Project description:We performed single cell RNA sequencing of human haemopoietic lympho-myeloid progenitors with the aim to separate functionally and transcriptionally distinct progenitors within the heterogeneous GMP, LMPP and MLP populations. We performed RNA sequencing in a total of 415 single cells. From donors 1 (CB369) and 2 (CB431), 163/166 and 157/249 cells passed quality control.We further analysed the 320 single cells that passed quality control from two different donors (157 and 163 from each donor; 91 LMPP, 110 MLP and 119 GMP). We performed clustering using Seurat package, which identified 3 clusters of cells. We identified genes that were differentially expressed among cells of the different clusters. The majority of the cells in cluster 1 were MLP, the majority of cluster 3 were GMP while cluster comprised of LMPP and GMP cells. Cluster 1 showed high expression of lymphoid-affiliated genes. Conversely, cluster 3 showed increased expression of myeloid genes, while cluster 2 showed a mixed transcriptional signature. PCA analysis revealed a transcriptional continuum of LMPP, MLP and GMP populations.
Project description:Genome wide expression profiling was used to detect cases of adult T-ALL lacking GATA3 expression. GATA3low T-ALL exhibited enrichment of myeloid/lymphoid progenitor (MLP) and granulocyte/monocyte progenitor (GMP) genes, while T-cell specific signatures were downregulated. Among upregulated genes FLT3 was identified and mutational analyses revealed a high rate of FLT3 mutations.
Project description:Regulation of lineage potential and transcriptional priming by Ikaros. New insight is provided into a bivalent regulation of lineage priming in the HSC and its lympho-myeloid restricted progeny the LMPP by the lymphoid lineage-determining factor Ikaros; Whereas Ikaros is responsible for the activation of a cascade of lymphoid expression programs and for the establishment of lymphoid potential from the HSC to the LMPP it is also responsible for the repression of stem cell and erythroid genetic programs that are incompatible with further lineage restrictions emanating from the LMPP Experiment Overall Design: Ikaros null versus wt
Project description:Genome wide expression profiling was used to detect cases of adult T-ALL lacking GATA3 expression. GATA3low T-ALL exhibited enrichment of myeloid/lymphoid progenitor (MLP) and granulocyte/monocyte progenitor (GMP) genes, while T-cell specific signatures were downregulated. Among upregulated genes FLT3 was identified and mutational analyses revealed a high rate of FLT3 mutations. Analysis of 83 samples of bone marrow mononuclear cells (39x HGU-133 2.0 plus chip; 44x HGU-133 A&B Set) from adult patients with acute T-cell lymphoblastic leukemia (T-ALL)
Project description:Cord blood (CB) samples from normal donors were obtained with informed consent. Fresh CB samples were processed within 18-34h after collection. Mononuclear cells were isolated and CD34+ fraction was separated. CB CD34+ enriched fraction was lineage depleted by staining with purified anti-human CD2, CD3, CD4, CD7, CD8a, CD11b, CD14, CD19, CD20, CD56, CD235a followed by Qdot 605 conjugated goat F(ab')2 anti-mouse IgG (H+L). Cells were also stained with anti-human CD38-FITC, CD45RA-PE or -BV650, CD123-PE Cy7, CD90-biotin, CD34- PerCP and CD10-APC. Finally, cells were incubated with streptavidin-conjugated APC-eF780 and Hoechst 33258 (Invitrogen, final concentration: 1 g/ml). Populations were defined, as follows: HSC - Lin-CD34+CD38-CD90+CD45RA-CD10-, MPP - Lin-CD34+CD38-CD90-CD45RA-CD10-, LMPP - Lin-CD34+CD38-CD90-/loCD45RA+CD10-, MLP - Lin-CD34+CD38-CD90-/loCD45RA+CD10+, GMP - Lin-CD34+CD38+CD123+CD45RA+CD10-, CMP - Lin-CD34+CD38+CD123+CD45RA-CD10-, MEP - Lin-CD34+CD38+CD123-CD45RA-CD10-.
