Project description:Mutations in genes intimately involved in the occurrence of haemotological cancers remains largely unknown. Here, we report a novel functional gene in hematopoiesis, which was discovered by screening mutant embryonic stem cells (ESCs). The gene, named “attenuated haematopoietic development” (Ahed), encoded an uncharacterised protein located in the nucleus. Ahed conditional knockout (cKO) mice were generated by mating with Vav1-cre transgenic mice. We found that Ahed cKO foetuses became anaemic after E14.5 and died before birth. Flow cytometric analysis showed that erythroid cells significantly decreased in E14.5 Ahed cKO livers. By contrast, the fetal liver contained a substantial number of lineage-Sca-1+c-KitHi (LSK) cells, whose phenotype corresponded to hematopoietic stem/progenitor cells. Transplantation experiments revealed that Ahed-deficient LSK cells were unable to reconstitute haematopoiesis in vivo. We confirmed the downregulation of expression levels of HSC-related genes, such as Gata2, Lmo2, and Runx1 in E14.5 FL LSK cells in Ahed cKO mice. In addition, upstream regulator analyses revealed that the GATA2 pathway was most significantly hampered in both FL and adult BM. Collectively, these data determined that Ahed is indispensable for functional haematopoietic stem/progenitor cells.
Project description:Haematopoietic stem cells can differentiate into all blood cell types. In this process, cells become progressively restricted to a single cell type. The order in which differentiating cells loose lineage potential, and the prospective isolation of cells with a defined potential remains a long-standing question. We performed gene expression analysis of haematopoietic cells from Gata1-EGFP reporter mice, leading to a model for hematopoiesis where the initial lineage decision consists of a seperation of erythroid/megakaryocyte/mast cell/eosinophil potential from lymphopoietic/monocyte/neutrophil potential Find unbiased heterogeneity in the preGM hematopoietic progenitor population
Project description:Hematopoietic stem cell (HSC) differentiation into mature lineages has been studied under physiological conditions in vivo by genetic barcoding-driven lineage tracing. HSC clones differ in output (differentiation-inactive versus differentiation-active), and in fates (multilineage versus lineage-restricted). Single-cell sequencing data revealed transcriptome diversity of HSC and progenitors, and suggested differentiation pathways. However, molecular hallmarks of functionally distinct HSC clones have not been resolved because existing lineage tracing experiments did not provide transcriptomes, and single cell RNA sequencing lacked information on precursor-product relationships, and hence fate. To close this gap, here we introduce PolyloxExpress, a Cre recombinase-dependent DNA substrate for in situ barcoding in mice that is expressed as mRNA. PolyloxExpress barcoding allows parallel readout of clonal HSC fates (via comparison of barcodes in HSC and mature lineages), and transcriptomes (via single-cell RNA sequencing and barcode matching). Analysing a total of 91 individual HSC clones, we show that differentiation-inactive versus differentiation-active HSC clones reside in different regions of the transcriptional landscape. Inactive HSC clones are closer to the origin of the transcriptional trajectory, yet are proliferatively not more quiescent than active clones. Multilineage versus myelo-erythroid fate-restricted HSC clones show very few transcriptional differences at the HSC stage, yet pronounced fate-specific profiles at the multipotent progenitor stage. Projecting HSC clones with defined fates onto transcriptional landscapes provides a basis for future studies into the molecular determinants for stem cell fate.
