scholarly journals The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3707-3707
Author(s):  
Adedamola Elujoba-Bridenstine ◽  
Lijian Shao ◽  
Katherine Zink ◽  
Laura Sanchez ◽  
Brian Cox ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Progenitor Cells ◽  
Gene Set Enrichment Analysis ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
And Behavior

Hematopoietic stem and progenitor cells (HSPCs) have multi-lineage potential and can be used in transplants as a curative treatment for various hematopoietic diseases. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. One level of regulation comes from the sympathetic nervous system that innervates the niche and releases neurotransmitters to stromal cells. However, the direct regulation of HSPCs via cell surface expression of neural receptors has not been functionally explored. Using imaging mass spectrometry, we detected strong and regionally specific gamma-aminobutyric acid (GABA) neurotransmitter signal in the endosteal region of mouse bone marrow. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation has not been investigated. Based on published mouse HSPC single cell RNA-seq data (Nestorowa et al., Blood 2016), we found that a subset of HSPCs expressed the GABA type B receptor subunit 1 (Gabbr1). We confirmed by surface receptor expression that a subset of mouse bone marrow HSPCs express Gabbr1 protein. Using the same single cell RNA-seq data as above, our own gene set enrichment analysis (GSEA) revealed positive correlation of Gabbr1 expression with genes involved in immune system processes, such as response to type I interferons. We generated a CRISPR-Cas9 Gabbr1 mutant mouse model on a C57/BL6 background suitable for hematopoietic studies. Analysis of Gabbr1 mutant bone marrow cells revealed a reduction in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but no change in the number of long-term hematopoietic stem cells (LT-HSCs). With further hematopoietic profiling, we discovered reduced numbers of white blood cells in peripheral blood that was primarily due to fewer B220+ cells. We show that Gabbr1 null HSPCs display reduced proliferative capacity, as well as diminished reconstitution ability when transplanted in a competitive setting. An in vitro differentiation assay revealed the impaired ability of Gabbr1 null HSPCs to produce B cell lineages. We tested our predicted association with type I interferon response by administration of poly(I:C) and found reduced HSPC proliferation in Gabbr1 null mice. Our results may translate well to humans, as a rare human SNP within the GABBR1 locus was found that correlates with altered leukocyte counts (Astle et al., Cell 2016). Our results indicate an important role for Gabbr1 in the regulation of HSPC proliferation and differentiation, highlighting Gabbr1 as an emerging factor in the modulation of HSPC function and behavior. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Meta-Analytic Single-Cell Atlas of Mouse Bone Marrow Hematopoietic Development

2021 ◽  
Author(s):  
Benjamin D Harris ◽  
John Lee ◽  
Jesse Gillis
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Progenitor Cells ◽  
Cell Types ◽  
Marker Genes ◽  
Mouse Bone Marrow ◽  
Clear Understanding ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Stem And Progenitor Cells

The clinical importance of the hematopoietic system makes it one of the most heavily studied lineages in all of biology. A clear understanding of the cell types and functional programs during hematopoietic development is central to research in aging, cancer, and infectious diseases. Known cell types are traditionally identified by the expression of proteins on the surface of the cells. Stem and progenitor cells defined based on these markers are assigned functions based on their lineage potential. The rapid growth of single cell RNA sequencing technologies (scRNAseq) provides a new modality for evaluating the cellular and functional landscape of hematopoietic stem and progenitor cells. The popularity of this technology among hematopoiesis researchers enables us to conduct a robust meta-analysis of mouse bone marrow scRNAseq data. Using over 300,000 cells across 12 datasets, we evaluate the classification and function of cell types based on discrete clustering, in silico FACS sorting, and a continuous trajectory. We identify replicable signatures that define cell types based on genes and known cellular functions. Additionally, we evaluate the conservation of signatures associated with erythroid and monocyte lineage development across species using co-expression networks. The co-expression networks predict the effectiveness of the signature at identifying erythroid and monocyte cells in zebrafish and human scRNAseq data. Together, this analysis provides a robust reference, particularly marker genes and functional annotations, for future experiments in hematopoietic development.

