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Next Generation Sequencing Facilitates Quantitative Analysis of db/m and db/db bone marrow derived LSK (Lineage negative Sca1-ckit positive cells) Transcriptomes I

ABSTRACT: The 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 transcriptome under the conditions of long-standing type 2 diabetes (T2D;>6 months) remains unexplored. We employed the strength of Next generation sequencing to to study the differential pattern of LSK transcriptome under chronic diabetes.

ORGANISM(S): Mus musculus

PROVIDER: GSE261501 | GEO | 2024/07/30

REPOSITORIES: GEO

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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:Purpose: the goal of this study is to investigate the consequences of USP3 deletion on gene expression in mouse LSK hematopoietic progenitors and in splenic B cells Methods: mRNA profiles of 8 weeks-old wild-type (WT) and ubiquitin specific protease 3 knockout (Usp3−/−) mice were generated by deep sequencing, in duplicate, using Illumina Hiseq2000. The sequence reads that passed quality filters were mapped with TopHat and the gene expressions were calculated using HTSeq-count. qRT–PCR validation was performed using SYBR Green assays Results: We assigned about 8-16 million reads per sample uniquely to a gene of the mouse reference genome (mm9). We identified 23,429 genes in the LSKs, naive B cells and activate B cells of WT and USP3−/− mice using TopHat in combination with HTSeq-count. Comparison of the RNAseq data from LSK with naive or activated B cells show that both the wt and the Usp3-/- LSKs largely exibited a gene expression profile that is specific for wt LSK and distinct from B cells (as supported by statistical significant difference between the transciptional profile of LSK versus naive or activated B cells, p value<0.0001 by Student t test). Comparison of normalized gene expression data for Wt LSKs versus naive B cells of one representative experiment shows Pearson coefficient of r=0.874, and R2=0.763. Distinct LSK-specific expressed genes (such as the MlI receptor and the Kit receptor) and B cells specific genes (such as the MS4A1/CD20 and Spi-B transcription factors) are identified. Expression of a set of 19 genes was assessed by RT-qPCR in three independent LSK mRNA per each genotype. qRT-PCR and the RNA-seq normalized expression data for these genes had a good linear relationship, validating the RNAseq analysis. Comparison of normalized gene expression data for Usp3-/- versus Wt LSK show Pearson coefficient r=0.986; R2=0.9738), naive B cells (Pearson coefficient r=0.987, R2=0.974) and LPS activated B cells (Pearson coefficient r=0.991, R2=0.983). RT-qPCR of a subset of hematopoietic stem cell genes, including Mlp2, ENg, Tek and Fdzl3, show no significant difference beteewn wt and Usp3-/- LSK cells. Less than 100 genes showed differential expression (up or down regulated) between the Wt and Usp3-/- LSK, with a fold change ≥1.5 and p value <0.05. Conclusions: Our results represent the first detailed analyis of the consequences of USP3 deletion on gene expression in hematopoietic populations such as LSKs progenitors and B cells by genome wide expression profiling in wt and Usp3-/- mice. RNAseq of two freshly isolated biological replicas of sorted LSKs from 8 weeks old Usp3-/- animals showed a very limited number of genes either slighly up or down regulated (<100 out of about 25.000) in Usp3-/- LSKs, none of which are reported to be directly involved in hematopoietic stem cell maintenance or to be linked to premature differentiation. We confirmed that Usp3-/- and wt LSKs express hematopoietic stem cell-specific genes to a similar extent. We conclude that young adult hematopoietic stem and progenitor cells (LSKs) perpetuated a stable gene expression program regardless of the homozygous deletion of USP3. mRNA profiles of 8 weeks-old wild type (WT) and Usp3-/- mice were generated by deep sequencing, in duplicate, using Illumina Hiseq2000. For each experiment wt n=4, Usp3-/- n=4 mice were analized. FACS sorted cells from from individual animals were pooled and subjected to deep sequencing. Cells were: LSK (Lin- Sca1+ cKit+) from bone marrow, sorted naive B cells from spleens (CD19+) and activated B cells harvested and FACS sorted after 4 days stimulation with lipopolysaccharide (LPS) in culture.

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.

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Next Generation Sequencing Facilitates Quantitative Analysis of db/m and db/db bone marrow derived LSK (Lineage negative Sca1-ckit positive cells) Transcriptomes I

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