Hematopoietic Stem Cell Function Research Articles

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

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

Hematopoietic stem cell quiescence and DNA replication dynamics maintained by the resilient β-catenin/Hoxa9/Prmt1 axis

AbstractMaintenance of quiescence and DNA replication dynamics are 2 paradoxical requirements for the distinct states of dormant and active hematopoietic stem cells (HSCs), which are required to preserve the stem cell reservoir and replenish the blood cell system in response to hematopoietic stress, respectively. Here, we show that key self-renewal factors, β-catenin or Hoxa9, largely dispensable for HSC integrity, in fact, have dual functions in maintaining quiescence and enabling efficient DNA replication fork dynamics to preserve the functionality of hematopoietic stem and progenitor cells (HSPCs). Although β-catenin or Hoxa9 single knockout (KO) exhibited mostly normal hematopoiesis, their coinactivation led to severe hematopoietic defects stemmed from aberrant cell cycle, DNA replication, and damage in HSPCs. Mechanistically, β-catenin and Hoxa9 function in a compensatory manner to sustain key transcriptional programs that converge on the pivotal downstream target and epigenetic modifying enzyme, Prmt1, which protects the quiescent state and ensures an adequate supply of DNA replication and repair factors to maintain robust replication fork dynamics. Inactivation of Prmt1 phenocopied both cellular and molecular phenotypes of β-catenin/Hoxa9 combined KO, which at the same time could also be partially rescued by Prmt1 expression. The discovery of the highly resilient β-catenin/Hoxa9/Prmt1 axis in protecting both quiescence and DNA replication dynamics essential for HSCs at different key states provides not only novel mechanistic insights into their intricate regulation but also a potential tractable target for therapeutic intervention.

IL-1β promotes MPN disease initiation by favoring early clonal expansion of JAK2-mutant hematopoietic stem cells

AbstractJAK2-V617F is the most frequent somatic mutation causing myeloproliferative neoplasm (MPN). JAK2-V617F can be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the prevalence of MPNs. The factors controlling the conversion of JAK2-V617F CHIP to MPN are largely unknown. We hypothesized that interleukin-1β (IL-1β)–mediated inflammation can favor this progression. We established an experimental system using bone marrow (BM) transplantations from JAK2-V617F and GFP transgenic (VF;GFP) mice that were further crossed with IL-1β−/− or IL-1R1−/− mice. To study the role of IL-1β and its receptor on monoclonal evolution of MPN, we performed competitive BM transplantations at high dilutions with only 1 to 3 hematopoietic stem cells (HSCs) per recipient. Loss of IL-1β in JAK2-mutant HSCs reduced engraftment, restricted clonal expansion, lowered the total numbers of functional HSCs, and decreased the rate of conversion to MPN. Loss of IL-1R1 in the recipients also lowered the conversion to MPN but did not reduce the frequency of engraftment of JAK2-mutant HSCs. Wild-type (WT) recipients transplanted with VF;GFP BM that developed MPNs had elevated IL-1β levels and reduced frequencies of mesenchymal stromal cells (MSCs). Interestingly, frequencies of MSCs were also reduced in recipients that did not develop MPNs, had only marginally elevated IL-1β levels, and displayed low GFP-chimerism resembling CHIP. Anti–IL-1β antibody preserved high frequencies of MSCs in VF;GFP recipients and reduced the rate of engraftment and the conversion to MPN. Our results identify IL-1β as a potential therapeutic target for preventing the transition from JAK2-V617F CHIP to MPNs.

Deciphering the differential impact of thrombopoietin/MPL signaling on hematopoietic stem/progenitor cell function in bone marrow and spleen

Thrombopoietin (TPO) and its receptor MPL play crucial roles in hematopoietic stem cell (HSC) function and platelet production. However, the precise effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches remain unclear. Here, we investigated the effects of TPO/MPL ablation on marrow and splenic hematopoiesis in TPO-/- and MPL-/- mice during aging. Despite severe thrombocytopenia, TPO-/- and MPL-/- mice did not develop marrow failure during a 2-year follow-up. Marrow and splenic HSCs exhibited different responses to TPO/MPL ablation and exogenous TPO treatment. Splenic niche cells compensated for marrow HSC loss in TPO-/- and MPL-/- mice by upregulating CXCL12 levels. These findings provide new insights into the complex regulation of HSCs by TPO/MPL and reveal a previously unknown link between TPO and CXCL12, two key growth factors for HSC maintenance. Understanding the distinct regulatory mechanisms between marrow and spleen hematopoiesis will help to develop novel therapeutic approaches for hematopoietic disorders.

Schistosome Infection Impacts Hematopoiesis.

Helminth infections are common in animals. However, the impact of a helminth infection on the function of hematopoietic stem cells (HSCs) and other hematopoietic cells has not been comprehensively defined. In this article, we describe the hematopoietic response to infection of mice with Schistosoma mansoni, a parasitic flatworm that causes schistosomiasis. We analyzed the frequency or number of hematopoietic cell types in the bone marrow, spleen, liver, thymus, and blood and observed multiple hematopoietic changes caused by infection. Schistosome infection impaired bone marrow HSC function after serial transplantation. Functional HSCs were present in the infected liver. Infection blocked bone marrow erythropoiesis and augmented spleen erythropoiesis, observations consistent with the anemia and splenomegaly prevalent in schistosomiasis patients. This work defines the hematopoietic response to schistosomiasis, a debilitating disease afflicting more than 200 million people, and identifies impairments in HSC function and erythropoiesis.

