and AMD3100, where such analyses are lacking. These compounds are widely used for hematopoietic stem cell mobilization of healthy donors or patients. Using albumin-deficient (Alb−/−) mice, we studied the contribution of albumin to mobilization outcomes. Mobilization with the bicyclam CXCR4 antagonist AMD3100 was attenuated in Alb−/− mice compared to wild-type littermates. By contrast, mobilization with recombinant human G-CSF (rhG-CSF), administered twice daily over a five-day course, was significantly increased in Alb−/− mice. In terms of a mechanism, we show that rhG-CSF bioavailability in the bone marrow is significantly improved in Alb−/− mice, compared to wild-type (WT) littermates, where rhG-CSF levels dramatically drop within a few hours of the injection. These observations likely explain the favorable mobilization outcomes with split-dose versus single-dose administration of rhG-CSF to healthy donors.

scholarly journals Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo

2019 ◽  
Vol 116 (4) ◽  
pp. 1447-1456 ◽  
Author(s):  
Rong Lu ◽  
Agnieszka Czechowicz ◽  
Jun Seita ◽  
Du Jiang ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Lineage Commitment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Different Levels

While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.

Modeling Anemia of Aging in Inbred Mouse Strains

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3444-3444
Author(s):  
Luanne L. Peters ◽  
Shirng-wern Tsaih ◽  
Rong Yuan
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Kidney Function ◽  
Inbred Mouse ◽  
Mouse Strains ◽  
Inbred Mouse Strains ◽  
Hematopoietic Stem ◽  
Age Related ◽  
Mouse Phenome Database

Abstract Anemia of aging is now recognized as a significant medical problem. The National Health and Nutrition Examination Survey (NHANES III) revealed a steady increase in anemia in both males and females after the age of 50. Based upon the WHO definition of anemia (<13 g/dL hemoglobin (Hgb) in men; <12 g/dL in women), ~10% of the community dwelling population ≥ 65 years of age are anemic. Underlying causes fall into three broad groups, each representing ~1/3 of cases: nutritional deficits/blood loss; inflammation, kidney disease and myelodysplasia; and unexplained anemia. Although anemia of aging is usually mild, it is no longer considered a normal part of aging. It is associated with poor health and increased vulnerability to adverse outcomes in a multitude of circumstances, placing an enormous burden on the healthcare system that will only grow as the population continues to age. As part of The Jackson Laboratory Aging Center (http://agingmice.jax.org/), we are performing an extensive phenotypic analysis of multiple traits related to aging in 32 inbred mouse strains. All data are, or will be upon completion, publicly available via the Mouse Phenome Database (MPD, www.jax.org/phenome). Complete blood counts were obtained at 6, 12, 18, and 24 months of age in 30 strains. Two-way ANOVA reveals that both strain and age significantly impact Hgb in mice. A highly significant strain-by-age interaction is also seen. Substantial inter-strain and within strain sex variability in the decline in Hgb levels with age is seen among the strains analyzed, suggesting genetic influences. Significant declines in Hgb levels in females at 18 and/or 24 months vs. 6 months occurred in 21 of the 30 strains and, in males, 17 strains. Haplotype association mapping (HAM) using a dense SNP panel identified multiple distinct, age-related loci influencing Hgb levels. For example, a locus on chromosome (Chr) 13 significantly associated with Hgb levels at 12 months of age in males was not detected even at the suggestive level at 18 months of age where two new highly significant loci emerged (Chrs 14, 17). Only two strains show a statistically significant increase in percent circulating reticulocytes with age, indicative of a proliferative anemia. Failure of a significant reticulocyte response in all other strains suggests that an age-related compromise in bone marrow function (hematopoiesis-restricted anemia) predominates in aged, anemic mice. The ratio of urinary albumin to creatinine (ACR) is commonly used as an indicator of kidney damage in mice. In females, the ACR is stable and does not rise significantly with age in the majority of strains, suggesting that declining kidney function is not a major cause of anemia of aging in female inbred mice. Significant increases in IL-6 and TNFα are seen in strains 129SvImJ, C3H/HeJ, and DBA/2J, suggesting a pro-inflammatory state. From this preliminary analysis of a large ongoing project, we can conclude: Hgb levels in mice vary significantly by strain and sex, and decline significantly with age in many strains. Other baseline hematological traits (e.g., red blood cell counts, platelet counts) likewise vary by strain, age and sex. These data are available via the Mouse Phenome Database (project Peters4). The anemia of aging seen in most strains correlates most closely with restricted hematopoiesis, as indicated by the failure of the reticulocyte count to increase in response to declining Hgb levels. There is growing evidence that decrements in hematopoietic stem cell number and function play a role in the aging process in humans. Notably, hematopoietic stem cell numbers and bone marrow cellularity data will be available on the MPD as these analyses are completed. HAM analysis suggests that distinct age-related loci influence Hgb levels in mice. In a small subset of strains, anemia of aging may reflect declining kidney function, as occurs in humans. Preliminary data suggests an increase in cytokine levels in some strains, again mimicking the aging human population. Increased IL-6 levels as a cause of anemia of aging is of particular interest due to its inhibition of hepcidin and thus iron availability. Overall, the data indicate that anemia of aging occurs in mice and models that seen in elderly human populations. Additional data including iron levels, T4, BUN, and more on aging inbred mouse strains will be posted to the MPD in the near future.

