Specification of hematopoietic stem cells in mammalian embryos: a rare or frequent event?

Hematopoietic stem cells (HSCs) are the blood-forming stem cells thought to be responsible for supporting the blood system throughout life. Transplantability has long been the flagship assay used to define and characterize HSCs throughout ontogeny. However, it has recently become clear that many cells emerge during ontogeny that lack transplantability yet nevertheless are fated to ultimately contribute to the adult HSC pool. Here, we explore recent advances in understanding the numbers and kinetics of cells that emerge during development to support lifelong hematopoiesis; these advances are made possible by new technologies allowing interrogation of lifelong blood potential without embryo perturbation or transplantation. Illuminating the dynamics of these cells during normal development informs efforts to better understand the origins of hematologic disease and engineer HSCs from differentiating pluripotent stem cells.

Diversity in the bone marrow niche: Classic and novel strategies to uncover niche composition.

Our view on the role and composition of the bone marrow (BM) has dramatically changed over time from a simple nutrient for the bone to a highly complex multicellular tissue that sustains haematopoiesis. Among these cells, multipotent haematopoietic stem cells (HSCs), which are predominantly quiescent, possess unique self-renewal capacity and multilineage differentiation potential and replenish all blood lineages to maintain lifelong haematopoiesis. Adult HSCs reside in specialised BM niches, which support their functions. Much effort has been put into deciphering HSC niches due to their potential clinical relevance. Multiple cell types have been implicated as HSC-niche components including sinusoidal endothelium, perivascular stromal cells, macrophages, megakaryocytes, osteoblasts and sympathetic nerves. In this review we provide a historical perspective on how technical advances, from genetic mouse models to imaging and high-throughput sequencing techniques, are unveiling the plethora of molecular cues and cellular components that shape the niche and regulate HSC functions.

Monkeypox: a new differential diagnosis when addressing genital ulcer disease.

We describe a case of genital ulcer and inguinal adenopathies that were attributable to monkeypox virus infection. We suggest clinicians adopt a low threshold for suspicion, particularly when evaluating genital ulcer disease.

The global clonal complexity of the murine blood system declines throughout life and after serial transplantation.

Although many recent studies describe the emergence and prevalence of "clonal hematopoiesis of indeterminate potential" in aged human populations, a systematic analysis of the numbers of clones supporting steady-state hematopoiesis throughout mammalian life is lacking. Previous efforts relied on transplantation of "barcoded" hematopoietic stem cells (HSCs) to track the contribution of HSC clones to reconstituted blood. However, ex vivo manipulation and transplantation alter HSC function and thus may not reflect the biology of steady-state hematopoiesis. Using a noninvasive in vivo color-labeling system, we report the first comprehensive analysis of the changing global clonal complexity of steady-state hematopoiesis during the natural murine lifespan. We observed that the number of clones (ie, clonal complexity) supporting the major blood and bone marrow hematopoietic compartments decline with age by ∼30% and ∼60%, respectively. Aging dramatically reduced HSC in vivo-repopulating activity and lymphoid potential while increasing functional heterogeneity. Continuous challenge of the hematopoietic system by serial transplantation provoked the clonal collapse of both young and aged hematopoietic systems. Whole-exome sequencing of serially transplanted aged and young hematopoietic clones confirmed oligoclonal hematopoiesis and revealed mutations in at least 27 genes, including nonsense, missense, and deletion mutations in Bcl11b, Hist1h2ac, Npy2r, Notch3, Ptprr, and Top2b.

Nfix Promotes Survival of Immature Hematopoietic Cells via Regulation of c-Mpl.

Hematopoietic stem and progenitor cells (HSPCs) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently reported that nuclear factor I/X (Nfix) promotes HSPC survival post-transplant. Here, we report that ectopic expression of Nfix in primary mouse HSPCs extends their ex vivo culture from about 20 to 40 days. HSPCs overexpressing Nfix display hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation relative to controls. Ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody negated the antiapoptotic effect of Nfix overexpression on cultured HSPCs. Furthermore, NFIX was capable of binding to and transcriptionally activating a proximal c-Mpl promoter fragment. In sum, these data suggest that NFIX-mediated upregulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo. Stem Cells 2018;36:943-950.