Isolation of Murine Neonatal and Adult Osteomacs to Examine Their Role in the Hematopoietic Niche.

Maintenance of hematopoietic stem cell (HSC) function is an orchestrated event between multiple cell types, and crosstalk between these cell types is an essential part of HSC regulation. Among the cell groups of the niche involved in this process are a group of bone-resident macrophages known as osteomacs (OM). Previously, it was demonstrated that OM and osteoblasts contained within neonatal calvarial cells are critical to maintain hematopoietic function. Additionally, interactions between neonatal calvarial cells and megakaryocytes further enhance this hematopoietic activity. In this chapter, we explore one such interaction involving OM and osteoblasts in the hematopoietic niche. We describe a protocol to isolate OM from both neonatal and adult mice, and subsequently use colony-forming assays to demonstrate their interaction with osteoblasts in maintaining HSC function.

Lipotoxicity-Related Hematological Disorders in Obesity.

Lipotoxicity can mediate endothelial dysfunction in obesity. Altered endothelial cell phenotype during the pathobiological course of the lipotoxicity may lead to hemostatic abnormalities, which is a hallmark of several hematological disorders. Impaired hemostasis could also be directly related to numerous metabolic diseases such as hypertension, diabetes, and atherosclerosis. On the other hand, the local hematopoietic bone marrow (BM) renin-angiotensin system (RAS) contributes to the development of atherosclerosis via acting on the lipotoxicity processes. Local BM RAS, principally an autocrine/paracrine/intracrine hematological system, is located at the crossroads of cellular regulation, molecular interactions, and lipotoxicity-mediated vascular endothelial dysfunction. The positive regulatory role of plasma LDL on AT1 receptor-mediated hematopoietic stem cell (HSC) differentiation and the production of pro-atherogenic monocytes have been described. LDL-regulated HSC function may explain in part hypercholesterolemia-induced inflammation as well as the anti-inflammatory and anti-atherosclerotic effects of AT1 receptor blockers. The role of local adipose tissue RAS is directly related to the pathogenesis of metabolic derangements in obesity. There may be a crosstalk between local BM RAS and local adipose tissue RAS at the genomics and transcriptomics levels. This chapter aims to review hematological alterations propagating the pathological influences of lipotoxicity on the vascular endothelium.

Chapter 3 - The hematopoietic niche and the autoreactive memory in autoimmune disorders

Hematopoiesis is a complex process that takes place inside the bone marrow, where a specialized structure, the bone marrow niche, participates in the maintenance of hematopoietic stem cell functionality. Inflammatory conditions, such as autoimmune diseases, could alter this equilibrium leading to pathologic consequences. Immune cells, which also reside in the bone marrow, directly participate in sustaining the inflammatory state in autoimmune diseases. In particular, memory lymphocytes are key players in the long-term maintenance of the immune response against self-antigens, causing tissue damage and bone marrow alterations.

Linking GATA2 to myeloid dysplasia and complex cytogenetics in adult myelodysplastic neoplasm and acute myeloid leukemia

AbstractGATA binding protein 2 (GATA2) is a conserved zinc finger transcription factor that regulates the emergence and maintenance of complex genetic programs driving development and function of hematopoietic stem and progenitor cells (HSPCs). Patients born with monoallelic GATA2 mutations develop myelodysplastic neoplasm (MDS) and acute myeloid leukemia (AML), whereas acquired GATA2 mutations are reported in 3% to 5% of sporadic AML cases. The mechanisms by which aberrant GATA2 activity promotes MDS and AML are incompletely understood. Efforts to understand GATA2 in basic biology and disease will be facilitated by the development of broadly efficacious antibodies recognizing physiologic levels of GATA2 in diverse tissue types and assays. Here, we purified a polyclonal anti-GATA2 antibody and generated multiple highly specific anti-GATA2 monoclonal antibodies, optimized them for immunohistochemistry on patient bone marrow bioosy samples, and analyzed GATA2 expression in adults with healthy bone marrow, MDS, and acute leukemia. In healthy bone marrow, GATA2 was detected in mast cells, subsets of CD34+ HSPCs, E-cadherin–positive erythroid progenitors, and megakaryocytes. In MDS, GATA2 expression correlates with bone marrow blast percentage, positively correlates with myeloid dysplasia and complex cytogenetics, and is a nonindependent negative predictor of overall survival. In acute leukemia, the percent of GATA2+ blasts closely associates with myeloid lineage, whereas a subset of lymphoblastic and undifferentiated leukemias with myeloid features also express GATA2. However, the percent of GATA2+ blasts in AML is highly variable. Elevated GATA2 expression in AML blasts correlates with peripheral neutropenia and complex AML cytogenetics but, unlike in MDS, does not predict survival.