scholarly journals The IL-33 Receptor/ST2 acts as a positive regulator of functional mouse bone marrow hematopoietic stem and progenitor cells

2020 ◽  
Vol 84 ◽  
pp. 102435 ◽  
Author(s):  
Maegan L. Capitano ◽  
Brad Griesenauer ◽  
Bin Guo ◽  
Scott Cooper ◽  
Sophie Paczesny ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Positive Regulator ◽  
Stem And Progenitor Cells

scholarly journals Single-cell RNA-seq reveals a distinct transcriptome signature of aneuploid hematopoietic cells

Blood ◽  
2017 ◽  
Vol 130 (25) ◽  
pp. 2762-2773 ◽  
Author(s):  
Xin Zhao ◽  
Shouguo Gao ◽  
Zhijie Wu ◽  
Sachiko Kajigaya ◽  
Xingmin Feng ◽  
...  
Keyword(s):  
Immune Response ◽  
Single Cell ◽  
Progenitor Cells ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Key Points ◽  
Stem And Progenitor Cells ◽  
Diploid Cells ◽  
Transcriptome Signature

Key Points We distinguished aneuploid cells from diploid cells within the hematopoietic stem and progenitor cells using scRNA-seq. Monosomy 7 cells showed downregulated pathways involved in immune response and maintenance of DNA stability.

Gabp Transcription Factor Is Required for Self-Renew and Proliferation of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1284-1284
Author(s):  
Zhongfa Yang ◽  
Karen Drumea ◽  
James Cormier ◽  
Junling Wang ◽  
Xuejun Zhu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Myeloid Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Stem And Progenitor Cells ◽  
Ets Domain

Abstract Abstract 1284 GABP is an ets transcription factor that regulates genes which are required for normal hematopoietic development. In myeloid cells, GABP is an essential component of a retinoic acid-inducible enhanceosome that mediates granulocytic gene expression and, in lymphoid cells, GABP regulates expression of IL7-R and the essential transcription factor, Pax5. GABP is a tetrameric complex that includes GABPa, which binds DNA via its ets domain, and GABPb, which contains the transcription activation domain. Genetic disruption of mouse Gabpa caused early embryonic lethality. We created mice in which loxP recombination sites flank exons that encode the Gabpa ets domain, and bred them to mice that bear the Mx1Cre recombinase; injection with pIC induced Cre expression and efficiently deleted Gabpa in hematopoietic cells. One half of the Gabpa knock-out (KO) mice died within two weeks of pIC injection in association with widespread visceral hemorrhage. Gabpa KO mice exhibited a rapid loss of mature granulocytes, and residual myeloid cells exhibited myelodysplasia due, in part, to regulation by Gabp of the transcriptional repressor, Gfi-1. We used bone marrow transplantation to demonstrate that the defect in Gabpa null myeloid cells is cell intrinsic. Although hematopoietic progenitor cells in Gabpa KO bone marrow were decreased more than 100-fold compared to pIC treated control mice, there was not a statistically significant difference in the numbers of Lin−c-kit+Sca-1− hematopoietic stem cells (HSCs) between KO and control mice. Genetic disruption of Gfi-1 disruption in HSCs caused increased cell cycle activity – an effect that is diametrically opposite of the effect of Gabpa KO; this suggests that the effect of Gabpa on HSCs is not due to its control of Gfi-1. In contrast, Gabpa KO HSCs exhibited a marked decrease in cell cycle activity, but did not demonstrate increased apoptosis. The defects in S phase entry of Gabpa null HSCs are reminiscent of the cell cycle defects in Gabpa null fibroblasts, in which expression of Skp2 E3 ubiquitin ligase, which controls degradation of the cyclin dependent kinase inhibitors (CDKIs) p21 and p27, was markedly reduced following Gabpa disruption. We showed that Gabpa KO cells express reduced levels of Skp2. We propose that GABP controls self-renewal and proliferation of mouse bone marrow stem and progenitor cells, in part, through its regulation of Skp2. Thus, Gabpa is a key regulator of myeloid differentiation through its control of Gfi-1, but it is required for cell cycle activity of HSCs, by a distinct effect that may be due to its control of Skp2 and CDKIs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single-cell analysis reveals the KIT D816V mutation in hematopoietic stem and progenitor cells in systemic mastocytosis: Supplementary data

