Hematopoietic stem cells (HSCs) are characterized by their ability to execute a wide range of cell fate choices, including self-renewal, quiescence, and differentiation into the many different mature ...blood lineages. Cell fate decision making in HSCs, as indeed in other cell types, is driven by the interplay of external stimuli and intracellular regulatory programs. Given the pivotal nature of HSC decision making for both normal and aberrant hematopoiesis, substantial research efforts have been invested over the last few decades into deciphering some of the underlying mechanisms. Central to the intracellular decision making processes are transcription factor proteins and their interactions within gene regulatory networks. More than 50 transcription factors have been shown to affect the functionality of HSCs. However, much remains to be learned about the way in which individual factors are connected within wider regulatory networks, and how the topology of HSC regulatory networks might affect HSC function. Nevertheless, important progress has been made in recent years, and new emerging technologies suggest that the pace of progress is likely to accelerate. This review will introduce key concepts, provide an integrated view of selected recent studies, and conclude with an outlook on possible future directions for this field.
Mouse heart development arises from
-expressing cardiovascular progenitors (CPs) that are specified during gastrulation. The molecular processes that control early regional and lineage segregation of ...CPs have been unclear. We performed single-cell RNA sequencing of wild-type and
-null CPs in mice. We showed that populations of
CPs are molecularly distinct and span the continuum between epiblast and later mesodermal cells, including hematopoietic progenitors. Single-cell transcriptome analysis of
-deficient CPs showed that Mesp1 is required for the exit from the pluripotent state and the induction of the cardiovascular gene expression program. We identified distinct populations of
CPs that correspond to progenitors committed to different cell lineages and regions of the heart, identifying the molecular features associated with early lineage restriction and regional segregation of the heart at the early stage of mouse gastrulation.
Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new ...single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice.
•An expression map of HSPC differentiation from single-cell RNA sequencing of HSPCs provides insights into blood stem cell differentiation.•A user-friendly Web resource provides access to single-cell gene expression profiles for the wider research community.
How transplanted haematopoietic stem cells (HSCs) behave soon after they reside in a preconditioned host has not been studied due to technical limitations. Here, using single-cell RNA sequencing, we ...first obtained the transcriptome-based classifications of 28 haematopoietic cell types. We then applied them in conjunction with functional assays to track the dynamic changes of immunophenotypically purified HSCs in irradiated recipients within the first week after transplantation. Based on our transcriptional classifications, most homed HSCs in bone marrow and spleen became multipotent progenitors and, occasionally, some HSCs gave rise to megakaryocytic-erythroid or myeloid precursors. Parallel in vitro and in vivo functional experiments supported the paradigm of robust differentiation without substantial HSC expansion during the first week. Therefore, this study uncovers the previously inaccessible kinetics and fate choices of transplanted HSCs in myeloablated recipients at early stage, with implications for clinical applications of HSCs and other stem cells.
Limited knowledge of cellular and molecular mechanisms underlying hematopoietic stem cell and multipotent progenitor (HSC/MPP) expansion within their native niche has impeded the application of stem ...cell-based therapies for hematological malignancies. Here, we constructed a spatiotemporal transcriptome map of mouse fetal liver (FL) as a platform for hypothesis generation and subsequent experimental validation of novel regulatory mechanisms. Single-cell transcriptomics revealed three transcriptionally heterogeneous HSC/MPP subsets, among which a CD93-enriched subset exhibited enhanced stem cell properties. Moreover, by employing integrative analysis of single-cell and spatial transcriptomics, we identified novel HSC/MPP 'pocket-like' units (HSC PLUS), composed of niche cells (hepatoblasts, stromal cells, endothelial cells, and macrophages) and enriched with growth factors. Unexpectedly, macrophages showed an 11-fold enrichment in the HSC PLUS. Functionally, macrophage-HSC/MPP co-culture assay and candidate molecule testing, respectively, validated the supportive role of macrophages and growth factors (MDK, PTN, and IGFBP5) in HSC/MPP expansion. Finally, cross-species analysis and functional validation showed conserved cell-cell interactions and expansion mechanisms but divergent transcriptome signatures between mouse and human FL HSCs/MPPs. Taken together, these results provide an essential resource for understanding HSC/MPP development in FL, and novel insight into functional HSC/MPP expansion ex vivo.
•Single-cell gene expression profiling is widely used to profile hematopoietic cells.•The best strategies for identifying stem cells currently rely on flow cytometry.•Location of HSCs in single-cell ...data with no flow cytometry information is challenging.•The hscScore method provides fast identification of HSCs from single-cell RNA-seq data.