Hematopoietic Stem Cells from Mice and Individuals with Sickle Cell Disease Display Premature Senescence and Loss of Function That Is Targetable By Senolytic Therapy

Sickle cell disease (SCD) is an inherited anemia caused by mutations in the β-globin gene, resulting in aberrant hemoglobin polymerization driving hemolysis, vasooclusion, and systemic inflammation. Allogeneic hematopoietic stem cell (HSC) transplantation is potentially curative, and novel autologous therapies involving gene-editing of mobilized HSCs are being developed. However, many SCD patients are unable to undergo autologous gene-editing due to failure to mobilize sufficient numbers of HSCs. Further, the damaging effects of SCD on the HSC pool remain uncharacterized. Therefore, we interrogated young and aged cohorts of humanized SCD mice and non-SCD controls for HSC frequency and function. Immunophenotypic HSC frequency was mildly elevated (1.3-fold, p=0.00316) in 2-month-old SCD mice, relative to controls (n=5-6/group). In contrast, HSC numbers were halved in 6 and 12-month-old SCD mice (n=8-10/group, p=0.00358 and p=0.0148, respectively). Competitive transplantation of whole bone marrow (WBM) from 2 and 6-month-old SCD mice displayed a profound loss of peripheral blood repopulation, relative to age-matched control mice. Quantitative limiting dilution transplantation of WBM from 2 and 6-month-old SCD mice revealed a 4 and 6-fold loss of functional HSCs, relative to control mice. Thus, despite elevated numbers of phenotypic HSCs in young SCD mice, the HSC pool is depleted of functional repopulating cells during SCD. To understand the loss of functional HSCs, we performed bulk RNA-sequencing of purified HSCs from 6-month-old SCD mice. We observed downregulation of 37 genes encoding ribosomal and histone proteins, suggesting impaired histone/ribosome biogenesis, a phenotype associated with senescence. Consistently, pathway enrichment analysis also revealed perturbed p53 activity and a high degree of association with published molecular signatures of senescence. To explore this further, HSCs isolated from 6-month-old SCD mice or age-matched controls were interrogated for senescence associated β-galactosidase (SA-β-gal) activity. On average 19.5% of SCD HSCs were SA-β-gal+ versus only 7.06% of HSCs isolated from controls (n=10-11/group, p=0.004). HSCs isolated from SCD mice also displayed elevated ROS, DNA damage, and expression of molecular markers of senescence (e.g. p21, BCLXL). Importantly, 23.14 ± 5.61% of Lin-CD34+CD38- bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) isolated from pediatric individuals with SCD were SA-β-gal+. In contrast, only 4.97 ± 2.97% of HSPCs were SA-β-gal+ in age-matched, non-SCD control individuals (n=8 control and n=5 SCD individuals, p=<0.0001). Further, 47.46 ± 17.49% of Lin-CD34+CD38-CD90+CD45RA- BM cells, which are highly enriched for human HSCs, were SA-β-gal+ in young individuals with SCD, relative to only 4.41 ± 2.37% in the BM of age-matched control individuals (n=4 control and n=5 SCD individuals, p=0.0019). We also detected elevated expression of p16, p21, and BCL2 in BM HSPCs isolated from pediatric individuals with SCD, relative to controls (n=6 control and n=9 SCD individuals). In total, these data reveal that SCD pathophysiology aberrantly invokes senescence programs and damages HSC function in mice and humans. Clearance of senescent HSCs from mouse BM has previously been shown to restore aging-related HSC dysfunction. We therefore tested if clearance of senescent HSCs from the BM could restore function to the SCD HSC pool. 2-month-old SCD and control mice were treated with two weekly cycles of 75 mg/kg/day of the senolytic, ABT-263, via oral gavage. 14 days after treatment, WBM was isolated and tested for HSC function and frequency. Senolytic therapy significantly increased HSC numbers in SCD mice BM, relative to vehicle-treated controls (n=5/group). Importantly, the in vivo hematopoietic repopulating activity of WBM from ABT-263-treated SCD mice was comparable to non-SCD controls, revealing a correction of our previously observed severe transplantation defect (n=12/group). In sum, these data reveal premature onset of senescence as a previously unappreciated phenotype of the HSC pool during SCD. This work provides the first proof-of-principle that treatment of individuals with SCD using senolytics may restore function to the HSC pool, and ongoing preclinical work aims to test if senolytic therapy enhances the yield and function of SCD HSCs mobilized for autologous therapy.

Deciphering the Differential Impact of Thrombopoietin/MPL Signaling on Hematopoietic Stem Cell Function in Bone Marrow and Spleen