2018 ◽  
Author(s):  
Jennine Grootens ◽  
Johanna S Ungerstedt ◽  
Maria Ekoff ◽  
Elin Rönnberg ◽  
Monika Klimkowska ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Single Cell ◽  
Progenitor Cells ◽  
Systemic Mastocytosis ◽  
Cell Subset ◽  
Tyrosine Kinase Receptor ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Background: Systemic mastocytosis (SM) is a hematological disease characterized by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34+ hematopoietic progenitors can carry the mutation, however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single hematopoietic stem and progenitor cells. Methods: Fluorescence-activated single-cell index sorting and D816V mutation assessment were applied to analyze mast cells and more than 10,000 CD34+ bone marrow progenitors across 10 hematopoietic progenitor subsets. In vitro assays verified cell-forming potential. Findings: We found that in SM 60-99% of the mast cells harbored the D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the hematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the hematopoietic landscape of SM patients, from hematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that FcεRI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time. Interpretation: The KIT D816V mutation arises in early hematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.

scholarly journals Single-Cell RNA-Seq to Assess Differential Responses to Tnfα in Human Hematopoietic Stem and Progenitor Cells in Myeloproliferative Neoplasm

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2518-2518
Author(s):  
Brianna Craver ◽  
Quy Nguyen ◽  
Gajalakshmi Ramanathan ◽  
Angela G. Fleischman
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Transcriptional Profiling ◽  
Unaffected Individual ◽  
Rna Seq ◽  
Apoptotic Response ◽  
Hematopoietic Stem ◽  
Expression Of Genes ◽  
Stem And Progenitor Cells ◽  
Lower Expression

Somatic mutations in hematopoietic stem and progenitor cells (HSPCs) leading to constitutive activation of thrombopoietin receptor signaling result in myeloproliferative neoplasms (MPN). The most common mutation found in MPN patients occurs in the Janus kinase 2 gene (JAK2V617F). We have previously found that JAK2V617F hematopoietic progenitors are resistant to tumor necrosis factor alpha (TNFα), however this mechanism is not well defined. We hypothesize that resistance to TNFα in JAK2V617F hematopoietic stem and progenitors is a driver of the competitive advantage over non-malignant clones. Here, we used droplet-based single-cell RNA-sequencing to investigate transcriptional profiling in primary human HSPCs. First, we harvested white blood cells from fresh bone marrow aspirates from one MPN patient (Polycythemia Vera with 71% JAK2V617F allele burden) as well as one unaffected individual then sorted Lin-/CD34+/CD38- hematopoietic progenitors. Immediately following sorting, half of the cells were stimulated with TNFα for 4 hours while the other half of cells were used as unstimulated controls. We then utilized the 10X Chromium platform to generate single-cell droplets for the 8,129 total cells from the unaffected individual and 33,299 total cells from the MPN patient. We ran alignment using the CellRanger pipeline then performed analysis using the Seurat package in R. Expression profiles of untreated HSPCs in both normal and MPN cells revealed high expression of genes involved in important pathways for hematopoiesis (Polycomb repressive complexes, chromatin regulation, the ubiquitin proteasome system etc.). Expression of CD34 was confirmed in both MPN and non-MPN cells, though CD34 expression was reduced following TNFα stimulation. Expression of stem (i.e. THY1, ITGA6) and progenitor (i.e. PTPRC) genes were detected within both individuals, which highlights the heterogeneity within Lineage-/CD34+/CD38- cells. Following stimulation with TNFα, we observed expression of genes in canonical pathways downstream of TNF including NF-κB, Mitogen-Activated Protein Kinase (MAPK), and Transforming Growth Factor Beta (TGFβ). Indeed, we observed a baseline level of expression of TGFβ-related genes in both normal and MPN cells. Upon inflammatory stimulation, normal HSPCs upregulated SMAD expression which are involved in the TGFβ pathway. Strikingly, we did not observe an increase in SMAD expression in MPN cells following TNF. This suggests a dampened response via the TGFβ pathway to TNF in MPN cells. Additionally, we found that TNF-stimulated HPSCs from the unaffected individual expressed canonical genes of the TNF pathway that encode for chemokines, cytokines, transcription factors and negative feedback regulators. In normal TNF-stimulated cells, we identified highly expressed genes involved in the caspase cascade, suggesting a robust apoptotic response in normal HPSCs. However, there was a lower expression of caspases in stimulated MPN cells, suggesting a dampened apoptotic response to TNF. One observation that was unique to TNF-stimulated cells from the MPN individual was the expression of glycoproteins involved in angiogenesis and platelet aggregation. Taken together, these data serve as a proof of principle for transcriptional profiling of primary human hematopoietic stem and progenitor cells and that this cell population rapidly and robustly alter their gene expression program upon TNFα stimulation. In conclusion, we show that HSPCs from an MPN patient exhibit a dampened response to TNF compared to normal HSPCs. Specifically, we observed a lower expression of genes involved with apoptosis and TGFβ signaling in MPN cells compared to normal cells following TNF stimulation. The finding of a dampened apoptotic response to TNF is consistent with the hypothesis that JAK2V617F cells gain a selective advantage over normal cells under inflammatory stress. To our knowledge, this is the first report of single-cell RNA-seq analysis on primary human HSPCs following FACS and inflammatory stimulation. Disclosures Fleischman: incyte: Speakers Bureau.