Project description:Hematopoietic stem cell (HSC) differentiation into mature lineages has been studied under physiological conditions in vivo by genetic barcoding-driven lineage tracing. HSC clones differ in output (differentiation-inactive versus differentiation-active), and in fates (multilineage versus lineage-restricted). Single-cell sequencing data revealed transcriptome diversity of HSC and progenitors, and suggested differentiation pathways. However, molecular hallmarks of functionally distinct HSC clones have not been resolved because existing lineage tracing experiments did not provide transcriptomes, and single cell RNA sequencing lacked information on precursor-product relationships, and hence fate. To close this gap, here we introduce PolyloxExpress, a Cre recombinase-dependent DNA substrate for in situ barcoding in mice that is expressed as mRNA. PolyloxExpress barcoding allows parallel readout of clonal HSC fates (via comparison of barcodes in HSC and mature lineages), and transcriptomes (via single-cell RNA sequencing and barcode matching). Analysing a total of 91 individual HSC clones, we show that differentiation-inactive versus differentiation-active HSC clones reside in different regions of the transcriptional landscape. Inactive HSC clones are closer to the origin of the transcriptional trajectory, yet are proliferatively not more quiescent than active clones. Multilineage versus myelo-erythroid fate-restricted HSC clones show very few transcriptional differences at the HSC stage, yet pronounced fate-specific profiles at the multipotent progenitor stage. Projecting HSC clones with defined fates onto transcriptional landscapes provides a basis for future studies into the molecular determinants for stem cell fate.
Project description:Aged hematopoietic stem cells (HSC) display diminished self-renewal and a myeloid differentiation bias. However, the physiological drivers and molecular processes that underpin this fundamental switch are not understood. HSCs produce formaldehyde and are protected from this metabolite by two tiers of protection: the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. Using single cell RNA sequencing, we find that the HSC and progenitor cells in young Aldh2-/- Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition and myeloid-biased progenitors. In addition, the p53 response is vigorously activated in Aldh2-/- Fancd2-/- HSCs, whilst p53 deletion rescued this aged transcriptomic signature and telomere attrition. Transplantation of single Aldh2-/- Fancd2-/- HSCs also reveals a predominantly myeloid output, which is reversed upon p53 deletion. To further define the origins of the myeloid differentiation bias, a GFP genetic reporter which detects Vwf+ myeloid primed HSCs was crossed into Aldh2-/- Fancd2-/- mice, revealing a striking enrichment of these lineage-biased Vwf+ HSCs. These results indicate that metabolism derived formaldehyde causes endogenous DNA damage which stimulates the p53 response in HSCs, which then accelerates their aging, resulting in a myeloid lineage biased output.
Project description:Current gene-expression databases for the haematopoietic system provide information on gene expression profiles present in bulk populations. Although informative, these studies lack the resolution that can be gained at a single-cell level. In particular, population-average data assumes homogeneity within the population and may as such obscure the ability to detect the heterogeneity of decision-making processes in individual cells. Here we report 1656 single cell transcriptomes analysed by single-cell RNA sequencing. Cells were FACS sorted on broad gates encompassing haematopoietic stem and progenitor populations (HSPCs), with index sorting data collected to permit retrospective identification of populations by surface marker expression. Our dataset thus represents the gene expression landscape of HSPCs at single-cell resolution, capturing the heterogeneity in and between cell populations. Pseudotime analysis visualized haematopoietic stem (HSC) to progenitor transitions, identified HSC as well as lineage-specific transcriptional programs, and also highlighted putative lineage branching points. To provide access to the wider scientific community, a user-friendly website was developed with intuitive search and display functionality. Single cell RNA sequencing of haematopoeitic stem and progenitor cells
Project description:We compare the transcriptome of two different clones of multipotent adult progenitor cells (MAPCs) using Affymetrix arrays. Experiment Overall Design: Three mRNA samples (biological replicates) per cell type taken at different passage number were compared. Two independently isolated MAPC clones were used.
Project description:It remains controversial whether human induced pluripotent stem cells (hiPSCs) are distinct from human embryonic stem cells (hESCs) in their molecular signatures and differentiation properties. We examined the gene expression and DNA methylation of 49 hiPSC and 10 hESC lines and identified no molecular signatures that distinguished hiPSCs from hESCs. Comparisons of the in vitro directed neural differentiation of 40 hiPSC and four hESC lines showed that most hiPSC clones were comparable to hESCs. However, in seven hiPSC clones, significant amount of undifferentiated cells persisted even after neural differentiation and resulted in teratoma formation when transplantated into mouse brains. These differentiation-defective hiPSC clones were marked by higher expression of several genes, including those expressed from long terminal repeats of human endogenous retroviruses. These data demonstrated that many hiPSC clones are indistinguishable from hESCs, while some defective hiPSC clones need to be eliminated prior to their application for regenerative medicine.