Bone Marrow Hematopoietic Cells Research Articles

Long-term combination therapy with metformin and oxymetholone in a Fanconi anemia mouse model.

Fanconi anemia (FA) is a disease caused by defective deoxyribonucleic acid (DNA) repair that manifests as bone marrow failure, cancer predisposition, and developmental defects. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wildtype controls with either MET alone, OXM alone, MET+OXM, or placebo diet from age 3weeks to 18months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p=.01) and hemoglobin levels (p<.05). In addition, the percentage of quiescent hematopoietic stem cell (HSC) (LSK [Lin-Sca+c-Kit+]) was significantly increased (p=.001) by long-term treatment with MET alone. The combination of metformin and oxymetholone did not result in a significant synergistic effect in any hematopoietic parameter. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration. We conclude that androgen therapy is not a contraindication to concurrent metformin administration in clinical trials. HIGHLIGHTS: Long-term coadministration of metformin in combination with oxymetholone is well tolerated by Fancd2-/- mice. Hematopoietic stem cell quiescence in mutant mice was enhanced by treatment with metformin alone. Metformin treatment caused a partial normalization of gene expression in the livers of mutant mice.

The malignant transformation potential of the oncogene STYK1/NOK at early lymphocyte development in transgenic mice

B-cell Chronic Lymphocytic Leukemia (B-CLL) is a malignancy caused by the clonal expansion of mature B lymphocytes bearing a CD5+CD19+ (B1) phenotype. However, the origin of B-CLL remains controversial. We showed previously that STYK1/NOK transgenic mice develop a CLL-like disease. Using this model system in this study, we attempt to define the stage of CLL initiation. Here, we show that the phenotype of STYK1/NOK-induced B-CLL is heterogeneous. The expanded B1 lymphocyte pool was detected within peripheral lymphoid organs and was frequently associated with the expansions of memory B cells. Despite this immunophenotypic heterogeneity, suppression of B cell development at an early stage consistently occurred within the bone marrow (BM) of STYK1/NOK-tg mice. Overall, we suggest that enforced expression of STYK1/NOK in transgenic mice might significantly predispose BM hematopoietic stem cells (HSCs) towards the development of B-CLL.

The Essence of Quiescence.

Historically hematopoietic stem cells are believed to be predominantly dormant but could be induced into active cell cycle under specific conditions. This review, coupled with years of research from our laboratory, challenges this belief by demonstrating a significant portion of hematopoietic stem cells are actively cycling rather than quiescent. This addresses a major heuristic error in the understanding of hematopoietic stem cells that has shaped this field for decades. By evaluating the cycle status of engraftable hematopoietic stem cells in whole unseparated bone marrow, we demonstrated that a significant portion of these cells are actively cycling, and further confirmed by tritiated thymidine suicide and bromodeoxyuridine labeling assays. Moreover, by analyzing both whole unseparated bone marrow and purified lineage-negative hematopoietic stem cells in murine models, our findings indicate that lineage-positive cells, usually discarded during purification, actually contain actively cycling stem cells. Taken together, our findings highlight that hematopoietic stem cells are characterized as actively cycling and expressing differentiation epitopes. This corrects a basic mistake in stem cell biology. Furthermore, these findings provide valuable insights for a better understanding of the actively cycling hematopoietic stem cells in the field of stem cell biology.

Dietary restriction plus exercise change gene expression of Cxcl12 abundant reticular cells in female mice.

Low energy availability due to excessive exercise lowers bone mass and impairs various physiological functions, including immunity and hematopoiesis. We focused on Cxcl12 abundant reticular (CAR) cells, which are bone marrow mesenchymal stem cells and are essential for the maintenance of hematopoietic and immune cells in bone marrow. We examine the functional changes in CAR cells resulting from dietary restriction combined with exercise. Five-week-old wild-type female mice were divided into an ad libitum group (CON), a 60% dietary restriction group (DR), an ad libitum with exercise group (CON + ex), and a 60% dietary restriction with exercise group (DR + ex). Blood parameters, bone structure parameters, and bone marrow fat volume were evaluated after 5weeks. In addition, bone marrow CAR cells were isolated by cell sorting and analyzed for gene expression by RT-qPCR. Bone mineral density (BMD) was significantly decreased in DR and DR + ex compared to CON and CON + ex. Especially, cortical bone mass and thickness were significantly decreased in DR and DR + ex groups, whereas trabecular bone mass was significantly increased. Bone marrow fat volume was significantly increased in DR and DR + ex groups compared to CON and CON + ex. The number of leukocytes in the blood was significantly decreased in the DR + ex group compared to the other three groups. RT-qPCR showed a significant decrease in gene expression of both Foxc1 and Runx2 in CAR cells of the DR + ex group compared to CON. Dietary restriction combined with exercise promotes CAR cell differentiation into bone marrow adipocyte and suppresses osteoblast differentiation.

Landscape of FLT3 Variations Associated with Structural and Functional Impact on Acute Myeloid Leukemia: A Computational Study.