Reaccion acneiforme noduloquistica secundaria a vemurafenib con buena respuesta a isotretinoina oralSevere acneiform eruption associated with vemurafenib with response to isotretinoin.

Vemurafenib, a kinase inhibitor that targets tumors with the BRAF V600E mutation, is a promising option for unresectable or metastatic melanoma. Cutaneous side-effects have been reported including alopecia, photosensitivity, squamous cell carcinoma, keratoacanthomas, keratosis pilaris-like eruption, and palmoplantar hyperkeratosis. Acneiform eruptions have been reported in 3%-6% of the patients treated with BRAF inhibitors,and 5 cases are described in the literature. Although they responded well to topical therapies, oral antibiotics, or observation, one case required oral etretinate and the withdrawal of vemurafenib because the adverse event reached grade 3. We report one case of a severe acneiform eruption associated with vemurafenib with a good response to isotretinoin allowing continuation of the BRAF inhibitor.

Hematopoietic stem cells under pressure.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) and progenitors are tasked with maintaining hematopoietic homeostasis in the face of numerous insults and challenges, including infection, inflammation, and exsanguination. HSCs possess the remarkable ability to reconstitute the entire hematopoietic system of an organism whose own hematopoietic system has been ablated. This ability is exploited routinely in the clinic via HSC transplantation (HSCT). Here, we focus on the physiological and molecular bottlenecks overcome by HSCs during transplantation. RECENT FINDINGS: During transplantation, HSCs encounter a damaged bone marrow niche, characterized molecularly by increases in oxygen concentrations and an altered cytokine milieu. New mechanisms and pathways have been recently implicated during HSCT, including transplanted HSC-dependent secretion of conditioning molecules that facilitate engraftment and pathways that protect HSCs from perturbed organelle homeostasis. SUMMARY: Better understanding the molecular processes HSCs employ to withstand the stress of transplant will illuminate novel targets for further improving conditioning regimens and engraftment during HSCT.

Genetic inactivation of Cdk7 leads to cell cycle arrest and induces premature aging due to adult stem cell exhaustion.

Cyclin-dependent kinase (Cdk)7, the catalytic subunit of the Cdk-activating kinase (CAK) complex has been implicated in the control of cell cycle progression and of RNA polymerase II (RNA pol II)-mediated transcription. Genetic inactivation of the Cdk7 locus revealed that whereas Cdk7 is completely dispensable for global transcription, is essential for the cell cycle via phosphorylation of Cdk1 and Cdk2. In vivo, Cdk7 is also indispensable for cell proliferation except during the initial stages of embryonic development. Interestingly, widespread elimination of Cdk7 in adult tissues with low proliferative indexes had no phenotypic consequences. However, ablation of conditional Cdk7 alleles in tissues with elevated cellular turnover led to the efficient repopulation of these tissues with Cdk7-expressing cells most likely derived from adult stem cells that may have escaped the inactivation of their targeted Cdk7 alleles. This process, a physiological attempt to maintain tissue homeostasis, led to the attrition of adult stem cell pools and to the appearance of age-related phenotypes, including telomere shortening and early death.

Haematopoietic stem cell health in sickle cell disease and its implications for stem cell therapies and secondary haematological disorders.

Gene modification of haematopoietic stem cells (HSCs) is a potentially curative approach to sickle cell disease (SCD) and offers hope for patients who are not eligible for allogeneic HSC transplantation. Current approaches require in vitro manipulation of healthy autologous HSC prior to their transplantation. However, the health and integrity of HSCs may be compromised by a variety of disease processes in SCD, and challenges have emerged in the clinical trials of gene therapy. There is also concern about increased susceptibility to haematological malignancies during long-term follow up of patients, and this raises questions about genomic stability in the stem cell compartment. In this review, we evaluate the evidence for HSC deficits in SCD and then discuss their potential causation. Finally, we suggest several questions which need to be addressed in order to progress with successful HSC manipulation for gene therapy in SCD.

NEUROBEACHIN regulates hematopoietic progenitor differentiation and survival by modulating NOTCH activity.