Lnk Controls Hematopoietic Stem Cell Self-Renewal through Direct Interactions with JAK2 and Contributes to Oncogenic JAK2-Induced Myeloproliferative Diseases in Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 895-895
Author(s):  
Alexey Bersenev ◽  
Chao Wu ◽  
Joanna Balcerek ◽  
Wei Tong
Keyword(s):  
Stem Cell ◽  
Signaling Pathways ◽  
Hematopoietic Stem Cell ◽  
Adaptor Protein ◽  
Janus Kinase ◽  
Myelogenous Leukemia ◽  
Cell Surface Receptor ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Myeloproliferative Diseases

Abstract Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2) that triggers a cascade of downstream signal transduction pathways that regulates many aspects of cell development. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. Surprisingly, we found that Lnk−/− HSCs have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeated 5-Florouracil (5-FU) treatments in vivo compared to wild type HSCs. We further provided genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal predominantly through Mpl. Consistent with this observation, Lnk deficiency in HSCs potentiates JAK2 activation in response to TPO. Biochemical experiments reveal that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Dysregulation of cytokine receptor signaling pathways leads to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML). Recently, the JAK2 V617F mutation has been observed at high frequencies in several myeloproliferative diseases (MPDs). The JAK2V617F mutant retains Lnk binding ability, suggesting Lnk status could modify MPD development. Indeed, we found that loss of Lnk accelerates oncogenic JAK2- induced CML/MPD in the mouse transplant models. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may contribute to leukemic transformation conferred by oncogenic JAK2.

A Novel MBT-Containing Protein, Hemp, Plays Essential Roles In Skeletal Formation and Hematopoietic Stem Cell Function.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1597-1597
Author(s):  
Phyo Wai Htun ◽  
Keiyo Takubo ◽  
Hideaki Oda ◽  
Feng Ma ◽  
Kenjiro Kosaki ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Conditional Knockout ◽  
Thoracic Vertebrae ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Epigenetic Regulator ◽  
Skeletal Formation

