Comprehensive analysis of single-cell RNA sequencing data from healthy human marrow hematopoietic cells.
ABSTRACT: OBJECTIVE:Single cell methodology enables detection and quantification of transcriptional changes and unravelling dynamic aspects of the transcriptional heterogeneity not accessible using bulk sequencing approaches. We have applied single-cell RNA-sequencing (scRNA-seq) to fresh human bone marrow CD34+ cells and profiled 391 single hematopoietic stem/progenitor cells (HSPCs) from healthy donors to characterize lineage- and stage-specific transcription during hematopoiesis. RESULTS:Cells clustered into six distinct groups, which could be assigned to known HSPC subpopulations based on lineage specific genes. Reconstruction of differentiation trajectories in single cells revealed four committed lineages derived from HSCs, as well as dynamic expression changes underlying cell fate during early erythroid-megakaryocytic, lymphoid, and granulocyte-monocyte differentiation. A similar non-hierarchical pattern of hematopoiesis could be derived from analysis of published single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), consistent with a sequential relationship between chromatin dynamics and regulation of gene expression during lineage commitment (first, altered chromatin conformation, then mRNA transcription). Computationally, we have reconstructed molecular trajectories connecting HSCs directly to four hematopoietic lineages. Integration of long noncoding RNA (lncRNA) expression from the same cells demonstrated mRNA transcriptome, lncRNA, and the epigenome were highly homologous in their pattern of gene activation and suppression during hematopoietic cell differentiation.
Project description:Long noncoding RNAs (lncRNAs) are regulators of cell differentiation and development. The lncRNA transcriptome in human hematopoietic stem and progenitor cells is not comprehensively defined. We investigated lncRNAs in 979 human bone marrow-derived CD34+ cells by single cell RNA sequencing followed by de novo transcriptome reconstruction. We identified 3,173 lncRNAs in total, among which 2,365 were previously unknown, and we characterized lncRNA stem, differentiation, and maturation signatures. lncRNA expression exhibited high cell-to-cell variation, which was only apparent in single cell analysis. lncRNA expression followed a lineage-specific and highly dynamic pattern during early hematopoiesis. lncRNAs in hematopoietic cells closely correlated with protein-coding genes of known functions in the regulation of hematopoiesis and cell fate decisions, and the potential regulatory roles of lncRNAs in hematopoiesis were imputed by projection from protein-coding genes with a "guilt-by-association" approach. We characterized lncRNAs preferentially expressed in hematopoietic stem cells and in various downstream differentiated lineage progenitors. We also profiled lncRNA expression in single cells from patients with myelodysplastic syndromes and in aneuploid cells in particular. Our study provides a global view of lncRNAs in human hematopoietic stem and progenitor cells. We observed a highly ordered pattern of lncRNA expression and participation in regulation of early hematopoiesis, and coordinate aberrant messenger RNA and lncRNA transcriptomes in dysplastic hematopoiesis. (Registered at clinicaltrials.gov with identifiers: 00001620, 00001397).
Project description:Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.
Project description:The balance of self-renewal and differentiation in long-term repopulating hematopoietic stem cells (LT-HSC) must be strictly controlled to maintain blood homeostasis and to prevent leukemogenesis. Hematopoietic cytokines can induce differentiation in LT-HSCs; however, the molecular mechanism orchestrating this delicate balance requires further elucidation. We identified the tumor suppressor GADD45G as an instructor of LT-HSC differentiation under the control of differentiation-promoting cytokine receptor signaling. GADD45G immediately induces and accelerates differentiation in LT-HSCs and overrides the self-renewal program by specifically activating MAP3K4-mediated MAPK p38. Conversely, the absence of GADD45G enhances the self-renewal potential of LT-HSCs. Videomicroscopy-based tracking of single LT-HSCs revealed that, once GADD45G is expressed, the development of LT-HSCs into lineage-committed progeny occurred within 36 hr and uncovered a selective lineage choice with a severe reduction in megakaryocytic-erythroid cells. Here, we report an unrecognized role of GADD45G as a central molecular linker of extrinsic cytokine differentiation and lineage choice control in hematopoiesis.
Project description:Hematopoietic Stem Cells (HSCs) generate blood and immune cells through a hierarchical process of differentiation. Genes that regulate this process are of great interest for understanding normal and also malignant hematopoiesis. Surprisingly, however, very little is known about long-non-coding RNAs (lncRNA) in HSCs. Neat1 is a lncRNA that plays a major role in the formation of sub-nuclear structures called paraspeckles, and was reported to regulate proliferation and differentiation in other cells types. We detected Neat1 expression using RNA-seq data and RT-qPCR in HSCs, progenitors and effector immune cells, by specific detection of its isoforms. Neat1 is highly expressed in stem and progenitor cells, yet it shows significant reduction in granulocytes. Microscopically, Neat1 is detected as sharp nuclear foci. Paraspeckle proteins NONO and PSPC1 are detected as aggregated nuclear foci in fresh primary hematopoietic cells, and in cultured cells. Induction of differentiation in vitro was found to enhance Neat1 expression. Taken together, our data demonstrate for the first time the expression of Neat1 and paraspeckles formation in HSCs and along hematopoiesis.
