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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Uhrf1-/- fetal liver hematopoietic stem cell (FL-HSCs) Transcriptomes

ABSTRACT: To investigate the molecular mechanism of Uhrf1 in controlling the self-renewal versus differentiation of FL-HSCs, high-throughput sequencing was performed to analyze the transcriptomes of WT and Uhrf1-deficient FL-HSCs.Consistent with the erythroid-biased differentiation of Uhrf1-deficient FL-HSCs, genes involved in erythrocyte differentiation were significantly enriched in Uhrf1-deficient FL-HSCs according to the Gene Ontology (GO) enrichment analysis. We generated gene signatures specific for stemness of HSCs (stem signature) or myeloerythroid progenitors (MEP signature) by subtracting the genes expressed in WT HPC(LK)s from those expressed in WT FL-HSCs or vice versa. Of the stem signature genes, 71.28% showed lower expression and 28.72% showed higher expression in Uhrf1-deficient FL-HSCs compared with WT FL-HSCs. Interestingly, among the MEP signature genes, genes enriched in erythroid differentiation (27.54%) were up-regulated in the absence of Uhrf1, whereas the remaining genes enriched in myeloid-specific genes (72.46%) were suppressed consistent with previous research. Particularly, in comparison to the WT control, Uhrf1-deficient FL-HSCs up-regulated certain erythrocyte differentiation-related genes (e.g., Gata1, Gata2, Gfi1b, Car1, Zfpm1 and Itga2b), most of which were physiologically up-regulated in HPC(LK)s, whereas some genes (Id2, Satb1, Hmga2) that play critical roles in HSC maintenance were down-regulated. These results suggested that Uhrf1 controls the self-renewal versus differentiation of FL-HSCs by suppressing the expression of the erythroid-specific genes and maintaining the expression of HSC stemness genes.

ORGANISM(S): Mus musculus

PROVIDER: GSE85450 | GEO | 2016/11/30

SECONDARY ACCESSION(S): PRJNA338482

REPOSITORIES: GEO

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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.

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.

Project description:MiRNAs have the potential to regulate cellular differentiation programs. However, miRNA-deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions unanswered. To address this issue, we deleted Dicer1, which encodes an essential RNaseIII enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein alpha (C/EBPA)-positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA-positive progenitor cells. Analysis of gene-expression profiles from wild type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multi-potent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating a regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages and caused myeloid dysplasia with morphological features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion towards myeloid differentiation in GMPs. To generate Cebpa-Cre;R26-LSL-Eyfp;Dicer1wt/fl/Dicer1fl/fl mice, we crossed mice that contain floxed Dicer1 alleles (Dicer1fl) with Cebpa-Cre;R26-LSL-Eyfp reporter mice 2. Fetal livers were obtained on embryonic day (E) 13.5. Routine genotyping of Dicer1; Cebpa-Cre;R26-LSL-Eyfp mice was performed by PCR assays of DNA from tail or toe biopsies. For transplantation, 6 to 8-week-old recipient mice (C57Bl/6, Jackson Laboratories) were irradiated (8.5 Gy) and tail-vein injected with fetal liver single-cell suspensions. Typically, cells from each fetal liver were transplanted into two recipient mice. Hematopoietic tissues were analyzed 6-10 weeks post transplantation. EYFP positive GMPs from the bone marrow of Dicer wt control (n=3), Dicer -/wt (n=3 and Dicer fl/fl (n=3) were sorted and analyzed for gene expression profiles.

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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Uhrf1-/- fetal liver hematopoietic stem cell (FL-HSCs) Transcriptomes

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