Dataset Information

Deficiency in Fpr2 results in reduced numbers of Lin^-cKit⁺Sca1⁺ myeloid progenitor cells.

ABSTRACT: The Lin^-c-Kit⁺ Sca-1⁺ cell population in the bone marrow (BM) serves as the direct precursor for differentiation of myeloid cells. In this study, we report that deficiency in Fpr2, a G protein-coupled chemoattractant receptor in mice, is associated with reduced BM nucleated cells, including CD31⁺Ly6C⁺ (granulocytes and monocytes), CD31^-/Ly6C^int (granuloid cells), and CD31^-/Ly6C^high (predominantly monocytes) cells. In particular, the number of Lin^-c-Kit⁺Sca-1⁺ (LKS) cells was reduced in Fpr2^-/- mouse BM. This was supported by observations of the reduced incorporation of intraperitoneally injected bromodeoxyuridine by cells in the c-Kit⁺ population from Fpr2^-/- mouse BM. Purified c-Kit⁺ cells from Fpr2^-/- mice showed reduced expansion when cultured in vitro with stem cell factor (SCF). SCF/c-Kit-mediated phosphorylation of P38, STAT1, Akt (Thr-308), and Akt (Ser-473) was also significantly reduced in c-Kit⁺ cells from Fpr2^-/- mice. Furthermore, Fpr2 agonists enhanced SCF-induced proliferation of c-Kit⁺ cells. Colony-forming unit assays revealed that CFU-granulocyte-macrophage formation of BM cells from Fpr2^-/- mice was significantly reduced. After heat-inactivated bacterial stimulation in the airway, the expansion of c-kit⁺ Sca-1⁺ cells in BM and recruitment of Ly6G⁺ cells to the lungs and CD11b⁺Ly6C⁺TNF?⁺ cells to the spleen of Fpr2^-/- mice was significantly reduced. These results demonstrate an important role for Fpr2 in the development of myeloid lineage precursors in mouse BM.

SUBMITTER: Chen K

PROVIDER: S-EPMC6120191 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Deficiency in Fpr2 results in reduced numbers of Lin-cKit+Sca1+ myeloid progenitor cells.

Chen Keqiang K Tang Peng P Bao Zhiyao Z He Tianzhen T Xiang Yi Y Gong Wanghua W Yoshimura Teizo T Le Yingying Y Tessarollo Lino L Chen Xin X Wang Ji Ming JM

The Journal of biological chemistry 20180717 35

The Lin-c-Kit+ Sca-1+ cell population in the bone marrow (BM) serves as the direct precursor for differentiation of myeloid cells. In this study, we report that deficiency in Fpr2, a G protein-coupled chemoattractant receptor in mice, is associated with reduced BM nucleated cells, including CD31+Ly6C+ (granulocytes and monocytes), CD31-/Ly6Cint (granuloid cells), and CD31-/Ly6Chigh (predominantly m ...[more]

PMID: 30018139

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Project description:The RUNX genes encode for transcription factors involved in development and human disease. RUNX1 and RUNX3 are frequently associated with leukemias, yet the basis for their involvement in leukemogenesis is not fully understood. Here we show that Runx1;Runx3 double knockout (DKO) mice exhibited lethal phenotypes due to bone marrow failure and myeloproliferative disorder. These contradictory clinical manifestations are reminiscent of human inherited bone marrow failure syndromes like Fanconi anemia (FA), caused by defective DNA repair. Indeed, Runx1;Runx3 DKO cells showed mitomycin C hypersensitivity, due to impairment of monoubiquitinated-FANCD2 recruitment to DNA damage foci, although FANCD2 monoubiquitination in the FA pathway was unaffected. RUNX1 and RUNX3 interact with FANCD2 independent of CBFM-NM-2, suggesting non-transcriptional role for RUNX in DNA repair. These findings suggest that RUNX dysfunction causes DNA repair defect, besides transcriptional misregulation, and promotes development of leukemias and other cancers. 6 mice were analyzed in this study. 3 Runx1;Runx3 double knockout cKit+Sca1+Lin- hematopoietic stem/progenitor cells were compared with their wild type littermate controls. RNA was isolated from 3 independent Runx1;Runx3 WT KSL samples, each pooled from 3 Runx1;Runx3 WT mice, and 3 independent Runx1;Runx3 DKO KSL samples, using the RNeasy Micro Kit (QIAgen). RNA integrity and quantity was assessed using the Agilent 2000 Bioanalyzer system. 3 M-NM-<g to 5 M-NM-<g RNA was processed using WT-Ovation Pico RNA Amplification System (NuGEN) paired with the WT-Ovation Exon Module and FL-Ovation cDNA Biotin Module (NuGEN). A detailed protocol in the userM-bM-^@M-^Ys guide kit was used without modification. cDNA were prepared for hybridization on GeneChip Mouse Gene 1.0 ST Arrays (Affymetrix) according to the instructions in GeneChip Hybridization Wash and Stain Kit for ST arrays (Affymetrix). Microarray hybridization, scanning and preliminary MAS 5.0 normalizations were completed at the A*STAR Biopolis Shared Facilities (BSF).