Acute myeloid leukemia (AML) is hallmarked by the clonal proliferation of myeloid blasts. Mutations that result in the constitutive activation of the fms-like tyrosine kinase 3 (FLT3) gene, coding for a class III receptor tyrosine kinase, are significantly associated with this heterogeneous hematologic malignancy. The fms-related tyrosine kinase 3 ligand binds to the extracellular domain of the FLT3 receptor, inducing homodimer formation in the plasma membrane, leading to autophosphorylation and activation of apoptosis, proliferation, and differentiation of hematopoietic cells in bone marrow. In the present study, we evaluated the association of FLT3 as a significant biomarker for AML and tried to comprehend the effects of specific variations on the FLT3 protein's structure and function. We also examined the effects of I836 variants on binding affinity to sorafenib using molecular docking. We integrated multiple bioinformatics tools, databases, and resources such as OncoDB, UniProt, COSMIC, UALCAN, PyMOL, ProSA, Missense3D, InterProScan, SIFT, PolyPhen, and PredictSNP to annotate the structural, functional, and phenotypic impact of the known variations associated with FLT3. Twenty-nine FLT3 variants were analyzed using in silico approaches such as DynaMut, CUPSAT, AutoDock, and Discovery Studio for their impact on protein stability, flexibility, function, and binding affinity. The OncoDB and UALCAN portals confirmed the association of FLT3 gene expression and its mutational status with AML. A computational structural analysis of the deleterious variants of FLT3 revealed I863F mutants as destabilizers of the protein structure, possibly leading to functional changes. Many single-nucleotide variations in FLT3 have an impact on its structure and function. Thus, the annotation of FLT3 SNVs and the prediction of their deleterious pathogenic impact will facilitate an insight into the tumorigenesis process and guide experimental studies and clinical implications.

A T-cell receptor as a promising immunotherapeutic target in acute myeloid leukemia: expression of T cell immunoglobulin and mucin-containing-3 on bone marrow hematopoietic cells

Background The emergence of immunotherapy has changed the treatment landscape of various types of cancers. However, limited target antigen remains to be a challenge for its application in acute myeloid leukemia (AML). T cell immunoglobulin and mucin-containing-3 (Tim-3) is an immune checkpoint receptor that plays a major role in the escape of host immune surveillance in AML. Thus, its role in cancer treatment needs to be thoroughly investigated. Methods Tim-3 expression on blasts, lymphocytes, and monocytes was analyzed by flow cytometry in bone marrow samples of 60 newly diagnosed AML patients and 30 matched non-neoplastic controls. Its association with different prognostic clinicopathological parameters was also evaluated. ROC curve confirmed the value of Tim-3 expression in discriminating AML patients from healthy controls and the role of Tim-3 in disease aggressiveness was also investigated. Results The median percentage of myeloblasts positive for Tim-3% and MFI were significantly higher in AML than normal controls (64.27% and 1.34 in patients vs. 41.89% and 1.12 in controls) and it was markedly associated with poor overall survival of the patients. Besides, the upregulation of Tim-3 on AML blasts was significantly higher than on lymphocytes and monocytes in denovo AML patients (P &lt;0.001). Conclusion This study indicates Tim-3 as a poor prognostic marker in AML and highlights Tim-3 as a new therapeutic target antigen for immunotherapy with low off-target toxicity.

The Mechanism of Setd2 in Mesenchymal Stromal Cell in MDS-Associated Ineffective Hematopoiety

Myelodysplastic syndrome (MDS) is a heterogeneous, clonal, hematopoietic stem progenitor cell malignant disease. Bone marrow microenvironment participates in the occurrence and development of MDS and plays a key role. Histone methyltransferase Setd2 is the only enzyme that can catalyze the methylation of mammalian histone H3K36Me3. Previous studies have confirmed that Setd2 is involved in the the transformation of MDS to leukemia, and the expression of S100A9 decreases after Setd2 knockout. This study is based on NHD13 mice with mesenchymal stem cell (MSC) targeted knockout of Setd2. Through experimental methods such as flow cytometry, peripheral blood routine, Wright staining, HE staining, and Gomori staining, it finds that Setd2 targeted knockout mice undergo faster leukemia transformation compared to NHD13 mice; Peripheral blood white blood cells increase, while hemoglobin and platelets decrease; The differentiation of hematopoietic stem progenitor cells in bone marrow is abnormal, with abnormal hematopoiesis and increased blast cells; Spleen fibrosis aggravates and megakaryocyte infiltrates; and Extramedullary hematopoiesis in liver. Through flow cytometry,β- Galactosidase staining and other experimental methods, we finds that compared with NHD13 mice, the MSC morphology of knockout mice is irregular, and the support function for hematopoietic cells is weakened. This study demonstrates that MSC targeted knockout of Setd2 has a promoting effect on the progression of MDS. We used mass spectrometry to identify proteins that are significantly upregulated in the targeted knockout mouse bone marrow supernatant compared to the NHD13 mouse bone marrow supernatant, and and select the protein Uba5 related to hematopoiesis from them. The expression of Uba5 in the bone marrow supernatant of knockout mice is significantly higher than that in the NHD13 group through WB validation. It is speculated that the hematopoietic changes in Setd2 targeted knockout mice may be related to the downstream target protein Uba5 of Setd2. This study takes the change of bone marrow microenvironment as an entry point to explore the possible mechanism of MDS ineffective hematopoiesis, hoping to provide microenvironment regulation therapy for some MDS patients with genes that hematopoietic stem cell transplantation that cannot cure(such as TP53, NRAS, KRAS), and provide new ideas for MDS treatment. There are still many limitations in this study, further clinical validation and vivo and vitro rescue experiments on downstream target genes and proteins are still needed in the future.