Introduction Thrombopoietin (TPO) and its receptor MPL are key regulators of hematopoietic stem cell (HSC) function. In humans, ablation of either TPO or MPL leads to bone marrow failure in the early years of life. In contrast, mice lacking TPO or MPL display marked thrombocytopenia and severely reduced HSC numbers but manage to maintain normal survival up to one year of age. In this study, we investigated whether these mice eventually develop marrow failure with longer follow up, with a specific focus on understanding the effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches. Methods Marrow and spleen hematopoiesis in the TPO -/- mice and MPL -/- mice were examined during a 2-yr follow-up. Results At 2 yrs of age, TPO -/- and MPL -/- mice exhibited not only a severely decreased platelet count (TPO -/- vs. MPL -/- vs. control: 38 vs. 107 vs. 1255 x 10 9/L) but also a moderate reduction in neutrophil count compared to age-matched wild-type controls (TPO -/- vs. MPL -/- vs. control: 1.1 vs. 1.7vs.3.3 x 10 3/ul). Femur marrow cell count or spleen cell count was not significantly different between TPO -/- or MPL -/- mice and control mice. There was no significant difference in overall survival between the TPO -/- or MPL -/- mice and wild-type controls. Marrow Lin -cKit +Sca1 +CD150 +CD48 - HSC cell numbers were significantly decreased in young (4-6mo) TPO -/- and MPL -/- mice (~4-fold) compared to age-matched control mice. This HSC loss was further worsened in old (~24mo) TPO -/- (25-fold) and MPL -/- (12-fold) mice compared to control mice. In contrast, there was no significant difference in splenic HSC cell numbers between young TPO -/- or MPL -/- mice and control mice, with only a moderate decrease of HSCs in old TPO -/- and MPL -/- (~4-fold) mice compared to control mice. I n vivo using BrdU labeling revealed an increased proliferation of marrow HSCs in TPO -/- and MPL -/- mice compared to age-matched control mice. In contrast, there was no significant increase in splenic HSC proliferation in TPO -/- or MPL -/- mice. These findings suggest that TPO/MPL signaling has distinct effects on both the number and the function of HSCs in the marrow and spleen. Competitive repopulation assays revealed that HSCs from 2yr old TPO -/- or MPL -/- marrow failed to engraft the recipient mice at 12 weeks post-transplantation. We also conducted competitive repopulation assays in which spleen cells from old TPO -/- mice (CD45.2) were injected together with spleen cells from 2yr old wild-type competitor mice (CD45.1) into lethally irradiated wild-type recipients (CD45.1). At 12 days after transplantation, the TPO -/- spleen donor showed comparable engraftment in the Lin -cKit +Sca1 + stem/progenitor cell compartment, similar to the wild-type competitor spleen donor, suggesting that at least the short-term engraftment capacity of the TPO -/- spleen donor was not impaired. We are currently conducting further investigation to assess the long-term repopulation capacity of TPO -/- spleen HSCs. To investigate how TPO/MPL ablation impacts the hematopoietic niche in the marrow and spleen, we used quantitative flow cytometry and/or fluorescence imaging to examine these niche cells and their expression of niche factors. First, while there was no significant difference in the marrow vascular area (measured by in vivo VE-cadherin labeling) between TPO -/- or MPL -/- mice and control mice, we observed a significant increase in the splenic vascular area in TPO -/- and MPL -/- mice. Second, CXCL12 levels were significantly decreased in marrow endothelial cells (ECs; CD45 -CD31 +) from TPO -/- and MPL -/- mice compared to control mice; in contrast, CXCL12 levels were significantly increased in splenic ECs from TPO -/- and MPL -/- mice. Similar results were also observed with marrow and splenic perivascular stromal cells (CD45 -CD31 -Ter119 -Sca1 -). Therefore, TPO/MPL ablation enhanced both the quantity and quality of the splenic hematopoietic niche, which could contribute to the maintenance of splenic hematopoiesis in TPO -/- and MPL -/- mice. Last, consistent with findings from the TPO/MPL ablated mice, exogenous TPO treatment increased marrow HSC proliferation but did not alter splenic HSC proliferation. In addition, TPO treatment increased CXCL12 expression in marrow ECs, but decreased CXCL12 levels in splenic ECs. Taken together, our study revealed the complexity of TPO/MPL signaling in HSC regulation which differs between the marrow and spleen

MYCT1 Controls Environmental Sensing to Maintain Self-Renewal in Human Hematopoietic Stem Cells

Hematopoietic stem cells (HSC) sustain life-long blood production and have the ability to recapitulate the hematopoietic system upon transplantation. This makes them therapeutically invaluable for the treatment of hematopoietic malignancies and genetic disorders. However, the regulatory processes governing HSC functional competence and engraftment ability are poorly understood and challenging to recapitulate ex vivo, making robust expansion of human HSCs difficult to achieve. We have identified a novel HSC regulatory protein, MYCT1 (MYC target 1), that is highly enriched in self-renewing human HSC but lost during differentiation and culture. Knockdown (KD) experiments in human fetal liver and cord blood revealed a critical requirement for MYCT1 in human HSPC ex vivo expansion and engraftment across HSC ontogeny. Single cell RNAseq of human HSPCs upon MYCT1 KD linked MYCT1 loss in HLF+ HSCs to a profound dysregulation of cellular programs essential for HSC stemness, including mitochondrial activity, proteostasis and splicing. Because MYCT1 becomes drastically downregulated during HSC ex vivo culture, we investigated if restoring MYCT1 expression in culture could improve HSC function. Indeed, maintaining MYCT1 levels during culture via lentiviral expression resulted in improved “stemness” transcriptional signatures, greater expansion of the most undifferentiated EPCR+ITGA3+ human HSPCs, and enhanced engraftment ability upon transplantation. Notably, we also uncovered that the novel albumin free culture conditions reported by Sakurai et al. 2023 to robustly expand human HSCs can also maintain higher levels of MYCT1 expression. This reinforces our findings that the loss of MYCT1 expression is a major contributor to the functional incompetence of human HSCs cultured ex vivo. Nonetheless, the MYCT1 regulated processes and why they are critical for HSC competence are not known. Immunofluorescence and IP-mass spectrometry revealed that MYCT1 localizes in the endosomal membrane and interacts with vesicle trafficking machinery and receptor signaling components essential for HSC function. MYCT1 KD in human HSCs and endothelial cells (EC) led to hyperactive endocytosis and caused exaggerated signaling responses to cytokines in the culture microenvironment. We found that ex vivo culture gradually increased endocytosis specifically in CD34+ EPCR+ HSPCs in parallel with the culture-induced downregulation of MYCT1 in these cells, but not in the more differentiated progeny. On the other hand, restoring MYCT1 expression during HSC culture dampened the excessive endocytosis. Furthermore, maintaining low endocytosis during culture, either by the endocytosis inhibitor chlorpromazine or by sorting HSPCs with lower endocytic internalization of fluorescent dextran, results in the enrichment of immunophenotypic LT-HSCs in culture. Our data show that the moderation of environmental sensing through MYCT1-controlled endocytosis is essential for preserving human HSC self-renewal and engraftment ability. As MYCT1 expression is downregulated in cultured human HSPC, our findings imply that loss of the molecular machinery required for proper sensing of microenvironment signals has far-reaching effects into essential cellular functions required for stemness, and is a key contributor to the functional incompetence of cultured human HSC.