Abstract Abstract 1597 Hemp (hematopoietic expressed mammalian polycomb, also denoted as mbt-containing 1) gene was originally identified in the hematopoietic stem cell (HSC)-enriched fraction of the mouse fetal liver (FL). It encodes a protein containing a putative Cys2-Cys2 zinc-finger region, followed by four tandem malignant brain tumor (MBT) repeats, which is frequently observed in polycomb gene (PcG) proteins. The structural characteristics strongly suggest that Hemp functions as an epigenetic regulator, but its biological role remains unknown. To address this issue, we generated hemp-deficient (hemp–/–) mice. Hemp–/– mice died soon after birth. Although no abnormalities were detected in internal organs, skeletal analysis exhibited a variety of malformations. Severe deformities were observed in the thoracic cavity, strongly suggesting that hemp–/– mice died of respiratory failure. Interestingly, they showed malformations of cervical and thoracic vertebrae, which were different from typical homeotic transformations observed in PcG-deficient mice. These results suggest that Hemp governs downstream target genes in distinct manners from conventional PcG proteins. The hematopoietic analysis of hemp in the FL showed that hemp is preferentially expressed in CD150+LSK and CD150–LSK HSC fractions in the hematopoietic hierarchy. Hemp–/– FL contained a significantly reduced number of hematopoietic cells and produced fewer number of hematopoietic colonies as compared to hemp+/+ FL. The decreases correlated with reduced number of CD150+LSK HSCs in hemp–/– FL, which generated much fewer hematopoietic colonies in the HPP-CFC assay. In addition, the competitive repopulation assay exhibited that the hematopoietic reconstitution ability of hemp–/– FL CD150+LSK HSCs was significantly impaired. Moreover, microarray analysis revealed that expression levels of several genes, such as Prdm16, Sox4, and Erdr1 were altered in hemp–/– FL HSCs. Since hemp–/– mice died at neonate, the role of Hemp in adult hematopoiesis remains to be elucidated. To address this issue, we generated hemp conditional knockout (cKO) mice. Acquired deletion of Hemp in the hematopoietic tissues was successfully achieved by crossing hempflox/flox mice with MxCre mice and stimulating the compound mice with pIpC. Analysis of the hematopoietic tissues revealed that the cell numbers of Mac+Gr1– and Mac+Gr1+ fractions in the hemp cKO bone marrow (BM) were significantly increased and decreased, respectively, as compared to those of the wild-type BM. However, no apparent differences have so far been observed between hemp cKO and wild-type littermates in functional analyses, such as colony forming activity and competitive repopulation ability of the BM cells. Here, we report that a novel MBT-containing protein, Hemp, plays essential roles in skeletal formation and HSC function during embryogenesis and also contributes to myeloid differentiation in adult hematopoiesis. Since Hemp likely functions as an epigenetic regulator, further studies will be required to clarify whether and what methylated lysine residues Hemp interacts with through the MBT repeats, what kind of genes are direct targets of Hemp, and how Hemp exerts its biological activity. Disclosures: No relevant conflicts of interest to declare.

The F-Box Protein NIPA Regulates the Hematopoietic Stem Cell Pool

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2330-2330
Author(s):  
Stefanie Kreutmair ◽  
Anna Lena Illert ◽  
Rouzanna Istvanffy ◽  
Melanie Sickinger ◽  
Christina Eckl ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Pool ◽  
F Box Protein ◽  
Stem Cell Pool

Abstract Abstract 2330 Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation and the expression of numerous cell cycle regulators must be tightly controlled. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. To examine the function of NIPA on vivo, we generated NIPA deficient animals, which are viable but sterile due to a defect in recombination and testis stem cell maintenance. To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in NIPA deficient animals. Peripheral blood counts taken at different ages revealed no apparent difference between NIPA knockout and wild type mice in numbers and differentiation. In contrast, looking at the hematopoietic stem cell pool, FACS analyses of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA deficient animals, where LSKs were reduced to 40% of wild type littermates (p=0,0171). This effect was only apparent in older animals, where physiologically higher LSK numbers have to compensate for the exhaustion of the stem cell pool. Additionally, older NIPA deficient mice have only half the amount of multi myeloid progenitors (MMPs) in contrast to wild type animals. To examine efficient activation of stem cells to self-renew in response to myeloid depression, we treated young and old mice with the cytotoxic drug (5-FU) four days before bone marrow harvest. As expected, 5-FU activated hematopoietic progenitors in wild type animals, whereas NIPA deficient progenitors failed to compensate to 5-FU depression, e.g. LSKs of NIPA knockout mice were reduced to 50% of wild type levels (p<0.001), CD150+CD34+ Nipa deficient cells to 20% of wild type levels (p<0.0001). Interestingly, these effects were seen in all NIPA deficient animals independent of age, allowing us to trigger the self-renewal phenotype by activating the hematopoietic stem cell pool. Using competitive bone marrow transplantation assays, CD45.2 positive NIPA deficient or NIPA wild type bone marrow cells were mixed with CD45.1 positive wild type bone marrow cells and transplanted into lethally irradiated CD45.2 positive recipient mice. Thirty days after transplantation, FACS analysis of peripheral blood and bone marrow showed reduced numbers of NIPA knockout cells in comparison to NIPA wild type bone marrow recipient mice. This result was even more severe with aging of transplanted mice, where NIPA deficient cells were reduced to less than 10% of the level of wild type cells in bone marrow of sacrificed mice 6 months after transplantation, pointing to a profound defect in repopulation capacity of NIPA deficient HSCs. Taken together our results demonstrate a unique and critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures: No relevant conflicts of interest to declare.

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