Hematopoietic stem cells (HSCs) are an essential source and reservoir for normal hematopoiesis, and their function is compromised in many blood disorders. HSC research has benefitted from the recent development of single-cell molecular profiling technologies, where single-cell RNA sequencing (scRNA-seq) in particular has rapidly become an established method to profile HSCs and related hematopoietic populations. The classic definition of HSCs relies on transplantation assays, which have been used to validate HSC function for cell populations defined by flow cytometry. Flow cytometry information for single cells, however, is not available for many new high-throughput scRNA-seq methods, thus highlighting an urgent need for the establishment of alternative ways to pinpoint the likely HSCs within large scRNA-seq data sets. To address this, we tested a range of machine learning approaches and developed a tool, hscScore, to score single-cell transcriptomes from murine bone marrow based on their similarity to gene expression profiles of validated HSCs. We evaluated hscScore across scRNA-seq data from different laboratories, which allowed us to establish a robust method that functions across different technologies. To facilitate broad adoption of hscScore by the wider hematopoiesis community, we have made the trained model and example code freely available online. In summary, our method hscScore provides fast identification of mouse bone marrow HSCs from scRNA-seq measurements and represents a broadly useful tool for analysis of single-cell gene expression data.
The sympathetic nervous system has been evolutionary selected to respond to stress and activates haematopoietic stem cells via noradrenergic signals. However, the pathways preserving haematopoietic ...stem cell quiescence and maintenance under proliferative stress remain largely unknown. Here we found that cholinergic signals preserve haematopoietic stem cell quiescence in bone-associated (endosteal) bone marrow niches. Bone marrow cholinergic neural signals increase during stress haematopoiesis and are amplified through cholinergic osteoprogenitors. Lack of cholinergic innervation impairs balanced responses to chemotherapy or irradiation and reduces haematopoietic stem cell quiescence and self-renewal. Cholinergic signals activate α7 nicotinic receptor in bone marrow mesenchymal stromal cells leading to increased CXCL12 expression and haematopoietic stem cell quiescence. Consequently, nicotine exposure increases endosteal haematopoietic stem cell quiescence in vivo and impairs hematopoietic regeneration after haematopoietic stem cell transplantation in mice. In humans, smoking history is associated with delayed normalisation of platelet counts after allogeneic haematopoietic stem cell transplantation. These results suggest that cholinergic signals preserve stem cell quiescence under proliferative stress.
Inhibition of glycogen synthase kinase-3 (Gsk3) supports mouse embryonic stem cells (ESCs) by modulating Tcf3, but the critical targets downstream of Tcf3 are unclear. We analyzed the intersection ...between genome localization and transcriptome data sets to identify genes repressed by Tcf3. Among these, manipulations of Esrrb gave distinctive phenotypes in functional assays. Knockdown and knockout eliminated response to Gsk3 inhibition, causing extinction of pluripotency markers and loss of colony forming capability. Conversely, forced expression phenocopied Gsk3 inhibition or Tcf3 deletion by suppressing differentiation and sustaining self-renewal. Thus the nuclear receptor Esrrb is necessary and sufficient to mediate self-renewal downstream of Gsk3 inhibition. Leukaemia inhibitory factor (LIF) regulates ESCs through Stat3, independently of Gsk3 inhibition. Consistent with parallel operation, ESCs in LIF accommodated Esrrb deletion and remained pluripotent. These findings highlight a key role for Esrrb in regulating the naive pluripotent state and illustrate compensation among the core pluripotency factors.
► Esrrb is the principal target of Tcf3 repression in the pluripotency network ► Esrrb is essential for self-renewal downstream of Gsk3 inhibition ► Esrrb potently suppresses differentiation and sustains ESC self-renewal ► Esrrb is a core pluripotency factor but can be compensated by LIF/Stat3
Inhibition of glycogen synthase kinase 3 keeps embryonic stem cells in a naïve ground state. Surprisingly this effect is mediated mainly by upregulating expression of a single transcription factor Esrrb. Esrrb function can be compensated by independent activation of Stat3, however, demonstrating the plasticity of the core network underpinning pluripotency.
Haematopoietic stem cells (HSC) are situated at the apex of the haematopoietic differentiation hierarchy, ensuring the life-long supply of mature haematopoietic cells and forming a reservoir to ...replenish the haematopoietic system in case of emergency such as acute blood loss. To maintain a balanced production of all mature lineages and at the same time secure a stem cell reservoir, intricate regulatory programs have evolved to control multi-lineage differentiation and self-renewal in haematopoietic stem and progenitor cells (HSPCs). Leukaemogenic mutations commonly disrupt these regulatory programs causing a block in differentiation with simultaneous enhancement of proliferation. Here, we briefly summarize key aspects of HSPC regulatory programs, and then focus on their disruption by leukaemogenic fusion genes containing the mixed lineage leukaemia (MLL) gene. Using MLL as an example, we explore important questions of wider significance that are still under debate, including the importance of cell of origin, to what extent leukaemia oncogenes impose specific regulatory programs and the relevance of leukaemia stem cells for disease development and prognosis. Finally, we suggest that disruption of stem cell regulatory programs is likely to play an important role in many other pathologies including ageing-associated regenerative failure.
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