The Effect of SCD-1 Inhibition on Human Hematopoietic Stem Cell Mitochondrial Metabolism, Cell Proliferation, and Differentiation Potential

Hematopoietic stem cells (HSC) give rise to all blood lineages and sustain the production of blood cells throughout life. Due to their inherently high regenerative potential, HSC are used in a variety of clinical settings, including bone marrow transplantation (BMT) directly to cure a variety of hematologic and oncologic disorders often with gene therapy. During regeneration, HSC are activated into cycle. For this, HSC undergo drastic mitochondrial and metabolic remodeling to meet the bioenergetic and biosynthetic needs of activated HSC. A growing body of evidence indicates that HSC sustain injury during activation. This remodeling is important for optimal HSC function but is permanently changed after HSC activating stress. It is thus important to identify the metabolic needs of activated HSC to improve HSC functions for therapeutic purposes. In both murine and human HSC, numerous metabolic enzymes get upregulated during activation, including those involved in de novo lipid synthesis. This metabolism is poorly understood in human HSC. Our overarching question is to understand the metabolic needs of HSC within the human system. Stearoyl-co-A-desaturase 1 (SCD-1), an enzyme responsible for conversion of stearic to oleic acid within the de novo lipid synthesis pathway, is suspected to play a role in metabolic reprogramming after stress. Using CD34+ mobilized human peripheral blood, we analyzed the effect of SCD-1 inhibition on HSC and progenitor proliferation, mitochondrial metabolism, and lineage differentiation potential. Cells were cultured either in vehicle control conditions or with SCD-1 inhibitor (SCDi). Subsequently, cells were counted and analyzed by flow cytometry on Days 4-5 and 10-15. More primitive or stem cell markers were utilized on Days 0 and 5 whereas differentiation markers were utilized on Day 10-15. Both tetramethylrhodamine-ethyl ester dye (TMRE) and MitoSOX reagents were used to assess mitochondrial membrane potential and mitochondrial ROS, respectively. Our results demonstrate that the size of the HSC population generated by SCDi-treated CD34+ cells within 5 days of culture was similar to vehicle treated cells and was composed of similar proportion of CD34+CD38+, CD34+CD38-, and CD38+CD34- cells, indicating that SCD-1 inhibition does not alter HSC expansion under these culture conditions. However, TMRE levels were lower in all HSC populations whereas mitoSOX levels were unchanged by SCD-1 inhibition compared to vehicle. In differentiation conditions, SCDi cultures were composed of a larger proportion of myeloid progenitors and a smaller proportion of erythroid cells after 10-15 days, compared to vehicle-treated cultures. These findings suggest that de novo lipid synthesis is necessary for HSC differentiation but dispensable for proliferation/expansion in vitro. We then used a xenotransplant model to assess the effect of SCDi on HSC regenerative potential in vivo. CD34+ mobilized human peripheral blood cells were cultured for 3 days under conditions that maintain HSC functions in vitro and transplanted into immunodeficient, sublethally irradiated NSG mice. Peripheral blood of xenotransplanted mice was analyzed monthly for human cell chimerism and mature blood lineage potential from 1 to 6 months post-transplant. Bone marrow analysis was performed at the 6 month time point as well, assessing for both HSC markers and lineage markers. Mice that received SCDi-treated cells exhibited human cell chimerism at a level similar to mice that were transplanted with vehicle treated cells, at about 3 to 30%. However, SCDi-treated cells gave rise to a lymphoid-biased graft, in particular T cells, both in the peripheral blood and in the bone marrow of xenotransplanted mice, compared to a more balanced myeloid-lymphoid graft from vehicle treated cells. In conclusion, these findings suggest that de novo lipid synthesis is critically important for HSC lineage fate and balanced differentiation in vitro and in vivo. This is especially clinically relevant as this work may implicate a possible therapeutic target as improving de novo lipid synthesis may aid in patients who suffer from persistent cytopenias after BMT and graft failure.