Genomic Determinants of Therapy Response in ETV6::RUNX1 Leukemia

ETV6::RUNX1 leukemia is the second most common childhood B-cell acute lymphoblastic leukemia subtype. Although it has a low overall relapse risk, a significant proportion of relapses occur within this subtype due to its relatively high incidence. No consistent genomic biomarkers have been identified that predict therapy response beyond early therapy response as measured by minimal residual disease at the end of induction. In this study, our aim was to use multiomic data from 175 ETV6::RUNX1 leukemias treated according to the NOPHO ALL2000 and ALL2008 protocols to identify genomic features that predict therapy response at presentation. Cases were classified into three responder groups based on MRD at end-of-induction (EOI) therapy: fast responders with negative MRD, intermediate responders with positive but &amp;lt;0.1% MRD, and slow responders with &amp;gt;0.1% MRD. Response data was also collected at mid-induction (day 15) and end-of-consolidation (EOC), when available. Diagnostic and remission DNA were analyzed using array-based copy number data (n=142) or whole genome sequencing (WGS) (n=33), and diagnostic bulk RNA sequencing (n=51) was complemented with single cell multiomics (RNA, surface protein and VDJ sequencing) data from both healthy (n=8) and leukemic bone marrow cells upon chemotherapy (n=8). Overall, the frequency of somatic genetic changes was not significantly associated with treatment response suggesting that genetic diversification through elevated random mutagenesis events may not underlie the drug resistant phenotype. However, analysis of mutational signatures revealed that fast-responding leukemic blasts frequently exhibited the APOBEC mutational signature and had higher cell cycle activity (Figure 1A). Accordingly, we show that expression of A3B is highly specific to cycling cells also in healthy bone marrow hematopoietic progenitor cells, pro- and pre-B cells. We also examined RAG-mediated genetic instability that manifests in focal structural variations and has highest activity in ETV6::RUNX1leukemia (Figure 1B). We confirmed several recurrent RAG off-target sites, including significant enrichment of the known risk locus TBX1LR1 in slow responding cases. Strikingly, slow-responding leukemias had also frequent rearrangements in the immunoglobulin kappa genes and the kappa-deleting elements, despite their pro-B-like transcriptome state upon diagnosis. Based on the single cell multiomics profiles, we found that upon treatment the blast phenotype shifts towards an immature-B-like transcriptome, with concomitant expression of Ig light chains with productive sequence, thereby permitting activation of BCR signaling that provides a survival advantage. At CNV level, we found that the chromosomal alterations related to the translocation event associated with treatment response. Specifically, slow responders had more often the amplification of der(21)t(12;21) chromosome, impacting expression of USP5, TIGAR and SLC2A3, associated in earlier studies to chemoresistance and tumorigenesis. In comparison, fast responders were often missing a region of chromosome 12p with lower expression of several genes including oncogenic KRAS. In summary, combining multiomic and response data, we characterized genetic alterations and mutational processes that affect the treatment response in ETV6::RUNX1leukemia. Our findings highlight the genetic and transcriptomic heterogeneity of this common pediatric leukemia subtype and its implications for treatment sensitivity, paving the way for better classification of the disease at presentation.

Rapid Degradation of mDia2 Protein during Terminal Erythropoiesis Via an In Vivo Aid System: An Alternative Approach for Loss-of-Function Studies