Hematopoietic stem cells (HSCs) can generate all blood cells. This ability is exploited in HSC transplantation (HSCT) to treat hematologic disease. A clear understanding of the molecular mechanisms that regulate HSCT is necessary to continue improving transplant protocols. We identified the BEACH-domain containing protein (BDCP), NEUROBEACHIN (NBEA), as a putative regulator of HSCT. Here, we demonstrated that NBEA and related BDCPs, including LRBA, NBEAL1 and LYST, are required during HSCT to efficiently reconstitute the hematopoietic system of lethally irradiated mice. Nbea knockdown in mouse HSCs induced apoptosis and a differentiation block post-transplantation. Nbea deficiency in hematopoietic progenitor cells perturbed the expression of genes implicated in vesicle trafficking and led to changes in NOTCH receptor localization. This resulted in perturbation of the NOTCH transcriptional program, which is required for efficient HSC engraftment. In sum, our findings reveal a novel role for NBEA in the control of NOTCH receptor turnover in hematopoietic cells and supports a model where BDCP regulated vesicle trafficking is required for efficient HSCT.

Murine foetal liver supports limited detectable expansion of life-long haematopoietic progenitors.

Current dogma asserts that the foetal liver (FL) is an expansion niche for recently specified haematopoietic stem cells (HSCs) during ontogeny. Indeed, between embryonic day of development (E)12.5 and E14.5, the number of transplantable HSCs in the murine FL expands from 50 to about 1,000. Here we used a non-invasive, multi-colour lineage tracing strategy to interrogate the embryonic expansion of murine haematopoietic progenitors destined to contribute to the adult HSC pool. Our data show that this pool of fated progenitors expands only two-fold during FL ontogeny. Although Histone2B-GFP retention in vivo experiments confirmed substantial proliferation of phenotypic FL-HSC between E12.5 and E14.5, paired-daughter cell assays revealed that many mid-gestation phenotypic FL-HSCs are biased to differentiate, rather than self-renew, relative to phenotypic neonatal and adult bone marrow HSCs. In total, these data support a model in which the FL-HSC pool fated to contribute to adult blood expands only modestly during ontogeny.

Inflammatory exposure drives long-lived impairment of hematopoietic stem cell self-renewal activity and accelerated aging.

Hematopoietic stem cells (HSCs) mediate regeneration of the hematopoietic system following injury, such as following infection or inflammation. These challenges impair HSC function, but whether this functional impairment extends beyond the duration of inflammatory exposure is unknown. Unexpectedly, we observed an irreversible depletion of functional HSCs following challenge with inflammation or bacterial infection, with no evidence of any recovery up to 1 year afterward. HSCs from challenged mice demonstrated multiple cellular and molecular features of accelerated aging and developed clinically relevant blood and bone marrow phenotypes not normally observed in aged laboratory mice but commonly seen in elderly humans. In vivo HSC self-renewal divisions were absent or extremely rare during both challenge and recovery periods. The progressive, irreversible attrition of HSC function demonstrates that temporally discrete inflammatory events elicit a cumulative inhibitory effect on HSCs. This work positions early/mid-life inflammation as a mediator of lifelong defects in tissue maintenance and regeneration.

Clones assemble! The clonal complexity of blood during ontogeny and disease.

Hematopoietic stem and progenitor cells (HSPCs) govern the daily expansion and turnover of billions of specialized blood cells. Given their clinical utility, much effort has been made toward understanding the dynamics of hematopoietic production from this pool of stem cells. An understanding of hematopoietic stem cell clonal dynamics during blood ontogeny could yield important insights into hematopoietic regulation, especially during aging and repeated exposure to hematopoietic stress-insults that may predispose individuals to the development of hematopoietic disease. Here, we review the current state of research regarding the clonal complexity of the hematopoietic system during embryogenesis, adulthood, and hematologic disease.

Murine hematopoietic stem cell activity is derived from pre-circulation embryos but not yolk sacs.