scholarly journals Mouse Erythroid Cells Originate from a Megakaryocyte Precursor in Common Myeloid Progenitors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 337-337
Author(s):  
Elisabeth F Heuston ◽  
Bethan Psaila ◽  
Cheryl A Keller ◽  
NISC Comparative SequencingProgram ◽  
Stacie M Anderson ◽  
...  
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Qpcr Assay ◽  
Gene Set Enrichment Analysis ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Myeloid Progenitors

The hierarchical model of hematopoiesis posits that hematopoietic stem and progenitor cells produce common myeloid progenitors (CMP). CMP can become granulocyte/monocyte progenitors (GMP) or bipotential megakaryocyte/erythroid progenitors (MEP). MEP can produce megakaryocytic (Mk) or erythroid (Ery) cells. However, we and others have shown that early mouse and human progenitor populations express many Mk genes (Heuston, Epig. Chrom., 2016), while single cell studies have identified lineage-specific colony forming cells in progenitor populations thought to be multipotent (Psaila, Genome Biol., 2016). To identify the earliest mouse Ery and Mk cells, we performed single cell RNASeq on 10000 stem and progenitor cells (Lin-Sca1+Kit+), 12000 CMP (Lin-Sca1-Kit+CD16/32-CD34+), 6000 MEP (Lin-Sca1-Kit+CD16/32-CD34-) and 8000 GMP (Lin-Sca1-Kit+CD16/32+CD34+). TSNE analysis of expression in the 4 populations identified 33 clusters, which were correlated to biological functions using gene set enrichment analysis. In LSK, no cells with an Ery RNA profile were found, while 56% of cells co-expressed Mk-associated (e.g., Meis1, Fli1) and lymphoid genes. In CMP, 12% of the cells co-expressed Ery (e.g., Gata1, Fog1) and Mk (e.g., Pf4, Cd41) genes, while 23% had an Mk-specific profile (e.g., Fli1, Cd41) enriched for platelet biology processes (p< 3E-18). Unlike traditional models, over 94% of MEP had Ery RNA profiles enriched for ribosome synthesis and heme-biology processes (p< 4E-10). To establish developmental relationships, we performed pseudotime analysis using the Monocle and Scanpy software packages. These programs model differentiation by mapping similar transcriptomes together. Map nodes indicate lineage commitment points and cells further from a node are more differentiated. Combined analysis of LSK, CMP, and MEP generated a model with a single node and 2 trajectories. LSK with Mk and lymphoid RNA profiles diverged at the node, as did 14% of CMP. 31% of CMP with an Mk RNA profile were downstream of the node. Further downstream were cells with mixed Ery/Mk profiles, and furthest from the node were MEP with Ery profiles. A separate pseudotime analysis of CMP only 2 trajectories: one with decreasing Mk- and increasing Ery RNA profiles, and a second with an early Mk endomitotic RNA profile. Pseudotime analysis of MEP only identified a linear trajectory: cells at one end expressed early Ery RNA profiles, and cells at the other end had RNA profiles similar to those of burst-forming unit-erythroid (BFU-E). We generated a predictive set of RNAs for each TSNE cluster. We used index-sorting with 11 markers (Kit, Sca1, CD34, CD16/32, CD36, CD41, CD48, CD123, CD150, CD9, Flk2) to isolate single cells for custom high-throughput multiplex qPCR. This allowed confirmation of cell frequency within TSNE clusters while identifying surface markers for prospective isolation