Loss-of-function manipulations are crucial methods for studying gene functions. Despite the availability of many chemical-induced genetic manipulation techniques, there are limitations associated with these technologies. Tamoxifen-induced conditional gene expression, gene knockdown, or cell tracking represent some of the most common genetic manipulations employed in mice. However, tamoxifen-induced gene or Cre expression in bone marrow cell populations is relatively weak, affecting only a low proportion of cells. Similar observations have been made with the in vivo doxycycline-inducible system. Given that inflammation could affect the dispersion of bone marrow hematopoietic cells, an inducible system based on the MX1 promoter and interferon-alpha is not suitable for long-term protein knockdown. In addition, the above-mentioned methods are not able to achieve rapid loss-of-function because it is largely determined by the half-life of the transcriptional and translational products of the targeted gene. Furthermore, none of these technologies could achieve reversible depletion of the protein of interest. Therefore, there is a need for enhanced techniques for inducible and reversible loss-of-function gene manipulations in the field of hematopoiesis. The capacity for quick, targeted protein elimination is particularly necessary when researching the functions of proteins that play roles in swift biological processes. The Auxin-inducible degron (AID) system is a chemically inducible tool, widely employed for targeted protein degradation to study protein functions in cultured mammalian cells. It holds significant potential as a powerful tool for studying protein functions in vivo within the bone marrow. The AID system comprises three components: the auxin signal, AID tag, and auxin receptor F-box proteins (AFBs). When an AID tag is fused to a protein of interest, the AFB proteins recognize the tag and, in the presence of the auxin signal, induce the protein's degradation via the proteasome. Additionally, due to its small molecular weight, auxin can potentially penetrate the bone marrow barrier and achieve the working concentration in the bone marrow, facilitating protein degradation. In this study, we aim to investigate the role of mDia2 in terminal erythropoiesis using AID system. The mDia family proteins are formin proteins that catalyze the assembly of F-actin. The loss of function of mDia2 achieved by Cre-flox system leads to deficiency in enucleation and anemia. We hypothesize that the mDia2 loss of function could be compensated by upregulated mDia1 in the Cre-flox mediated knockout system, and a rapid loss of mDia2 protein could lead to more severe phenotypes in vivo and ex-vivo. We knocked in minilAA7-eGFP to the end of the last exon of mDia2 gene to create an endogenous tagged mDia2 protein in C57/B6 mice. We also created an AFB protein transgenic mouse by knocking in the atAFB2 protein coding gene at the ROSA26 locus driven by the Vav1 promoter. After crossing the mDia2-minilAA7-eGFP transgenic mice with Vav1-atAFB2 mice, we obtained mDia2-minilAA7-eGFP and Vav1-atAFB2 double homozygous mice. We administered auxin via intraperitoneal (IP) injection at a concentration of 300 mg/kg and collected total bone marrow cells four hours post-injection. Quantification of the GFP signal through flow cytometry revealed a significant reduction following auxin administration compared to the control group. This protein degradation was further confirmed with Western blotting assays. To understand how an immediate loss of mDia2 influences terminal erythropoiesis, we isolated lineage-negative cells from auxin-administered mice and cultured them in an EPO-containing medium with 10 µM auxin for 48 hours. Compared to lineage-negative cells from VavCre-mDia2f/f mice, auxin-induced mDia2 degradation resulted in a significantly lower enucleation rate. Notably, auxin administration had no observable effects on either WT mice or atAFB2 homozygous mice. This suggests that rapid mDia2 loss, induced by the AID system, permits a better understanding of the targeted protein's function by avoiding potential compensatory effects from mDia1 during terminal enucleation. In conclusion, our established in vivo AID system enables rapid targeted protein degradation and serves as a powerful tool for studying protein functions potentially obscured by compensatory effects during erythropoiesis.

The role of GATA2 in adult hematopoiesis and cell fate determination.

The correct maintenance and differentiation of hematopoietic stem cells (HSC) in bone marrow is vital for the maintenance and operation of the human blood system. GATA2 plays a critical role in the maintenance of HSCs and the specification of HSCs into the different hematopoietic lineages, highlighted by the various defects observed in patients with heterozygous mutations in GATA2, resulting in cytopenias, bone marrow failure and increased chance of myeloid malignancy, termed GATA2 deficiency syndrome. Despite this, the mechanisms underlying GATA2 deficiency syndrome remain to be elucidated. The detailed description of how GATA2 regulates HSC maintenance and blood lineage determination is crucial to unravel the pathogenesis of GATA2 deficiency syndrome. In this review, we summarize current advances in elucidating the role of GATA2 in hematopoietic cell fate determination and discuss the challenges of modeling GATA2 deficiency syndrome.

Bone Marrow Microenvironment and Hematopoietic Cell Types Are Differentially Affected By Transcriptomic Quality Control Filtering Metrics in Single Cell Data