The ATF4-RPS19BP1 Axis Modulates Ribosome Biogenesis to Promote Erythropoiesis

The function of hematopoietic stem cells (HSCs) and the process of hematopoiesis are intricately governed by a complex interplay of intrinsic programs and extrinsic signals from the microenvironment (Crane et al., Nat Rev Immunol, 2017). Previous work reported by our group and others has demonstrated that ATF4, a basic region-leucine zipper transcription factor, plays pivotal roles in fetal liver hematopoiesis, HSC maintenance, bone formation and tumorigenesis (Zhao et al., Blood, 2015; Sun et al., Sci Adv, 2021; Yang et al., Cell, 2004). However, the precise function of ATF4 in the bone marrow niche and how ATF4 regulates adult hematopoiesis remain largely unknown. Here, we employ four cell-type-specific mouse Cre lines to conditionally knock out Atf4 in Cdh5 + endothelial cells (ECs), Prx1 + bone marrow stromal cells (BMSCs), Osx + osteo-progenitor cells, and Mx1 + hematopoietic cells, and uncover the role of Atf4 in niche cells and hematopoiesis. We found that Atf4 deletion in BMSCs reduced BMSC numbers and impaired their CFU-F activity as well as differentiation toward osteoblasts, but spares hematopoiesis and HSC function in adult mice. Furthermore, Atf4 depletion in the ECs or osteo-progenitors of adult mice has a very mild effect on the BM hematopoiesis and HSC maintenance. Intriguingly, Atf4 depletion in the hematopoietic system resulted in severe anemia. More than 70% of the Mx1cre; Atf4 fl/fl (Δ/Δ) mice gradually died from anemia. Atf4 deficiency markedly reduced the frequency of erythroid progenitor cells (EPCs) (including MEP, PreCFU-E, and CFU-E cells) and impaired their CFU-E and BFU-E colony-forming ability. Atf4 depletion severely impaired the repopulation ability of BM cells and the self-renewal capacity of HSCs. Single cell RNA-seq (scRNA-seq) analysis of BM Lin -cKit + cells confirmed the defects of erythropoiesis after Atf4 depletion. The absence of ATF4 compelled early erythroid progenitors to enter the S phase, which led to replication stress and in turn activated both the DNA damage response and apoptosis. Subsequently, we conducted an integrative analysis of the genes that were identified as downregulated in MEPs by RNA-seq, ATAC-seq, and H3K4me3 Cut&Tag and genes that were identified as downregulated in erythroid progenitors (i.e., PreMegE, Ery1, and Ery2) by scRNA-seq, which predicted Rps19bp1 as the top target gene of Atf4. Luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays confirmed that ATF4 was a transcriptional activator of Rps19bp1. Downregulation of Rps19bp1 caused by Atf4 deletion led to decreased assembles of 40S proteins, such as RPS3, RPS6 and RPS19. O-propargyl-puromycin (OP-Puro) incorporation and SUnSET assay further confirmed the global protein synthesis defect in Atf4-deficient MEP cells. The protein synthesis defects and impaired erythropoiesis were rescued upon RPS19BP1 overexpression in Atf4 knockdown MEL cell line or BM cKit + cells from Atf4-deficient mice. Ribosome profiling of CMP cells from Δ/Δ and fl/fl mice further demonstrated that the expression of proteins involved in ribosome biogenesis was significantly lower in Atf4-depleted than in wildtype CMP cells. The expression of gene sets linked to PreCFU-E signature was also lower in Atf4-depleted than in wildtype CMPs, suggesting that the reduction in ribosome biogenesis significantly affected the translation efficiency of erythroid-related pathway components, thereby impeding erythroid lineage commitment. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, we demonstrated that, unlike in the fetal liver, ATF4 governs adult HSC function and erythropoiesis in a cell-intrinsic manner. We revealed a novel role for ATF4 in erythropoiesis, which links it to RPS19BP1, ribosome biogenesis and protein translation. Our findings have highlighted the crucial importance of protein synthesis regulation during erythroid lineage commitment. This discovery will likely have extended implications for understanding and treating ribosomopathy-associated erythroid failure.

A Longitudinal Study to Assess Hematopoietic Stem Cell and Immune Cell Health in Astronauts Pre-, during, and Post-Mission

The International Space Station (ISS) allows research to be done in low earth orbit (LEO), offering a uniquely accelerating environment to model disease and study human health. Studies done in microgravity can simulate models of stress, inflammation, aging, immune dysfunction, and pre-malignant transformation of stem cells. The NASA Twins Study suggests that extended periods of spaceflight may affect hematopoietic stem cell and immune cell health and function through their multi-omics approach of one ground-based twin, Mark Kelly, and one on board the ISS, Scott Kelly (Garrett-Bakelman et al., Science, 2019). A recent study also suggests an adaptive response to immune cell activity related to pre- and post-flight long-term missions after transcriptomic analysis of astronaut blood samples (Stratis, et al., Frontiers in Immunology, 2023). These studies emphasize the importance of assessing astronaut hematopoietic stem cell and immune cell function to further understand the mechanisms of cellular and molecular changes associated with time spent in LEO. We designed a longitudinal study to collect (under an IRB-approved protocol) peripheral blood from Axiom Mission 2 (Ax-2) astronauts to assess their hematopoietic stem cell and immune cell health. Timepoints include Launch-45 days, Launch-2 days, inflight, Return+1 day, Return+50 days, and yearly for 5 years. Ax-2 was a 10-day mission which launched on May 21, 2023 and splashed down on May 31, 2023. This was a private crew-tended mission with a sample size of 4 astronauts. As part of the stem cell health and fitness assessment, we selected for CD34+ hematopoietic stem and progenitor cells (HSPCs) from the 4 private astronauts and performed functional survival and self-renewal assays from each timepoint. Preliminary results suggest that the self-renewal capacity of HSPCs decreases inflight, but upon Return+1 day, HSPCs regain their ability to clone themselves at a higher capacity than pre-flight. Furthermore, we collected HSPCs for whole genome sequencing, 10x Genomics single cell sequencing, and whole transcriptome sequencing, to analyze pre-, during, and post-flight genomic and transcriptomic changes. Analyses will be done to identify mutational burden and mutational signatures associated with each timepoint. These mutational signatures will be compared to previously identified C-to-T mutations to assess the pre-malignant status of HSPCs due to short-term spaceflight (Jiang et al., Cell Reports, 2021). In addition to assessing CD34+ HSPC health, we will also profile the immune landscape by sequencing in parallel the CD34- fraction and performing targeted FACS analysis based on initial sequencing results. We also seek to identify markers of inflammation and anti-viral response through analysis of cytokines from biobanked plasma. This investigation has the potential to provide valuable insight into stem cell and immune cell health and fitness associated with short-duration missions in LEO.