of cell subsets. We focused on 2 populations: CMP-E, which had an Ery RNA profile (10% of clustered CMP and 12% of CMP in the qPCR assay), and CMP-MkE, which had Mk and Ery RNA profiles (12% of clustered CMP and 13% of CMP in the qPCR assay). We prospectively isolated CMP-E and CMP-MkE to compare RNASeq profiles, ATACSeq profiles, and colony forming ability against those of bulk CMP, Ery, and Mk. In CMP-E, 54% of RNAs were expressed in both CMP and ERY, while 41% were expressed only in CMP (p < 6E-72). In contrast, 41% of CMP-E ATACSeq peaks were present in CMP and ERY, while 57% of CMP-E peaks were present only in CMP (p < 1E-3). We conclude that in CMP-E, the RNASeq profile is more erythroid than the ATACSeq profile. In CMP-MkE, 89% of RNAs were expressed in both CMP and Mk, while 7% were expressed only in CMP (p < 8E-190). Likewise, 88% of CMP-MkE ATACSeq peaks were present in both CMP and Mk, while 3% were present only in CMP (p < 1E-3). We conclude that in CMP-MkE, the RNASeq and ATACSeq profiles are equivalent. In soft agar assays, 21% of CMP-E and 3% of CMP-MkE colonies contained BFU-E, compared to 9% of control colonies. We conclude that the CMP-E and CMP-MkE populations are skewed towards the ERY and MK lineages, but are not erythro-megakaryocyte restricted. Our data support a model in which there are two megakaryocyte precursor populations and no erythroid populations in LSK. A third megakaryocyte population in CMP gives rise to erythroid cells. Finally, our data show that transcriptional changes precede chromatin accessibility changes in the earliest erythroid cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mechanism of Action of Diallyl Sulphide in Ameliorating the Hematopoietic Radiation Injury

2021 ◽  
Author(s):  
Omika Katoch ◽  
Mrinalini Tiwari ◽  
Namita Kalra ◽  
Paban K. Agrawala
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation ◽  
Stem And Progenitor Cells ◽  
Radiation Induced ◽  
Medicinal Properties ◽  
Marrow Cellularity

AbstractDiallyl sulphide (DAS), the pungent component of garlic, is known to have several medicinal properties and has recently been shown to have radiomitigative properties. The present study was performed to better understand its mode of action in rendering radiomitigation. Evaluation of the colonogenic ability of hematopoietic progenitor cells (HPCs) on methocult media, proliferation and differentiation of hematopoietic stem cells (HSCs), and transplantation of stem cells were performed. The supporting tissue of HSCs was also evaluated by examining the histology of bone marrow and in vitro colony-forming unit–fibroblast (CFU-F) count. Alterations in the levels of IL-5, IL-6 and COX-2 were studied as a function of radiation or DAS treatment. It was observed that an increase in proliferation and differentiation of hematopoietic stem and progenitor cells occurred by postirradiation DAS administration. It also resulted in increased circulating and bone marrow homing of transplanted stem cells. Enhancement in bone marrow cellularity, CFU-F count, and cytokine IL-5 level were also evident. All those actions of DAS that could possibly add to its radiomitigative potential and can be attributed to its HDAC inhibitory properties, as was observed by the reversal radiation induced increase in histone acetylation.

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