Project description:To gain insights into the global and local transcriptomic changes underlying defects in adult RUNX1in HSPCs, we then performed scRNA SEQ on sorted CD41a- and CD41a+ HSPCs from cultures expressing NT or RUNX1-targeting lentiviral shRNA.
Project description:Transciptome analysis of CD34+ enriched human HSPC lentivirally transduced with cohesin WT or mutant CD34+ enriched HSPCs from cord blood were transduced with a constitutive lentiviral vector expressing cohesin WT or mutant tagged to GFP. After 72hrs cells were GFP+ sorted and subjected to downstream microarray protocol.
Project description:The goal of this study was to compare the transcriptional changes evoked by Csnk2b-deficiency in Foxp3+ regulatory T cells Messenger RNA profiles of CD4+Foxp3+ regulatory T cells isolated from 8 week old wild type (WT) and Csnk2b-deficient mice were generated by deep sequencing in duplicate, using Illumina MiSeq
Project description:One of the long-standing goals in the field has been to establish a culture system that would allow maintenance of HSC properties ex vivo. In the absence of such system, the ability to model human hematopoiesis in vitro has been limited, and there has been little progress in the expansion of human HSCs for clinical application. To that end, we defined a mesenchyml stem cell co-culture system for expansion of clonally multipotent human HSPCs that are protected from apoptosis and immediate differentiation, and retain the HSPC phenotype. By performing a genome-wide gene expression analysis of purified HSPCs isolated at different stages of co-culture, we asked at the molecular level, to what degree hematopetic stem cell properties can be preserved during culture. This temporal gene expression data from in vivo derived- and ex vivo expanded human HSPCs will serve as a resource to identify novel regulatory pathways that control HSC properties, and to develop clinically applicable protocols for HSC expansion. Human CD34+ fetal liver cells were co-cultured on a subclone of OP9 stomal cells (OP9M2 sublemented with supportive cytokines (see below)). To distinguish between molecular changes acquired over prolonged culture versus immediately after exposure to culture, gene expression in isolated CD45+CD34+CD38-CD90+ HSPCs was assessed after 12 hours, 2 weeks and 5 weeks in culture. Cultured CD45+CD34+CD38-CD90+HSPCs were compared to freshly isolated CD45+CD34+CD38-CD90+HSPCs and their more differentiated CD45+CD34+CD38+CD90- downstream progenitor cells.
Project description:Our understanding of cell fate decisions in hematopoietic stem cells is incomplete. Here, we show that the transcription factor Helios is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), where it is required to suppress the separation of the platelet/megakaryocyte lineage from the HSPC pool. Helios acts mainly in quiescent cells, where it directly represses the megakaryocyte gene expression program in cells as early as the stem cell stage. Helios binding promotes chromatin compaction, notably at the regulatory regions of platelet-specific genes recognized by the Gata2 and Runx1 transcriptional activators, implicated in megakaryocyte priming. Helios null HSPCs are biased toward the megakaryocyte lineage at the expense of the lymphoid and partially resemble cells of aging animals. We propose that Helios acts as a guardian of HSPC pluripotency by continuously repressing the megakaryocyte fate, which in turn allows downstream lymphoid priming to take place. These results highlight the importance of negative and positive priming events in lineage commitment.
Project description:Multiome single nuclei RNA/ATAC-seq analysis was performed on sorted population of SLAM LSK HSPCs to uncover transcriptional determinants regulating HSPCs in distinct transitional states.