The RNA Helicase DHX16 Is Required for the Maintenance of Hematopoietic Stem Cells

Lifelong hematopoiesis relies on the homeostasis of hematopoietic stem cells(HSCs), but the underlying mechanisms remain incompletely understood. Here, we identified a critical role for DHX16, a member of DEAH-box RNA helicase, in the maintenance of HSCs. After inducing deletion of Dhx16 gene in mice, the knockout animals developed pancytopenia within 5-7 days and died within two weeks. Flow cytometric analyses demonstrated that Dhx16-deficient mice had significantly reduced hematopoietic stem and progenitor cells, altered cell cycle dynamics with decreased G0 phase and increased G2/M phase, and increased apoptosis of HSCs. Competitive BM transplantation assays convinced the impaired self-renewal ability of Dhx16 knockout mice. To identify Dhx16-dependent genes and molecular pathways involved in the regulation of HSC function, we performed the RNA-Seq and long-read Iso-Seq analysis to compare the transcriptome between the Dhx16-deficient HSCs and Dhx16 fl/fl HSCs. RNA-Seq results showed that signaling pathways in Dhx16-deficient hematopoietic stem cells, including ribosome and spliceosome assembly, G2/M checkpoint, and p53 pathway were significantly affected. While long-read Iso-Seq revealed 210 genes with splicing alterations. Through further study, we confirmed that Dhx16-deficiency led to the retention of intron 4 in Emg1, a gene essential for ribosome biogenesis, resulting in a premature termination and nonsense-mediated mRNA decay. Therewith, decreased Emg1 expression in HSCs impaired ribosome biogenesis, activated the p53 pathway, and resulted in the rapid exhaustion of HSCs. In conclusion, these findings reveal a critical role for DHX16 in HSC survival and function and uncover an unsuspected link between RNA helicases, alternative splicing, ribosome biogenesis, and HSC homeostasis.

Dynamic Expression of Erg Controls Fetal-to-Adult Maturation of the Hematopoietic System

Many blood diseases are biased in their age of onset toward infancy, childhood, or mature adulthood. This occurs in concert with underlying changes in the properties of hematopoietic stem and progenitor cells (HSPCs) over the course of development, maturation, and aging that tailor the output of effector blood cells to age-specific physiology. Understanding how blood formation changes over the course of a lifetime would form a foundation upon which to investigate age-related determinants of manifestations of blood diseases. A growing body of work has shown that the heterochronic Lin28b/ let-7 switch defines fetal versus adult definitive HSPC states. In previous work, we found that the Polycomb repressive complex 1 component Cbx2 is a downstream target of let-7 that functions to specify the temporal maturation state of the hematopoietic system. In our current study, we find that Cbx2 represses the master hematopoietic stem cell (HSC) transcription factor (TF) Erg in fetal HSPCs, providing a mechanism by which the Lin28b/ let-7 axis integrates with the core HSC TF circuitry. During developmental maturation, Erg is upregulated during the fetal-to-adult HSC transition in humans (5.5-fold), mice (3.8-fold), and zebrafish (4.9-fold). Moreover, by analyzing existing ATAC-seq datasets, we find that Erg motifs are highly enriched in chromatin regions specifically active in adult relative to fetal HSPCs (P < 0.0001). Together, these findings suggest age-biased role(s) for Erg in hematopoiesis. We therefore hypothesized that since Erg is a key regulator of self-renewal and lineage output in HSCs, Erg likely serves stage-specific roles in HSCs. To test this, we either suppressed Erg expression in adult HSPCs or ectopically activated Erg in fetal HSPCs to determine consequences on HSPC function. We found that ectopic expression of Erg in HSPCs reprogrammed to the fetal state by expression of LIN28B resulted in blunting of fetal-like lineage biases. In adult mice, loss of a gene dose of Erg resulted in persistence of hallmarks of the fetal HSC state among blood effector cells and fetal-like HSPC distributions into maturity. During the normal process of fetal-to-adult maturation, HSCs transition from a highly proliferative to a quiescent state. We find that early postnatal Erg +/- HSCs do not effectively transition to quiescence and maintain active fetal-like self-renewal, which is leads to impairment in HSC function and susceptibility to replicative exhaustion later in adulthood. Mechanistically, we find that adult Erg +/- HSPCs ectopically maintain fetal-like gene expression profiles and patterns of promoter-enhancer looping. This is associated with failure to repress self-renewal promoting TFs that are normally expressed only in fetal HSPCs. Together, these findings demonstrate that master hematopoietic TFs and their associated networks are tightly titrated to implement age-appropriate rates self-renewal and mature cell output in HSPCs. Our results confirm heterochronic roles for Erg in defining age-tailored hematopoiesis on schedule with normal development and maturation and form a basis for applying this understanding of temporal hematopoietic maturation to disease models.

