Prospective isolation of radiation induced erythroid stress progenitors reveals unique transcriptomic and epigenetic signatures enabling increased erythroid output
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ABSTRACT:
PROVIDER: PRJNA579342 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10341425.fastq.gz | Fastqsanger.gz | |||
| SRR10341426.fastq.gz | Fastqsanger.gz | |||
| SRR10341427.fastq.gz | Fastqsanger.gz | |||
| SRR10341428.fastq.gz | Fastqsanger.gz | |||
| SRR10341429.fastq.gz | Fastqsanger.gz |
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Project description:Massive expansion of erythroid progenitor cells is essential for surviving anemic stress. Research towards understanding this critical process, referred to as stress-erythropoiesis, has been hampered due to lack of specific marker-combinations enabling analysis of the distinct stress-progenitor cells capable of providing radioprotection and enhanced red blood cell production. Here we present a method for precise identification and in vivo validation of progenitor cells contributing to both steady-state and stress-erythropoiesis, enabling for the first time in-depth molecular characterization of these cells. Differential expression of surface markers CD150, CD9 and Sca1 defines a hierarchy of splenic stress-progenitors during irradiation-induced stress recovery in mice, and provides high-purity isolation of the functional stress-BFU-Es with a 100-fold improved enrichment compared to state-of-the-art. By transplanting purified stress-progenitors expressing the fluorescent protein Kusabira Orange, we have for the first time determined their kinetics in vivo and demonstrated that CD150+CD9+Sca1- stress-BFU-Es provide a massive but transient radioprotective erythroid wave, followed by multi-lineage reconstitution from CD150+CD9+Sca1+ multi-potent stem/progenitor cells. Whole genome transcriptional analysis revealed that stress-BFU-Es express gene signatures more associated with erythropoiesis and proliferation compared to steady-state BFU-Es, and are BMP-responsive. Evaluation of chromatin accessibility through ATAC sequencing reveals enhanced and differential accessibility to binding sites of the chromatin-looping transcription factor CTCF in stress-BFU-Es compared to steady-state BFU-Es. Our findings offer molecular insight to the unique capacity of stress-BFU-Es to rapidly form erythroid cells in response to anemia and constitute an important step towards identifying novel erythropoiesis stimulating agents.
2020-01-03 | GSE139336 | GEO
Project description:Intra-tumour heterogeneity caused by clonal evolution is a major problem in cancer treatment stratification. To address this problem, we performed label-free quantitative proteomics on primary acute myeloid leukemia (AML) samples. We identified 50 leukemia-enriched plasma membrane (PM) proteins enabling the prospective isolation of genetically distinct subclones from individual AML patients. Subclones differed in their regulatory phenotype, drug sensitivity, growth and engraftment behavior, as determined by RNA sequencing, DNaseI hypersensitive site mapping, transcription factor occupancy analysis, in vitro culture and xenograft transplantation. Finally, we show that these markers can be used to identify and longitudinally track distinct leukemic clones in patients in routine diagnostics. Our study describes a strategy for a major improvement in stratifying cancer diagnosis and treatment.
2018-07-26 | GSE117667 | GEO
Project description:Background: Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by âbulkâ assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined.Results: To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlated the surface immunophenotype, transcriptional profile and differentiation potential of individual MEP cells. Highly purified, single MEP cells (n=681) were analyzed using index fluorescence-activated cell sorting with parallel targeted transcriptional profiling of the same cells performed using a specifically designed panel of 87 genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrated that immunophenotypic MEP in fact comprise three distinct subpopulations: (1) âPre-MEPâ, enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity (2) âE-MEPâ, strongly biased towards erythroid differentiation, and (3) âMK-MEPâ, a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally-defined MEP are in fact a mixed population: a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally-primed to generate exclusively single-lineage output. Conclusions: Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders and erythromegakaryocytic leukemias. Multiplex RT-PCR gene expression profiling of 807 human megakaryocyte-erythroid progenitor cells (MEP) isolated from three healthy donors by apheresis following G-CSF treatment. Cells were excluded if more than 70 assays did not result in amplification or displayed Ct higer than 13 for B2M or higher than 15 for GAPDH. Furthermore cells with a mean non-dropout Ct value greater than 20 were removed. This resulted in a dataset of 681 cells, which were subsequently normalised to the mean of B2M and GAPDH expression.
2016-04-02 | E-GEOD-79331 | biostudies-arrayexpress
Project description:Background: Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by “bulk” assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined.Results: To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlated the surface immunophenotype, transcriptional profile and differentiation potential of individual MEP cells. Highly purified, single MEP cells (n=681) were analyzed using index fluorescence-activated cell sorting with parallel targeted transcriptional profiling of the same cells performed using a specifically designed panel of 87 genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrated that immunophenotypic MEP in fact comprise three distinct subpopulations: (1) “Pre-MEP”, enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity (2) “E-MEP”, strongly biased towards erythroid differentiation, and (3) “MK-MEP”, a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally-defined MEP are in fact a mixed population: a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally-primed to generate exclusively single-lineage output. Conclusions: Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders and erythromegakaryocytic leukemias.
2016-04-02 | GSE79331 | GEO
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: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:The transcription factor GATA1 has pleiotropic hematopoietic functions, particularly in erythroid and megakaryocytic ontogeny. While mechanistic investigations have uncovered many facets of GATA1 biology, how GATA1 co-regulates divergent cell fates remains incompletely characterized. We previously described that loss of Stag2, a member of the cohesin complex and a recurrent mutational target in myelodysplastic syndrome (MDS) and myeloid leukemia of Down Syndrome, results in altered chromatin accessibility, transcription factor function, and cell differentiation. Hence, we hypothesized that chromatin accessibility determines GATA1 cistrome specificity and lineage fate decisions. To understand the connection between chromatin accessibility and GATA1, we comprehensively studied erythropoiesis in Stag2∆ mice. Defects in Stag2-deficient hematopoiesis included reduced numbers of erythroid progenitors (EryPs), impaired terminal erythroid differentiation, increased number of MkPs, and increased megakaryocytes. RNA- and ATAC-sequencing of EryPs revealed altered patterns of Gata1 target gene expression with altered accessibility in conjunction with loss of expression of erythroid targets and gain of megakaryocyte targets. Despite unchanged Gata1 expression, Gata1 occupancy was reprogrammed from erythroid to megakaryocyte targets with Fli1 motifs enriched at Stag2∆ Gata1 binding sites. Functionally, we observed that Stag2-deficient EryPs have diminished erythroid output and augmented megakaryocyte output in orthogonal differentiation assays, which was partially reversed with Fli1 knockdown. Human models and primary MDS patients recapitulated the essential phenotypic and molecular features of our in vivo murine MDS model. Collectively, this study establishes chromatin accessibility as a determinant of transcription factor binding specificity, revealing an accessibility-driven Gata1 retargeting mechanism underlying MDS dyserythropoiesis.
2026-06-08 | GSE301108 | GEO
Project description:Impact on erythroid progenitor type on erythroid differntiation
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| PRJEB53187 | ENA