Project description:Adult hematopoiesis has been studied in terms of progenitor differentiation potentials, whereas its kinetics in vivo is poorly understood. We combined inducible lineage tracing of endogenous adult hematopoietic stem cells (HSCs) with flow cytometry and single-cell RNA sequencing to characterize early steps of hematopoietic differentiation in the steady-state. Labeled cells, comprising primarily long-term HSCs and some short-term HSCs, produced megakaryocytic lineage progeny within 1 wk in a process that required only two to three cell divisions. Erythroid and myeloid progeny emerged simultaneously by 2 wk and included a progenitor population with expression features of both lineages. Myeloid progenitors at this stage showed diversification into granulocytic, monocytic, and dendritic cell types, and rare intermediate cell states could be detected. In contrast, lymphoid differentiation was virtually absent within the first 3 wk of tracing. These results show that continuous differentiation of HSCs rapidly produces major hematopoietic lineages and cell types and reveal fundamental kinetic differences between megakaryocytic, erythroid, myeloid, and lymphoid differentiation.
Project description:A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age.
Project description:Hematopoiesis is a tightly regulated process resulting in the production of blood cells. Self-renewal and differentiation of hematopoietic stem cells (HSCs) are key processes in hematopoietic development. Disruption of these steps can lead to altered cell distribution and disease. To investigate the role of the nuclear factor-?B subunit RelA/p65 in the regulation of HSCs in vivo, we generated mice lacking RelA/p65 in the hematopoietic compartment. Using this model system, we show that loss of p65 severely impairs HSC function and occurs in conjunction with increased hematopoietic stem and progenitor cell cycling, extramedullary hematopoiesis, and differentiation defects. Gene array studies of phenotypic HSCs indicate the up-regulation of genes normally expressed in lineage restricted cells, as well as the down-regulation of genes involved in HSC maintenance and homeostasis. We hypothesize that changes in gene expression in p65-deficient cells lead to decreased self-renewal and differentiation efficiency of hematopoietic stem and progenitor cells. These studies demonstrate that p65 is an important regulator of hematopoiesis through the transcription of genes involved in HSC fate.
Project description:Hematopoietic stem cells (HSCs) have a unique capacity to undergo self-renewal and multi-lineage differentiation to provide a lifetime supply of mature blood cells. By using conditional knockout technology, we disrupted the c-myb proto-oncogene specifically in adult bone marrow (BM) to demonstrate that this transcription factor is a regulator of self-renewal and multi-lineage differentiation of adult HSCs. Targeted disruption of the c-myb gene resulted in a critical depletion of the HSC pool. In addition, BM hematopoiesis in adult mice was impaired, resulting in profound reductions of various hematopoietic lineages including neutrophilic, monocytic, B lymphoid, erythroid, and, unexpectedly, megakaryocytic cells. Serial BM transplantation into lethally irradiated recipient mice indicated an essential role for c-myb in the self-renewal process. Furthermore, in vitro functional assays demonstrated that deletion of the c-myb gene leads to a slightly reduced proliferative capacity and an aberrant and accelerated differentiation of HSCs. In addition to long-term HSCs, functional studies also show that c-myb plays a critical role in short-term HSCs and multi-potential progenitors. Collectively, our data indicate a critical role for c-myb in adult BM hematopoiesis and in self-renewal and multi-lineage differentiation of adult HSCs.
Project description:Self-renewal, replication, and differentiation of hematopoietic stem cells (HSCs) are regulated by cytokines produced by niche cells in fetal liver and bone marrow. HSCs must overcome stresses induced by cytokine deprivation during normal development. In this study, we found that ubiquitin-specific peptidase 10 (USP10) is a crucial deubiquitinase for mouse hematopoiesis. All USP10 knockout (KO) mice died within 1 year because of bone marrow failure with pancytopenia. Bone marrow failure in these USP10-KO mice was associated with remarkable reductions of long-term HSCs (LT-HSCs) in bone marrow and fetal liver. Such USP10-KO fetal liver exhibited enhanced apoptosis of hematopoietic stem/progenitor cells (HSPCs) including LT-HSCs but not of lineage-committed progenitor cells. Transplantation of USP10-competent bone marrow cells into USP10-KO mice reconstituted multilineage hematopoiesis. These results suggest that USP10 is an essential deubiquitinase in hematopoiesis and functions by inhibiting apoptosis of HSPCs including LT-HSCs.
Project description:Hematopoiesis during development is a dynamic process, with many factors involved in the emergence and regulation of hematopoietic stem cells (HSCs) and progenitor cells. Whereas previous studies have focused on developmental signaling and transcription factors in embryonic hematopoiesis, the role of well-known adult hematopoietic cytokines in the embryonic hematopoietic system has been largely unexplored. The cytokine interleukin-1 (IL-1), best known for its proinflammatory properties, has radioprotective effects on adult bone marrow HSCs, induces HSC mobilization, and increases HSC proliferation and/or differentiation. Here we examine IL-1 and its possible role in regulating hematopoiesis in the midgestation mouse embryo. We show that IL-1, IL-1 receptors (IL-1Rs), and signaling mediators are expressed in the aorta-gonad-mesonephros (AGM) region during the time when HSCs emerge in this site. IL-1 signaling is functional in the AGM, and the IL-1RI is expressed ventrally in the aortic subregion by some hematopoietic, endothelial, and mesenchymal cells. In vivo analyses of IL-1RI-deficient embryos show an increased myeloid differentiation, concomitant with a slight decrease in AGM HSC activity. Our results suggest that IL-1 is an important homeostatic regulator at the earliest time of HSC development, acting to limit the differentiation of some HSCs along the myeloid lineage.