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

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

Elevated Functional Hematopoietic Stem and Progenitor Cells in the Peripheral Blood of G-CSF-Mobilized Dnmt3a-Mutant Mice

Clonal hematopoiesis (CH) arising from somatic mutations in hematopoietic stem cells (HSCs) is associated with increased risk of developing several non-hematologic and hematologic diseases. In the context of stem cell transplantation, donor HSCs carrying CH-associated somatic mutations can be passed from donor to recipient during stem cell transplantation. Most literature supports that there is no negative impact on recipient survival or outcomes after allogeneic stem cell transplantation (allo-HSCT) from donors with CH. However, it is less clear how allo-HSCT donors with CH respond to common mobilization methods such as Neupogen (G-CSF). As hematopoietic stem and progenitor cells (HSPCs) with CH mutations have been found to respond to external stimuli such as inflammation in a manner distinct from wild-type HSPCs, it is plausible that CH-mutant HSPCs also distinctly respond to G-CSF mobilization. It is important to understand whether the process of mobilization differentially enriches for CH-mutant HSPCs in the mobilized peripheral blood product. To address this gap in knowledge, we used a murine model of Dnmt3a-mutant CH. After five consecutive days of Neupogen (G-CSF) administration, modeling the procedure for human HSC mobilization, we observed that Dnmt3a-mutant mice had significantly increased white blood cell, neutrophil and monocyte counts compared to control mice. We observed a significant increase in cell surface marker-defined HSPCs in the peripheral blood and spleen of G-CSF-treated Dnmt3a-mutant mice compared to G-CSF-treated control mice. Using in vitro colony-forming unit assays, the peripheral blood of G-CSF-treated Dnmt3a-mutant mice was found to contain a greater number of functional hematopoietic progenitors compared to G-CSF-treated control mice. To determine if the peripheral blood of G-CSF-treated Dnmt3a-mutant mice had a greater abundance of functional HSCs, we transplanted equal volumes of mobilized blood from Dnmt3a-mutant and control mice into primary and secondary lethally irradiated wild-type recipient mice. We observed increased long-term engraftment of mobilized blood from Dnmt3a-mutant mice compared to control mice in both primary and secondary recipient animals. At a mechanistic level, HSC mobilization is facilitated by myeloid cells that respond directly to G-CSF via the G-CSF receptor ( Csf1r). In Dnmt3a-mutant mice, we observed increased Csf1r expression in myeloid progenitor and mature myeloid cells compared to control mice, providing a putative mechanism by which increased mobilization is enabled. Together, our data support differential mobilization potential of Dnmt3a-mutant compared to control HSCs in response to G-CSF, which may have implications in the context of allo-HSCT.

Polycom Group Protein Mel18 Inhibits Hematopoietic Stem Cell Self-Renewal through Repressing the Expression of Genes Important for Cellular Senescence and Proliferation

Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1), that act in concert to initiate and maintain stable gene repression. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the canonical PRC1 complex. While Bmi1 is a positive regulator of hematopoietic stem cell (HSC) and leukemia stem cell (LSC) self-renewal, the role of Mel18 in normal and malignant hematopoiesis is not fully understood. To further determine the role of Mel18 in hematopoiesis, we have generated Mel18 conditional knockout mice ( Mel18 f/f-Mx1Cre +). Acute deletion of Mel18 in the hematopoietic compartment did not affect the frequency and survival of hematopoietic stem and progenitor cells (HSPCs). To determine the impact of Mel18 deficiency on HSPC proliferation, we performed serial replating assays and found that loss of Mel18 increases the replating potential of HSPCs in vitro. To determine the role of Mel18 in HSC self-renewal, we first performed serial bone marrow (BM) transplantation assays and found that Mel18 null BM cells show increased engraftment in both primary and secondary transplantation assays. Limiting dilution transplantation showed that there were more functional HSCs in the BM of Mel18 knockout mice. We then performed HSC transplantation assays and found that loss Mel18 enhances HSC self-renewal. Thus, we demonstrated that Mel18 inhibits HSC self-renewal and decreases the number of functional HSCs in vivo. To understand the mechanism by which Mel18 inhibits HSC self-renewal and proliferation, we performed RNA-seq using Mel18 +/+ and Mel18 -/- HSCs. We then performed gene set enrichment analysis (GSEA) to identify potential Mel18 target genes important for HSC behavior. We found that HSC gene signatures are enriched in Mel18 -/- HSCs. We also found that leukemia stem cell gene signatures and CBF-mutant-AML genes are significantly enriched in Mel18 -/- HSCs, suggesting that loss of Mel18 may prime normal HSCs for leukemic transformation. Notably, our RNA-seq assay showed that senescence-related genes, including S100a9 and S100a8, were down-regulated in Mel18 -/- HSPCs. Importantly, loss of Mel18 significantly decreased the number of senescent HSPCs. To understand how Mel18 regulates gene transcription, we performed ATAC-seq assays in Mel18 +/+ and Mel18 -/- HSPCs. Mel18 null HSPCs showed decreased chromatin accessibility near both S100a8 and S100a9 genes. We confirmed that both S100a8 and S100a9 are downregulated in Mel18 -/- HSPCs compared to Mel18 +/+ HSPCs. Given that cell cycle arrest is a common feature of cellular senescence, we also examined the expression of cell cycle related genes in Mel18 -/- HSPCs. The expression of cell cycle regulators, including CDK4 and CCND2, is increased in Mel18 -/- HSPCs. Mel18 can bind to a specific DNA sequence, 5‘-GACTNGACT-3‘, thereby repressing the expression of target genes. Mel18 directly binds to the CCND2 gene locus as revealed by ChIP-seq assays in embryonic stem cells, suggesting that Mel18 may directly repress CCND2 expression in HSPCs. In summary, we demonstrate that Mel18 inhibits HSC self-renewal via repressing the expression of genes that are important for cellular senescence and proliferation.