Transcriptome of EpoR-/- CFU-E cells
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PROVIDER: PRJNA428317 | ENA |
REPOSITORIES: ENA
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Project description:During erythropoiesis, erythroid progenitor cells proliferate in response to erythropoietin (Epo) and progressively differentiate, which encompasses an increasing accumulation of iron-bound hemoglobin. The impact of Epo on these dynamic processes remained to be resolved. By combining the development of a time-collapsed super SILAC method with transcriptome analysis we examined Epo-induced alterations in the proteome of erythroid progenitor cells at the colony-forming unit erythroid stage (CFU E). We showed that the timing of a major increase in the transcription factors GATA1 and TAL1 precedes the cessation of cell cycle progression but coincides with a massive upregulation of enzymes of the heme biosynthetic pathway. A failure of this upregulation due to the absence of the Epo receptor resulted in massive iron accumulation in the fetal liver and dilation of the maternal vasculature in the placenta. Thus, Epo-induced dynamic proteome adaptations in CFU E cells not only prevent anemia but also severe iron-intoxication in embryos.
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Project description:Burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45+GPA-IL-3R-CD34+CD36-CD71low and CD45+GPA-IL-3R-CD34-CD36+CD71high phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity ~90%. The BFU-E colony forming ability of CD45+GPA-IL-3R-CD34+CD36-CD71low cells required SCF and erythropoietin, while the CFU-E colony forming ability of CD45+GPA-IL-3R-CD34-CD36+CD71high cells required only erythropoietin. Bioinformatic analysis of the RNA-seq data revealed unique transcriptomes in each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematological disease. Our data provide important resource for future studies.
2014-10-30 | GSE61566 | GEO
Project description:Dormant Mycobacterium tuberculosis bacilli play important role in latent tuberculosis infection. Previously we have demonstrated that cultivation of M. tuberculosis in potassium-deficient media resulted in the generation of dormant ‘non-culturable’ cells. Addition of a moderate concentration of rifampicin enabled to kill a minor subpopulation of actively replicating bacilli and obtain a homogeneous ‘zero-CFU’ population of dormant cells characterized by total inability to produce colonies on solid media and by high potential to reactivate after reintroducing of potassium. An RNA-seq based transcriptome analysis of this dormant ‘zero-CFU’ population revealed a 30-50 fold decrease of the total level of mRNA in the cells, indicating global shift-down of gene expression level. The residual scarce protein-coding transcriptome of the dormant cells showed decreased abundance of mRNAs encoding ribosomal proteins and enzymes of TCA cycle and respiratory chain, and increased abundance of mRNAs encoding PE-PGRS proteins. Interestingly, the transcriptome of dormant cells showed little changes during several days of persistence. This stability of ‘dormant’ transcripts may reflect their readiness for translation upon resuscitation process. Transition of M. tuberculosis cells to dormancy was accompanied by the cleavage of 23S ribosomal RNA at a specific point, located outside the ribosome catalytic center. Another feature of the ‘dormant’ transcriptome was an increased abundance of non-coding transcripts. Enrichment of "dormant transcriptome" by small non-coding RNAs playing regulatory function in the cell probably indicates their role in transition to and maintenance of dormant ‘non-culturable’ state.
2015-06-01 | GSE66408 | GEO
Project description:In this study we analyzed the zebrafish embryonic host response induced by E. tarda (FL6-60) immersion. The E. tarda induced transcriptome profile was compared to those induced by either E. coli or Pseudomonas aeruginosa immersion using the same experimental setup. All infection experiments were performed using mixed egg clutches of Albino strain zebrafish. At 24 hpf embryos were dechorionated using 2mg/ml pronase and left to recover for one hour in egg water. Subsequently embryos were immersed in a bacterial suspension (E. tarda (1E8 CFU/ml), E. coli (1E8 CFU/ml), Pseudomonas aeruginosa PAO1 (1E9 CFU/ml) or P. aeruginosa PA14 (1E9 CFU/ml)) and incubated for 5 hours at 28 ˚C. After the incubation period the embryos were snap-frozen in liquid nitrogen. All treatment groups were analyzed using a common reference approach.
2011-09-30 | E-GEOD-28481 | biostudies-arrayexpress
Project description:Burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45+GPA-IL-3R-CD34+CD36-CD71low and CD45+GPA-IL-3R-CD34-CD36+CD71high phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity ~90%. The BFU-E colony forming ability of CD45+GPA-IL-3R-CD34+CD36-CD71low cells required SCF and erythropoietin, while the CFU-E colony forming ability of CD45+GPA-IL-3R-CD34-CD36+CD71high cells required only erythropoietin. Bioinformatic analysis of the RNA-seq data revealed unique transcriptomes in each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematological disease. Our data provide important resource for future studies. Transcription profiles of Human erythroid progenitors at distinct developmental stages were generated by deep sequencing, in triplicate, using IlluminaHiSeq 2000. The complete dataset comprises 4 sample types: CD34, BFU, CFU, and Pro (reanalysis of GSM1304777-GSM1304779).
2014-10-30 | E-GEOD-61566 | biostudies-arrayexpress
Project description:Time-resolved CFU-E proteome was analyzed by time collapsed super-SILAC with erythropoietin-stimulated BaF3-mEpoR proteome as internal heavy standard.
2022-09-28 | PXD004539 | Pride
Project description:Myeloid cells are important sites of lytic and latent infection by human cytomegalovirus (CMV). We previously showed that only a small subset of myeloid cells differentiated from CD34+ hematopoietic stem cells is permissive to CMV replication, underscoring the heterogeneous nature of these populations. The exact identity of susceptible and resistant cell types, and the cellular features characterizing permissive cells, however, could not be dissected using averaging transcriptional analysis tools such as microarrays and, hence, remained enigmatic. Here, we profile the transcriptomes of ~ 7000 individual cells at day one post-infection using the 10X genomics platform. We show that viral transcripts are detectable in the majority of the cells, suggesting that virion entry is unlikely to be the main target of cellular restriction mechanisms. We further show that viral replication occurs in a small but specific sub-group of cells transcriptionally related to, and likely derived from, a cluster of cells expressing markers of Colony Forming Unit – Granulocyte, Erythrocyte, Monocyte, Megakaryocyte (CFU-GEMM) oligopotent progenitors. Compared to the remainder of the population, CFU-GEMM cells are enriched in transcripts with functions in mitochondrial energy production, cell proliferation, RNA processing and protein synthesis, and express similar or higher levels of interferon-related genes. While expression levels of the former are maintained in infected cells, the latter are strongly down-regulated. We thus propose that the preferential infection of CFU-GEMM cells may be due to the presence of a pre-established pro-viral environment, requiring minimal optimization efforts from viral effectors, rather than to the absence of specific restriction factors. Together, these findings identify a potentially new population of myeloid cells susceptible to CMV replication, and provide a possible rationale for their preferential infection.
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