PhD student, University of Cambridge, Cambridge Stem Cell Institute
Study of human hematopoietic stem cells heterogeneity and quiescence exit regulation
The production of blood is constantly maintained by the concerted action of hematopoietic stem cells (HSCs) and progenitor cells. HSCs can undergo self-renewal, by which stem cells divide to make more stem cells, or they can differentiate into any of the mature blood cell types. HSCs divide infrequently and they reside in a specific state outside the cell cycle called “quiescence”. However, upon sensing specific signals, HSCs exit quiescence and enter the cell cycle in a process called “activation”. While the molecular networks of quiescent HSC are starting to be understood, very little is known about how these networks change when the cell leaves the quiescent status and enters the cell cycle. This information is nonetheless important because it is thought that cell fate decisions are made during this activation phase. It has also become clear that the HSC compartment is heterogeneous, comprising distinct subsets of HSCs with different self-renewal, cycling and differentiation properties. Understanding how this heterogeneous pool of HSC coordinates cell division, self-renewal and differentiation will help to develop protocols for HSCs expansion, improve clinical HSCs transplants and understand how leukemias, cancers of the blood, arise. My project aims to define the cellular heterogeneity and the molecular events associated with exit from quiescence and cell cycle progression in human HSCs, to understand how the cell cycle state contributes to cell fate decisions. Because of HSCs heterogeneity, this study makes use of high-throughput single cell techniques to discriminate differences between single cells. First, I used a system that allows distinction of events occurring during quiescence exit from those linked to cell cycle progression. Second, I optimised single cell RNA-sequencing for genome wide transcriptional analysis of quiescent and activated HSCs. Third, I analysed more than 4000 single HSCs at the functional level and I correlated their functions with cell surface markers expression, using index sort flow cytometry. Altogether I identified two new functionally distinct sub-populations within the HSC pool, as well as genes that are driving the exit from quiescence. Future work will validate the effect of these genes on HSC function, as a better understanding of their regulation may be useful clinically.
Abstract: Understanding how differentiation programs originate from the gene-expression 'landscape' of hematopoietic stem cells (HSCs) is crucial for the development of new clinical therapies. We mapped the transcriptional dynamics underlying the first steps of commitment by tracking transcriptome changes in human HSCs and eight early progenitor populations. We found that transcriptional programs were extensively shared, extended across lineage-potential boundaries and were not strictly lineage affiliated. Elements of stem, lymphoid and myeloid programs were retained in multilymphoid progenitors (MLPs), which reflected a hybrid transcriptional state. By functional single cell analysis, we found that the transcription factors Bcl-11A, Sox4 and TEAD1 (TEF1) governed transcriptional networks in MLPs, which led to B cell specification. Overall, we found that integrated transcriptome approaches can be used to identify previously unknown regulators of multipotency and show additional complexity in lymphoid commitment.
Pub.: 28 May '13, Pinned: 04 Jul '17
Abstract: The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.
Pub.: 30 Apr '14, Pinned: 04 Jul '17
Abstract: Regulated blood production is achieved through the hierarchical organization of dormant hematopoietic stem cell (HSC) subsets that differ in self-renewal potential and division frequency, with long-term (LT)-HSCs dividing the least. The molecular mechanisms underlying this variability in HSC division kinetics are unknown. We report here that quiescence exit kinetics are differentially regulated within human HSC subsets through the expression level of CDK6. LT-HSCs lack CDK6 protein. Short-term (ST)-HSCs are also quiescent but contain high CDK6 protein levels that permit rapid cell cycle entry upon mitogenic stimulation. Enforced CDK6 expression in LT-HSCs shortens quiescence exit and confers competitive advantage without impacting function. Computational modeling suggests that this independent control of quiescence exit kinetics inherently limits LT-HSC divisions and preserves the HSC pool to ensure lifelong hematopoiesis. Thus, differential expression of CDK6 underlies heterogeneity in stem cell quiescence states that functionally regulates this highly regenerative system.
Pub.: 24 Feb '15, Pinned: 04 Jul '17
Abstract: Maintenance of the blood system requires balanced cell fate decisions of haematopoietic stem and progenitor cells (HSPCs). Since cell fate choices are executed at the level of individual cells, new single cell profiling technologies offer exciting possibilities to map the dynamic molecular changes underlying HSPC differentiation. Here we have used single cell RNA-Seq to profile over 1,600 single HSPCs, where deep sequencing has enabled detection of an average of 6,558 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 datasets 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-megakaryocytic and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. Using external spike-in controls, we estimate absolute 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.
Pub.: 02 Jul '16, Pinned: 04 Jul '17
Abstract: In a classical view of hematopoiesis, the various blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. We developed a cell-sorting scheme to resolve myeloid (My), erythroid (Er), and megakaryocytic (Mk) fates from single CD34+ cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with intermingled My, Er, and Mk fates. However, few oligopotent progenitor intermediates were present in the adult bone marrow. Instead only two progenitor classes predominate, multipotent and unipotent, with Er-Mk lineages emerging from multipotent cells. The developmental shift to an adult “two-tier” hierarchy challenges current dogma and provides a revised framework to understand normal and disease states of human hematopoiesis.
Pub.: 05 Nov '15, Pinned: 04 Jul '17