Project description:Regulation of lineage potential and transcriptional priming by Ikaros. New insight is provided into a bivalent regulation of lineage priming in the HSC and its lympho-myeloid restricted progeny the LMPP by the lymphoid lineage-determining factor Ikaros Whereas Ikaros is responsible for the activation of a cascade of lymphoid expression programs and for the establishment of lymphoid potential from the HSC to the LMPP it is also responsible for the repression of stem cell and erythroid genetic programs that are incompatible with further lineage restrictions emanating from the LMPP Keywords: Ikaros null versus wt
Project description:The advent of single cell (Sc) genomics has challenged the dogma of haematopoiesis as a tree-like structure of stepwise lineage commitment through distinct and increasingly restricted progenitor populations. Instead, analysis of ScRNA-seq has proposed that the earliest events in human hematopoietic stem cell (HSC) differentiation are characterized by only subtle molecular changes, with hematopoietic stem and progenitor cells (HSPCs) existing as a continuum of low-primed cell-states that gradually transition into a specific lineage (CLOUD-HSPCs). Here, we combine ScRNA-seq, ScATAC-seq and cell surface proteomics to dissect the heterogeneity of CLOUD-HSPCs at different stages of human life. Within CLOUD-HSPCs, pseudotime ordering of both mRNA and chromatin data revealed a bifurcation of megakaryocyte/erythroid and lympho/myeloid trajectories immediately downstream a subpopulation with an HSC-specific enhancer signature. Importantly, both HSCs and lineage-restricted progenitor populations could be prospectively isolated based on correlation of their molecular signatures with CD35 and CD11A expression, respectively. Moreover, we describe the changes that occur in this heterogeneity as hematopoiesis develops from neonatal to aged bone marrow, including an increase of HSCs and depletion of lympho-myeloid biased MPPs. Thus, this study dissects the heterogeneity of human CLOUD-HSPCs revealing distinct HSPC-states of relevance in homeostatic settings such as ageing.
Project description:The advent of single cell (Sc) genomics has challenged the dogma of haematopoiesis as a tree-like structure of stepwise lineage commitment through distinct and increasingly restricted progenitor populations. Instead, analysis of ScRNA-seq has proposed that the earliest events in human hematopoietic stem cell (HSC) differentiation are characterized by only subtle molecular changes, with hematopoietic stem and progenitor cells (HSPCs) existing as a continuum of low-primed cell-states that gradually transition into a specific lineage (CLOUD-HSPCs). Here, we combine ScRNA-seq, ScATAC-seq and cell surface proteomics to dissect the heterogeneity of CLOUD-HSPCs at different stages of human life. Within CLOUD-HSPCs, pseudotime ordering of both mRNA and chromatin data revealed a bifurcation of megakaryocyte/erythroid and lympho/myeloid trajectories immediately downstream a subpopulation with an HSC-specific enhancer signature. Importantly, both HSCs and lineage-restricted progenitor populations could be prospectively isolated based on correlation of their molecular signatures with CD35 and CD11A expression, respectively. Moreover, we describe the changes that occur in this heterogeneity as hematopoiesis develops from neonatal to aged bone marrow, including an increase of HSCs and depletion of lympho-myeloid biased MPPs. Thus, this study dissects the heterogeneity of human CLOUD-HSPCs revealing distinct HSPC-states of relevance in homeostatic settings such as ageing.
Project description:A knockdown screen of chromatin regulatory genes in human THP1 MLL-AF9 AML cells identified EPC1 and EPC2 as required for cell survival. Normal myeloid progenitor cells did not require EPC1 or EPC2 for their function. To determine the transcriptional consequences of EPC knockdown, normal murine granulocyte-macrophage progenitor (GMP) cells, or murine MLL-AF9 AML GMP-like cells, were FACS purified from mice and infected with lentiviral vectors targeting EPC1 or EPC2 for knockdown, or a non-tageting control. 42 hours later knockdown or control cells were FACS purified and exon array analysis was performed on the sorted populations. For normal GMP cells, three biological replicates (three separate animals) each of control cells, EPC1 knockdown cells and EPC2 knockdown cells; for MLL-AF9 AML GMP-like cells cells, three biological replicates (three separate leukaemias) each of control cells, EPC1 knockdown cells and EPC2 knockdown cells.
Project description:A knockdown screen of chromatin regulatory genes in human THP1 MLL-AF9 AML cells identified EPC1 and EPC2 as required for cell survival. Normal myeloid progenitor cells did not require EPC1 or EPC2 for their function. To determine the transcriptional consequences of EPC knockdown, normal murine granulocyte-macrophage progenitor (GMP) cells, or murine MLL-AF9 AML GMP-like cells, were FACS purified from mice and infected with lentiviral vectors targeting EPC1 or EPC2 for knockdown, or a non-tageting control. 42 hours later knockdown or control cells were FACS purified and exon array analysis was performed on the sorted populations.