Project description:BackgroundThe Lin-Sca1+c-Kit+ (LSK) fraction of the bone marrow (BM) comprises multipotent hematopoietic stem cells (HSCs), which are vital to tissue homeostasis and vascular repair. While diabetes affects HSC homeostasis overall, the molecular signature of mRNA and miRNA transcriptomic under the conditions of long-standing type 2 diabetes (T2D;>6 months) remains unexplored.MethodsIn this study, we assessed the transcriptomic signature of HSCs in db/db mice, a well-known and widely used model for T2D. LSK cells of db/db mice enriched using a cell sorter were subjected to paired-end mRNA and single-end miRNA seq library and sequenced on Illumina NovaSeq 6000. The mRNA sequence reads were mapped using STAR (Spliced Transcripts Alignment to a Reference), and the miRNA sequence reads were mapped to the designated reference genome using the Qiagen GeneGlobe RNA-seq Analysis Portal with default parameters for miRNA.ResultsWe uncovered 2076 out of 13,708 mRNAs and 35 out of 191 miRNAs that were expressed significantly in db/db animals; strikingly, previously unreported miRNAs (miR-3968 and miR-1971) were found to be downregulated in db/db mice. Furthermore, we observed a molecular shift in the transcriptome of HSCs of diabetes with an increase in pro-inflammatory cytokines (Il4, Tlr4, and Tnf11α) and a decrease in anti-inflammatory cytokine IL10. Pathway mapping demonstrated inflammation mediated by chemokine, cytokine, and angiogenesis as one of the top pathways with a significantly higher number of transcripts in db/db mice. These molecular changes were reflected in an overt defect in LSK mobility in the bone marrow. miRNA downstream target analysis unveils several mRNAs targeting leukocyte migration, microglia activation, phagosome formation, and macrophage activation signaling as their primary pathways, suggesting a shift to an inflammatory phenotype.ConclusionOur findings highlight that chronic diabetes adversely alters HSCs' homeostasis at the transcriptional level, thus potentially contributing to the inflammatory phenotype of HSCs under long-term diabetes. We also believe that identifying HSCs-based biomarkers in miRNAs or mRNAs could serve as diagnostic markers and potential therapeutic targets for diabetes and associated vascular complications.

Project description:In vitro bioreactor-based cultures are being extensively investigated for large-scale production of differentiated cells from embryonic stem cells (ESCs). However, it is unclear whether in vitro ESC-derived progenitors have similar gene expression profiles and functionalities as their in vivo counterparts. This is crucial in establishing the validity of ESC-derived cells as replacements for adult-isolated cells for clinical therapies. In this study, we compared the gene expression profiles of Lin-ckit+Sca-1+ (LKS) cells generated in vitro from mouse ESCs using either static or bioreactor-based cultures, with that of native LKS cells isolated from mouse fetal liver (FL) or bone marrow (BM). We found that in vitro-generated LKS cells were more similar to FL- than to BM LKS cells in gene expression. Further, when compared to cells derived from bioreactor cultures, static culture-derived LKS cells showed fewer differentially expressed genes relative to both in vivo LKS populations. Overall, the expression of hematopoietic genes was lower in ESC-derived LKS cells compared to cells from BM and FL, while the levels of non-hematopoietic genes were up-regulated. In order to determine if these molecular profiles correlated with functionality, we evaluated ESC-derived LKS cells for in vitro hematopoietic-differentiation and colony formation (CFU assay). Although static culture-generated cells failed to form any colonies, they did differentiate into CD11c+ and B220+ cells indicating some hematopoietic potential. In contrast, bioreactor-derived LKS cells, when differentiated under the same conditions failed to produce any B220+ or CD11c+ cells and did not form colonies, indicating that these cells are not hematopoietic progenitors. We conclude that in vitro culture conditions significantly affect the transcriptome and functionality of ESC-derived LKS cells and although in vitro differentiated LKS cells were lineage negative and expressed both ckit and Sca-1, these cells, especially those obtained from dynamic cultures, are significantly different from native cells of the same phenotype.

Project description:Purpose: the goal of this study is to investigate the consequences of ubiquitin specific protease 15 (USP15) deletion on gene expression in mouse LSK hematopoietic progenitors. Methods: mRNA profiles of 8 weeks-old wild-type (WT) and ubiquitin specific protease 15 knockout (Usp15−/−) mice were generated by deep sequencing using Illumina Hiseq2500. The sequence reads that passed quality filters Alignments of the sequence reads that passed quality filters were performed using TopHat2.1, Genome build 38 and Ensembl gtf version 77 and genecounts have been generated using Itreecount. https://github.com/NKI-GCF/itreecount Results: We assigned about 30 million reads per sample uniquely to a gene of the mouse reference genome (mm10) We identified 21,219 genes in the LSKs of WT and Usp15−/− mice using TopHat2.1 in combination with Itreecount. https://github.com/NKI-GCF/itreecount. Distinct LSK-specific expressed genes (such as Eng, Tek, the MlI receptor and the Kit receptor) are identified. Comparison of normalized gene expression data for Usp15-/- versus WT LSK confirmed the loss of Usp15. Apart from Usp15, almost no other significantly deregurated genes were detected using a treshold of fold change ≥1.5 and p value <0.05, suggesting the maintenance of an overall stable identity of the cellular compartment in Usp15-/- mice. Conclusions: Our results represent the first detailed analyis of the consequences of USP15 deletion on gene expression in hematopoietic populations such as LSKs progenitors by genome wide expression profiling in WT and Usp15-/- mice. RNAseq of two freshly isolated biological replicas of sorted LSKs from 8 weeks old Usp15-/- animals confirmed the loss of Usp15, while showing almost no significant other up or down regulated genes among the 21,219 genes identified in Usp15-/- LSKs. We conclude that young adult hematopoietic stem and progenitor cells (LSKs) perpetuated a stable gene expression program regardless of the homozygous deletion of USP15.

Dataset Information

Deficiency in Fpr2 results in reduced numbers of Lin^-cKit⁺Sca1⁺ myeloid progenitor cells.

Publications

Deficiency in Fpr2 results in reduced numbers of Lin<sup>-</sup>cKit<sup>+</sup>Sca1<sup>+</sup> myeloid progenitor cells.

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