A crucial step prior to analysis of single cell transcriptomic data includes filtering out low-quality (e.g., dying) cells and artifacts (e.g., empty droplets). Choosing appropriate quality control parameters leads to correct cell assignment, so that the data for downstream analysis is high-quality and is representative of all processed cell types. These metrics include high and low cut-offs for mitochondrial content, total number of genes, number of different genes, and ribosomal content per cell. They have been found to vary based on cell and tissue type, as well biological variables such as age and disease, and technical variables such as sequencing technology. The aim of this study was to investigate the variation of these quality control metrics in a data set that included stem, progenitor, and differentiated cell types of the hematopoietic, mesenchymal, and endothelial lineages. Furthermore, we set out to determine whether applying standard cut-offs may differentially affect different cell types. We calculated individual filtering parameters for each cell type using the R package ddqc (Subramanian et al. Genome Biol. 2022). Thus, any biological variability based on mitochondrial, ribosomal, and gene counts was accounted for. Notably, distribution of measurements for each of the above quality control metrics varied by cluster, and certain cell-type means were adjacent to commonly used cut-off values. For instance, we observed higher content of mitochondrial genes in osteoblasts, sinusoidal endothelial cells, a subset of adipocyte-primed mesenchymal cells (MSPC), as well as hematopoietic progenitor cells, pre-B, and B cells. On the other hand, subsets of erythroblasts, neutrophils, as well as arteriolar endothelial cells had the lowest mitochondrial content. In addition, lower total gene counts and lower gene complexity were found in adipocyte-primed MSPCs, neutrophils, and megakaryocyte-erythrocyte progenitors, while higher counts were found in granulocyte-monocyte progenitor cells, megakaryocyte/erythroid-primed multipotent progenitor cells and subsets of erythroblasts. Applying universal cutoff values to this data set would greatly reduce, and some cases, eliminate cell types that have high mitochondrial content or lower gene counts and complexity, while leaving those cell types on the opposite spectrum unaffected. This would also skew proportions of cells in downstream analyses, thus affecting biological interpretation of the results. Overall, our results demonstrate biological variability of the quality control metric distributions in a single cell transcriptomic data set that includes diverse bone marrow niche and hematopoietic cell types. This reveals novel biological information regarding distinct cell types as well as cautions that applying standard filtering paraments on such a data set would eliminate some cell types and bias downstream analysis for others. Optimization of filtering criteria will be important when broadening single cell analysis to multiplexed approaches incorporating transcriptome, genome, epigenome, and proteome-level data.

Heterogeneous Intercellular Communication of Hematopoietic Stem Cells in the Mouse Bone Marrow

Introduction.Hematopoietic stem cells (HSCs) are responsible for maintaining the homeostasis of the blood and immune systems. This process must rapidly adapt to bleeding, infection, injury, and disease. HSCs reside in specialized microenvironments in the bone marrow, called “niche”. Several bone marrow cell types have been proposed as the key components of the niche. It is well established that HSCs rely on molecular signals from the niche cells to survive, migrate, proliferate, and differentiate. Recent studies suggest that HSCs differentiate heterogeneously. Additionally, the bone marrow is a heterogeneous environment consisting of more than thirty cell types. This study will test the hypothesis that individual HSCs are engaged in heterogeneous intercellular signaling in the bone marrow. Materials and Methods.We utilized CellChat (Jin et al., 2021), a database for ligand-receptor pairs and associated pathways, to predict cell-cell signaling at the single cell level. We used single cell RNA sequencing data from mouse hematopoietic stem and progenitor cells, blood and immune cells, and non-hematopoietic cells from the bone marrow to determine putative cell-cell interactions. The predicted signaling interactions were then adjusted based on the cell-cell spatial interaction revealed by PhenoCycler measurement (Akoya Biosciences). Mouse bone marrow transplantation experiments were performed to test the functional significance of the identified signaling pathway interactions. Results.We found that individual HSCs send and receive different levels of molecular signals across various signaling pathways. In addition to the expected interactions with the known HSC niche cells, we also found new interactions between HSCs and their progeny cells including myeloid progenitors and immune cells. These cell-cell signaling interactions are consistent with the spatial interaction frequencies of the corresponding cell types as revealed by our imaging data from the PhenoCycler analysis. Further, we found that some signaling pathways are positively or negatively correlated across individual HSCs, indicating complex intercellular signaling networks. The most significant negative correlation was found between HSCs sending MHC-I signals and sending PARs signals (Figure). Moreover, this MHC-I/PARs signaling correlation network also involves signaling from the HSC niche, including those that are known to play roles in HSC homing such as ESAM and VCAM. Therefore, we tested the engraftment efficiency of HSCs that receive PARs and those that do not using the HSC mouse transplantation model. We found that PAR1 + HSCs engraft much less efficiently. Finally, we present the HSC Interaction Data Explorer (HIDE), a database that comprehensively depicts the interactions between HSCs and other bone marrow cells including the involved signaling pathways, ligands and receptors of the pathways, and the pathway correlations. Conclusion.Our study uncovered novel interactions of HSCs with myeloid progenitor cells and immune cells, which were validated by spatial interaction data. These interactions indicate feedback signaling in regulating blood cell regeneration. Our findings further revealed signaling pathway correlation networks across individual HSCs, and we showed their functional significance using transplantation experiments. Finally, we present HIDE as a resource that informs the interactions between HSCs and various bone marrow cells, the relevant signaling pathways, and the pathway correlations. Figure Legend. HSC signaling correlation networks. Each node shows a signaling pathway and direction (e.g., HSCs sending MHC-I). The edges represent the spearman correlation score.

