Project description:The pleiotropic transcriptional regulator CITED2 is essential for lifelong maintenance of haematopoiesis. To understand the molecular roles of CITED2 in normal haematopoiesis, we performed RNA-sequencing on CD48−CD150+ haematopoietic stem cells (HSCs) derived from young mice.
Project description:Mass spectrometry was performed with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) interfaced with an UltiMate 3000 Binary RSLCnano System (Dionex). Proteome Discoverer v.1.4 (Thermo Scientific) with SEQUEST HT search engines was used for the spectra-preprocessing and HCD MS2 spectra were used for peptide identification and quantitation based on TMT reporter ions. TMT isobaric comparison of Ppia heterozygous (het) versus knockout (KO) haematopoietic stem and progenitor cells. Het 1 is sample 126 and KO 1 is sample 131 of dataset UTH_2. Het 2 and KO 2 are samples 126 and 127 of UTH_4. Het 3 and KO 3 are samples 130 and 127 of dataset UTH_3.
Project description:A knockout clone has been generated for both FAM50A and FAM50B; knockout of the other gene is then performed and the transcriptome is analysed to look at the effect of dual gene loss.
Project description:The 2-oxoglutarate dependent protein JMJD6 acts as a lysyl hydroxylase on specific residues of the splicing factor U2AF2. To understand the molecular roles of JMJD6 in normal haematopoiesis and identify any role of JMJD6 in splicing, we performed RNA-sequencing on Lin-Sca1+Kit+ (LSK) haematopoietic stem and progenitor cells (HSPCs) derived from young mice.
Project description:The underlying mechanisms which are responsible and govern early haematopoietic differentiation during development are poorly understood. Gene expression comparison between pluripotent human embryonic stem cells and earliest haematopoietic progenitors may reveal novel transcripts and pathways and provide crucial insight into early haematopoietic lineage specification and development. Understanding of transcriptional cues that direct differentiation of human embryonic stem cells (hESC) to defined and functional cell types is essential for their future clinical applications. In this study we have undertaken a comparative transcriptional approach of haematopoietic progenitors derived from hESC at various stages of a feeder and serum free differentiation method and have shown that the largest transcriptional changes occur during the first four days of differentiation. Data mining based on molecular function pointed to RhoGTPase signalling as key regulator of this differentiation. Inhibition of this pathway using a chemical inhibitor (Y26732) resulted in a significant downregulation of haematopoietic progenitors throughout the differentiation window, thus uncovering a previously unappreciated role for RhoGTPase signalling in differentiation of hESC to haematopoietic lineages. There are a total of 4 samples within this microarray experiment with 2 biological replicates for each sample. Pluripotent human embryonic stem cells (day 0) underwent haematopoietic differentiation and at various stages of development (day 4, day 6, day8) differentiated cells were FACS sorted for two key haemangioblast markers, CD31 and KDR.
Project description:Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1+ FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGF? and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.
Project description:The underlying mechanisms which are responsible and govern early haematopoietic differentiation during development are poorly understood. Gene expression comparison between pluripotent human embryonic stem cells and earliest haematopoietic progenitors may reveal novel transcripts and pathways and provide crucial insight into early haematopoietic lineage specification and development. Understanding of transcriptional cues that direct differentiation of human embryonic stem cells (hESC) to defined and functional cell types is essential for their future clinical applications. In this study we have undertaken a comparative transcriptional approach of haematopoietic progenitors derived from hESC at various stages of a feeder and serum free differentiation method and have shown that the largest transcriptional changes occur during the first four days of differentiation. Data mining based on molecular function pointed to RhoGTPase signalling as key regulator of this differentiation. Inhibition of this pathway using a chemical inhibitor (Y26732) resulted in a significant downregulation of haematopoietic progenitors throughout the differentiation window, thus uncovering a previously unappreciated role for RhoGTPase signalling in differentiation of hESC to haematopoietic lineages.