Elisa Laurenti

Hematopoietic Stem Cells Reversibly Switch from Dormancy to Self-Renewal during Homeostasis and Repair

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

The genetic basis of early T-cell precursor acute lymphoblastic leukaemia.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment

Proteins are identified that underlie the early commitment steps of human hematopoietic stem cell differentiation. Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates. Investigating the molecular state of HSCs is contingent on the ability to purify HSCs away from transiently engrafting cells. We demonstrated that human HSCs remain infrequent, using current purification strategies based on Thy1 (CD90) expression. By tracking the expression of several adhesion molecules in HSC-enriched subsets, we revealed CD49f as a specific HSC marker. Single CD49f+ cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting multipotent progenitors (MPPs). The demarcation of human HSCs and MPPs will enable the investigation of the molecular determinants of HSCs, with a goal of developing stem cell–based therapeutics.

Hematopoiesis: A Human Perspective

Despite its complexity, blood is probably the best understood developmental system, largely due to seminal experimentation in the mouse. Clinically, hematopoietic stem cell (HSC) transplantation represents the most widely deployed regenerative therapy, but human HSCs have only been characterized relatively recently. The discovery that immune-deficient mice could be engrafted with human cells provided a powerful approach for studying HSCs. We highlight 2 decades of studies focusing on isolation and molecular regulation of human HSCs, therapeutic applications, and early lineage commitment steps, and compare mouse and humanized models to identify both conserved and species-specific mechanisms that will aid future preclinical research.

Distinct routes of lineage development reshape the human blood hierarchy across ontogeny

Adjusting hematopoietic hierarchy In adults, more than 300 billion blood cells are replenished daily. This output arises from a cellular hierarchy where stem cells differentiate into a series of multilineage progenitors, culminating in unilineage progenitors that generate over 10 different mature blood cell types. Notta et al. mapped the lineage potential of nearly 3000 single cells from 33 different cell populations of stem and progenitor cells from fetal liver, cord blood, and adult bone marrow (see the Perspective by Cabezas-Wallscheid and Trumpp). Prenatally, stem cell and progenitor populations were multilineage with few unilineage progenitors. In adults, multilineage cell potential was only seen in stem cell populations. Science, this issue p. 10.1126/science.aab2116; see also p. 126 As humans age, progenitor cells take over from stem cells the task of producing a steady supply of blood cells. [Also see Perspective by Cabezas-Wallscheid and Trumpp] INTRODUCTION The hematopoietic road map is a compilation of the various lineage differentiation routes that a stem cell takes to make blood. This program produces greater than 10 blood cell fates and is responsible for generating more than 300 billion cells daily. On several occasions over the past six decades, the murine road map has been reconceived due to new information overturning dogma. However, the human road map has changed little. In the human model, blood differentiation initiates at the level of multipotent stem cells and passes through a series of increasingly lineage-restricted oligopotent and, finally, unipotent progenitor intermediates. One critical oligopotent intermediate is the common myeloid progenitor (CMP), believed to be the origin of all myeloid (My), erythroid (Er), and megakaryocyte (Mk) cells. Although murine studies challenge the existence of oligopotent progenitors, a comprehensive analysis of human My-Er-Mk differentiation is lacking. Moreover, whether the pool of oligopotent intermediates is fixed across human development (fetal to adult) is unknown. RATIONALE The differentiation road map taken by human hematopoietic stem cells (HSCs) is fundamental to our understanding of blood homeostasis, hematopoietic malignancies, and regenerative medicine. RESULTS We mapped the cellular origins of My, Er, and Mk lineages across three time points in human blood development: fetal liver (FL), neonatal cord blood (CB), and adult bone marrow (BM). Using a cell-sorting scheme based on markers linked to Er and Mk lineage specification (CD71 and CD110), we found that previously described populations of multipotent progenitors (MPPs), CMPs, and megakaryocyte-erythroid progenitors (MEPs) were heterogeneous and could be further purified. Nearly 3000 single cells from 11 cellular subsets from the CD34+ compartment of FL, CB, and BM (33 subsets in total) were evaluated for their My, Er, and Mk lineage potential using an optimized single-cell assay. In FL, the ratio of cells with multilineage versus unilineage potential remained constant in both the stem cell (CD34+CD38–) and progenitor cell (CD34+CD38+) enriched compartments. By contrast, in BM, nearly all multipotent cells were restricted to the stem cell compartment, whereas unilineage progenitors dominated the progenitor cell compartment. Oligopotent progenitors were only a negligible component of the human blood hierarchy in BM, leading to the inference that multipotent cells differentiate into unipotent cells directly by adulthood. Mk/Er activity predominantly originated from the stem cell compartment at all developmental time points. In CB and BM, most Mks emerged as part of mixed clones from HSCs/MPPs, indicating that Mks directly branch from a multipotent cell and not from oligopotent progenitors like CMP. In FL, an almost pure Mk/Er progenitor was identified in the stem cell compartment, although less potent Mk/Er progenitors were also present in the progenitor compartment. In a hematological condition of HSC loss (aplastic anemia), Mk/Er but not My progenitors were more severely depleted, pinpointing a close physiological connection between HSC and the Mk/Er lineage. CONCLUSION Our data indicate that there are distinct road maps of blood differentiation across human development. Prenatally, Mk/Er lineage branching occurs throughout the cellular hierarchy. By adulthood, both Mk/Er activity and multipotency are restricted to the stem cell compartment, whereas the progenitor compartment is composed of unilineage progenitors forming a “two-tier” system, with few intervening oligopotent intermediates. Roadmaps of human blood stem cell differentiation. The classical model envisions that oligopotent progenitors such as CMP are an essential intermediate stage from which My/Er/Mk differentiation originates. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation. 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.

Hematopoietic Stem Cell Function and Survival Depend on c-Myc and N-Myc Activity

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule Granzyme B, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.

Dormant and Self‐Renewing Hematopoietic Stem Cells and Their Niches

Abstract:  In the mouse, over the last 20 years, a set of cell‐surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long‐term self‐renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single‐cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c‐Kit, CD135, CD48, CD150, and CD34) on lineage‐depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin−Sca1+c‐Kit+ CD48−CD150+CD34−) that gives rise to an active self‐renewing CD34+ HSC population. HSC dormancy, as well as the balance between self‐renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.

Balancing dormant and self-renewing hematopoietic stem cells.

The mouse hematopoietic stem cell (HSC) is probably the best-understood somatic stem cell in higher organisms. Recent studies have shown that the highest self-renewal potential is most likely contained within an exceedingly small number of deeply dormant bone marrow HSCs. These stem cells are housed in individual niches that preserve their dormancy via signaling molecules such as Thrombopoietin, Angiopoietins, and Stem Cell Factor. In response to injury cues, dormant HSCs are efficiently activated and produce numerous progenitors and mature cells. A series of intracellular regulatory molecules including FoxOs, mTORC1, Fbw7, Egr1, Pbx1, pRb, c-Cbl, Myc, and Bmi1 mediate the processes of dormancy, cycling, self-renewal, differentiation, and survival, all of which control the behavior of HSCs.

The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.

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.

Enhanced c-Met activity promotes G-CSF–induced mobilization of hematopoietic progenitor cells via ROS signaling

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1(+)/c-Kit(+)/Lin(-) cells and interfered with their enhanced chemotactic migration to stromal cell-derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met-mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.