Metabolic Programming of Hematopoietic Stem Cell Function By Prenatal Folate

Hematopoietic stem cells (HSCs) are programmed by metabolic changes resulting in alterations to HSC function. Here, we discovered that varying prenatal folate status metabolically programs HSCs during development and affects adult HSC self-renewal and engraftment potential into adulthood. Folate status varies greatly in the global population based on nutritional intake, common genetic polymorphisms, and widespread supplementation. Folate-derived one-carbon metabolism (OCM) regulates cellular methylation, de novo nucleotide biosynthesis and mitochondrial metabolism, processes critical to HSC function and establishment. Despite immense therapeutic potential, there is limited understanding of mechanisms driving HSC developmental programming. We hypothesize that varying prenatal folate status affects risk for hematologic dysfunction by developmentally programming HSCs. To test how prenatal folate modulated HSC function, female mice were assigned to one of three experimental diets to model population-wide folate consumption: 0mg/kg (deficient, FD), 2mg/kg (control, FC) and 8mg/kg (supplemented, FS). Profiling of fetal liver (FL) hematopoietic cells revealed that FD diet caused expansion of long term (LT)-HSCs that was propagated across the hematopoietic hierarchy, including myeloid and lymphoid progenitors and mature myeloid cells. In contrast, FS decreased FL cell output. We next determined if these changes were driven by metabolic activity. FD fetal HSPCs exhibited higher mitochondrial membrane potential and lower glycolytic activity, whereas FS fetal HSPCs displayed both higher glycolytic activity and oxygen consumption despite impaired production of downstream hematopoietic cells. Together, these data suggest that HSPC metabolic programming initiates during fetal development and is folate-dose dependent. To determine if prenatal folate programs hematopoiesis into adulthood, we quantified bone marrow (BM) hematopoietic cells in adult offspring after weaning onto standard chow. Surprisingly, FD expanded lymphoid progenitors in adult offspring with no impact on mature lymphoid cell output. In contrast, FS diet expanded myeloid and lymphoid progenitors in the BM as well as downstream mature cells, including monocytes and B-cells. To determine if these effects were HSC cell-intrinsic, we performed competitive transplantation assays of LT-HSCs isolated from adult offspring. FD offspring HSCs showed worse engraftment by chimerism across all peripheral blood (PB) populations compared to FC. In comparison, FS offspring HSCs showed enhanced total cellular output by donor cells/uL across all PB mature lineages except platelets. Secondary whole BM transplantation revealed that FS HSCs retained enhanced total cellular output and superior engraftment compared to FC, whereas FD HSCs demonstrated complete exhaustion. These data indicate that adult HSC function is programmed by prenatal folate status. To investigate molecular alterations driving metabolic programming of adult hematopoiesis by prenatal folate, we performed single-cell RNA sequencing (Scseq) and metabolomics on adult offspring HSPCs. Scseq analysis revealed most differential gene expression (DE) in HSC clusters, with less DE in progenitors. Re-clustering of all HSCs revealed distinct heterogeneity within this population, where both FD and FS HSCs were identified as distinct clusters compared to FC. Metabolomic profiling further suggested that FD HSCs exhibited signs of mitochondrial dysfunction through, 1) accumulating glucose and lactate suggesting reliance on glycolysis and, 2) accumulating succinate and glutamine suggesting complex II impairment and inadequate utilization of mitochondrial metabolism. Surprisingly, the same changes associated with mitochondrial dysfunction in FD HSCs persisted downstream into lymphoid-biased multipotent progenitors. In contrast, limited metabolic changes were observed in FS HSCs, suggesting alternative pathways for sustained programming. These changes indicate that FD and FS offspring have distinct metabolic programming. Together, our data suggest that prenatal folate status programs hematopoietic stem cell function into adulthood and is driven by alterations to metabolic activity. Ongoing studies examine how prenatal folate status affects the adult hematological and immune response to challenge.

The immunometabolic landscape of bone marrow cells in multiple sclerosis.

In multiple sclerosis (MS), the bone marrow hematopoietic system supplies immune cells to orchestrate central nervous system (CNS) inflammation and autoimmunity. Understanding the metabolic processes within the bone marrow is essential for unraveling the phenotype and function of immune cells. However, a comprehensive exploration of the metabolic landscape and its association with systemic immune response in MS at the single-cell level has yet to be elucidated. Herein, we conducted an analysis of 70 289 bone marrow cells obtained from seven patients with MS and seven health controls (referenced as HRA001783) to address this question. Our focus was primarily on investigating the metabolic preferences of diverse immune cell populations and delineating their metabolic manifestations in the bone marrow microenvironment of MS. Through our analysis, we observed the activation of carbohydrate and amino acid metabolic pathways in the bone marrow cells of MS patients. Notably, we discovered significant metabolic alterations in cell-cell communication within the plasma cell population in the MS bone marrow. These findings shed light on the complex metabolic landscape within the bone marrow niche during MS and highlight the distinctive metabolic characteristics of plasma cells in this context, which may provoke novel understanding of MS pathogenesis and promote future design of immune therapies.