The embryonic site of definitive hematopoietic stem cell (dHSC) origination has been debated for decades. Although an intra-embryonic origin is well supported, the yolk sac (YS) contribution to adult hematopoiesis remains controversial. The same developmental origin makes it difficult to identify specific markers that discern between an intraembryonic versus YS-origin using a lineage trace approach. Additionally, the highly migratory nature of blood cells and the inability of pre-circulatory embryonic cells (i.e., 5-7 somite pairs (sp)) to robustly engraft in transplantation, even after culture, has precluded scientists from properly answering these questions. Here we report robust, multi-lineage and serially transplantable dHSC activity from cultured 2-7sp murine embryonic explants (Em-Ex). dHSC are undetectable in 2-7sp YS explants. Additionally, the engraftment from Em-Ex is confined to an emerging CD31+CD45+c-Kit+CD41- population. In sum, our work supports a model in which the embryo, not the YS, is the major source of lifelong definitive hematopoiesis.

Lifelong haematopoiesis is established by hundreds of precursors throughout mammalian ontogeny.

Current dogma asserts that mammalian lifelong blood production is established by a small number of blood progenitors. However, this model is based on assays that require the disruption, transplantation and/or culture of embryonic tissues. Here, we used the sample-to-sample variance of a multicoloured lineage trace reporter to assess the frequency of emerging lifelong blood progenitors while avoiding the disruption, culture or transplantation of embryos. We find that approximately 719 Flk1+ mesodermal precursors, 633 VE-cadherin+ endothelial precursors and 545 Vav1+ nascent blood stem and progenitor cells emerge to establish the haematopoietic system at embryonic days (E)7-E8.5, E8.5-E11.5 and E11.5-E14.5, respectively. We also determined that the spatio-temporal recruitment of endothelial blood precursors begins at E8.5 and ends by E10.5, and that many c-Kit+ clusters of newly specified blood progenitors in the aorta are polyclonal in origin. Our work illuminates the dynamics of the developing mammalian blood system during homeostasis.

Murine hemogenic endothelial precursors display heterogeneous hematopoietic potential ex vivo.

Hematopoietic stem and progenitor cells (HSPCs) sustain life-long hematopoiesis and are first detected in the embryo by transplantation at embryonic day 10.5 (E10.5). HSPCs are mesodermal in origin and ultimately emerge from a subset of arterial endothelium (i.e., hemogenic endothelium [HE]), which is highly concentrated in the aorta-gonad-mesonephros region of the midgestation embryo. Here, we used clonal ex vivo assays, in which endothelial cells isolated from the midgestation aorta and vitelline and umbilical arteries are co-cultured on supportive stroma, to show that only about 0.1%, 1.3%, and 0.29% of E9.5, E10.5, and E11.5 endothelium are functional HE, respectively. We further show high phenotypic and functional variability in the hematopoietic potential of individual hemogenic endothelial precursors. Using unique niche stroma capable of providing the signals necessary for definitive hematopoietic stem cell (dHSC) induction, we demonstrate that this variability in HE includes their potential to support phenotypic dHSCs. These data suggest the presence of a continuum of maturing HE with distinct hematopoietic potential or HE representative of a heterogeneous pool of precursors that give rise to HSPCs with disparate hematopoietic potential.

Functional screen identifies regulators of murine hematopoietic stem cell repopulation.

Understanding the molecular regulation of hematopoietic stem and progenitor cell (HSPC) engraftment is paramount to improving transplant outcomes. To discover novel regulators of HSPC repopulation, we transplanted >1,300 mice with shRNA-transduced HSPCs within 24 h of isolation and transduction to focus on detecting genes regulating repopulation. We identified 17 regulators of HSPC repopulation: Arhgef5, Armcx1, Cadps2, Crispld1, Emcn, Foxa3, Fstl1, Glis2, Gprasp2, Gpr56, Myct1, Nbea, P2ry14, Smarca2, Sox4, Stat4, and Zfp251. Knockdown of each of these genes yielded a loss of function, except in the cases of Armcx1 and Gprasp2, whose loss enhanced hematopoietic stem cell (HSC) repopulation. The discovery of multiple genes regulating vesicular trafficking, cell surface receptor turnover, and secretion of extracellular matrix components suggests active cross talk between HSCs and the niche and that HSCs may actively condition the niche to promote engraftment. We validated that Foxa3 is required for HSC repopulating activity, as Foxa3(-/-) HSC fails to repopulate ablated hosts efficiently, implicating for the first time Foxa genes as regulators of HSPCs. We further show that Foxa3 likely regulates the HSC response to hematologic stress. Each gene discovered here offers a window into the novel processes that regulate stable HSPC engraftment into an ablated host.