Clearing and replacing tissue-resident myeloid cells with an anti-CD45 antibody–drug conjugate

AbstractTissue-resident myeloid (TRM) cells in adults have highly variable lifespans, and may be derived from early embryonic yolk sac, fetal liver, or bone marrow. Some of these TRM cells are known pathogenic participants in congenital and acquired diseases. Myeloablative conditioning and hematopoietic stem cell transplantation can replace long-lived brain TRM cells, resulting in clinical improvements in metabolic storage diseases. With the advent of antibody–drug conjugate (ADC)-targeted cell killing as a cell-selective means of transplant conditioning, we assessed the impact of anti-CD45–ADC on TRM cells in multiple tissues. Replacement of TRM cells ranged from 40% to 95% efficiencies in liver, lung, and skin tissues, after a single anti-CD45–ADC dose and bone marrow hematopoietic cell transfer. Of note, the population size of TRM cells in tissues returned to pretreatment levels, suggesting a regulated control of TRM cell abundance. As expected, brain microglia were not affected, but brain monocytes and macrophages were 50% replaced. Anti-CD45–ADC and adoptive cell transfer were then tested in the chronic acquired condition, atherosclerosis exacerbated by Tet2 mutant clonal hematopoiesis. Plaque-resident myeloid cells were efficiently replaced with anti-CD45–ADC and wild-type bone marrow cells. Notably, this reduced existent atherosclerotic plaque burden. Overall, these results indicate that the anti-CD45–ADC clears both hematopoietic stem and TRM cells from their niches, enabling cell replacement to achieve disease modification in a resident myeloid cell–driven disease.

Does DNA methylation in the fetal brain leave an epigenetic memory in the blood?

Epigenetic memory is an emerging concept that refers to the process in which epigenetic changes occurring early-in life can lead to long-term programs of gene regulation in time and space. By leveraging neural network regression modeling of DNA methylation data in pigs, we show that specific methylations in the adult blood can reliably predict methylation changes that occurred in the fetal brain. Genes associated with these methylations represented known markers of specific cell types of blood including bone marrow hematopoietic progenitor cells, and ependymal and oligodendrocyte cells of brain. This suggested that methylation changes that occurred in the developing brain were maintained as an epigenetic memory in the blood through the adult life.

Depletion of Bone Marrow Hematopoietic Cells in Ebolavirus-Infected Rhesus Macaques: A Possible Cause of Hematologic Abnormalities in Ebolavirus Disease

The pathophysiology of long-recognized hematologic abnormalities in Ebolavirus (EBOV) disease (EVD) is unknown. From limited human sampling (of peripheral blood), it has been postulated that emergency hematopoiesis plays a role in severe EVD, but the systematic characterization of the bone marrow (BM) has not occurred in human disease or in nonhuman primate models. In a lethal rhesus macaque model of EVD, 18 sternal BM samples exposed to the Kikwit strain of EBOV were compared to those from uninfected controls (n = 3). Immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy showed that EBOV infects BM monocytes/macrophages and megakaryocytes. EBOV exposure was associated with severe BM hypocellularity, including depletion of myeloid, erythroid, and megakaryocyte hematopoietic cells. These depletions were negatively correlated with cell proliferation (Ki67 expression) and were not associated with BM apoptosis during disease progression. In EBOV-infected rhesus macaques with terminal disease, BM showed marked hemophagocytosis, megakaryocyte emperipolesis, and the release of immature hematopoietic cells into the sinusoids. Collectively, these data demonstrate not only direct EBOV infection of BM monocytes/macrophages and megakaryocytes but also that disease progression is associated with hematopoietic failure, notably in peripheral cytopenia. These findings inform current pathophysiologic unknowns and suggest a crucial role for BM dysfunction and/or failure, including emergency hematopoiesis, as part of the natural history of severe human disease.

Abstract 125: Elevated Bacterial Fatty Acid Metabolism And Fecal Calorific Values Are Associated With Reduced Diet-induced Adiposity And Weight Gain In Mice

Introduction: Activation of the sympathetic nervous system (SNS) leads to exaggerated immune responses in cardiometabolic conditions. We previously showed that ablation of beta-adrenergic receptors (ADRB) on the bone marrow (BM) hematopoietic cells reduces blood pressure, dampens the immune responses, and modifies gut microbiota, suggesting multisystem host-microbiota interactions. To investigate the role of the microbiota in these complex interactions, we tested the effects of high fat diet (HFD) on the gut microbiota and adiposity in BM mouse chimera characterized by reduced SNS-immune signaling. Methods: Male C5BL/6 wild type (WT) mice were irradiated and reconstituted with BM cells from either the C57BL/6 WT controls or the ADRB 1/2 knock-out mice (KD) to generate BM chimera. Following recovery, mice were fed with a control diet (CNT) or HFD for two weeks ad libitum . Food intake and body weights were measured weekly, and body fat (NMR) and visceral fat pad weights were measured at endpoint. At endpoint, 16S sequencing and metagenomic analyses (QIIME2, PiCRUST2) were performed in cecal samples, and residual fecal calorific values were determined using a simultaneous thermogravimetric analyzer differential calorimeter (TA Instruments). Results: No change in food intake was observed in either group. However, significant increases in body weight, total body fat, and visceral fat weight were observed in the WT compared to KD chimera on HFD ( p&lt;0.05) . HFD elevated gut bacterial Shannon α-diversity (p&lt; 0.05) in the WT but not the KD chimera, and we observed a decrease in g_Faecalibacterium and p_Actinobacteria in WT, and an increase in p_Bacteroidetes and o_Bacteroidales in the KD mice on HDF. Enrichment in fatty acid metabolic pathways and increased fecal calorific values were observed in the KD compared to WT chimera on HFD ( p&lt;0.05) . Conclusion: Reduced SNS-immune signaling is protective against diet-induced weight gain and body fat accumulation. This may in part be mediated by the gut microbiota, reflecting in increased bacterial fatty acid metabolism and elevated residual fecal calorific values, suggesting reduced fat absorption. We propose that selective enrichment of gut bacteria is a potential therapeutic target in diet-induces obesity.

Bletilla striata polysaccharide improves toxic and side effects induced by 5-FU: an untargeted metabolomics study

This study aimed to analyze the effect of Bletilla striata polysaccharide(BSP) on endogenous metabolites in serum of tumor-bearing mice treated with 5-fluorouracil(5-FU) by untargeted metabolomics techniques and explore the mechanism of BSP in alleviating the toxic and side effects induced by 5-FU. Male BALB/C mice were randomly divided into a normal group, a model group, a 5-FU group, and a 5-FU + BSP group, with eight mice in each group. Mouse colon cancer cells(CT26) were transplanted into the mice except for those in the normal group to construct the tumor-bearing mouse model by subcutaneous injection, and 5-FU chemotherapy and BSP treatment were carried out from the second day of modeling. The changes in body weight, diarrhea, and white blood cell count in the peripheral blood were recorded. The mice were sacrificed and sampled when the tumor weight of mice in the model group reached approximately 1 g. TUNEL staining was used to detect the cell apoptosis in the small intestine of each group. The proportions of hematopoietic stem cells and myeloid progenitor cells in bone marrow were measured by flow cytometry. Five serum samples were selected randomly from each group for untargeted metabolomics analysis. The results showed that BSP was not effective in inhibiting colon cancer in mice, but diarrhea, leukopenia, and weight loss caused by 5-FU chemotherapy were significantly improved after BSP intervention. In addition, apoptotic cells decreased in the small intestinal tissues and the percentages of hematopoietic stem cells and myeloid progenitor cells in bone marrow were significantly higher after BSP treatment. Metabolomics results showed that the toxic and side effects of 5-FU resulted in significant decrease in 29 metabolites and significant increase in 22 metabolites in mouse serum. Among them, 19 disordered metabolites showed a return to normal levels in the 5-FU+BSP group. The results of pathway enrichment indicated that metabolic pathways mainly involved pyrimidine metabolism, arachidonic acid metabolism, and steroid hormone biosynthesis. Therefore, BSP may ameliorate the toxic and side effects of 5-FU in the intestinal tract and bone marrow presumably by regulating nucleotide synthesis, inflammatory damage, and hormone production.

Perivascular localized cells commit erythropoiesis in PDGF-B-expressing solid tumors.

Tumors possess incessant growth features, and expansion of their masses demands sufficient oxygen supply by red blood cells (RBCs). In adult mammals, the bone marrow (BM) is the main organ regulating hematopoiesis with dedicated manners. Other than BM, extramedullary hematopoiesis is discovered in various pathophysiological settings. However, whether tumors can contribute to hematopoiesis is completely unknown. Accumulating evidence shows that, in the tumor microenvironment (TME), perivascular localized cells retain progenitor cell properties and can differentiate into other cells. Here, we sought to better understand whether and how perivascular localized pericytes in tumors manipulate hematopoiesis. To test if vascular cells can differentiate into RBCs, genome-wide expression profiling was performed using mouse-derived pericytes. Genetic tracing of perivascular localized cells employing NG2-CreERT2:R26R-tdTomato mouse strain was used to validate the findings in vivo. Fluorescence-activated cell sorting (FACS), single-cell sequencing, and colony formation assays were applied for biological studies. The production of erythroid differentiation-specific cytokine, erythropoietin (EPO), in TME was checked using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA, magnetic-activated cell sorting and immunohistochemistry. To investigate BM function in tumor erythropoiesis, BM transplantation mouse models were employed. Genome-wide expression profiling showed that in response to platelet-derived growth factor subunit B (PDGF-B), neural/glial antigen 2 (NG2)+ perivascular localized cells exhibited hematopoietic stem and progenitor-like features and underwent differentiation towards the erythroid lineage. PDGF-B simultaneously targeted cancer-associated fibroblasts to produce high levels of EPO, a crucial hormone that necessitates erythropoiesis. FACS analysis using genetic tracing of NG2+ cells in tumors defined the perivascular localized cell-derived subpopulation of hematopoietic cells. Single-cell sequencing and colony formation assays validated the fact that, upon PDGF-B stimulation, NG2+ cells isolated from tumors acted as erythroblast progenitor cells, which were distinctive from the canonical BM hematopoietic stem cells. Our data provide a new concept of hematopoiesis within tumor tissues and novel mechanistic insights into perivascular localized cell-derived erythroid cells within TME. Targeting tumor hematopoiesis is a novel therapeutic concept for treating various cancers that may have profound impacts on cancer therapy.