Hematopoietic stem cells under pressure.

"This is a non-final version of an article published in final form in Current Opinion in Hematology "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

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

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

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

Effect of developmental stage of HSC and recipient on transplant outcomes

The first hematopoietic stem cells (HSCs) that engraft irradiated adult mice arise in the aorta-gonad-mesonephros (AGM) on embryonic day 11.5 (E11.5). However, at this stage, there is a discrepancy between the apparent frequency of HSCs depicted with imaging and their rarity when measured with limiting dilution transplant. We have attempted to reconcile this difference using neonatal recipients, which are more permissive for embryonic HSC engraftment. We found that embryonic HSCs from E9.5 and E10.5 preferentially engrafted neonates, whereas developmentally mature, definitive HSCs from E14.5 fetal liver or adult bone marrow (BM) more robustly engrafted adults. Neonatal engraftment was enhanced after treating adult BM-derived HSCs with interferon. Adult BM-derived HSCs preferentially homed to the liver in neonatal mice yet showed balanced homing to the liver and spleen in adults. These findings emphasize the functional differences between nascent and mature definitive HSCs

Epigenetic memory in induced pluripotent stem cells.

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment

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

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

Signal Transmission in the Auditory System

Contains table of contents for Section 3 and reports on four research projects.National Institutes of Health Grant R01 DC00194National Institutes of Health Grant P01 DC00119National Science Foundation Grant IBN 96-04642W.M. Keck Foundation Career Development ProfessorshipNational Institutes of Health Grant R01 DC00238Thomas and Gerd Perkins Award ProfessorshipAlfred P Sloan Foundation Instrumentation GrantJohn F. and Virginia B. Taplin Award in Health Sciences and TechnologyNational Institutes of Health/National Institute of Deafness and Other Communication DisordersNational Institutes of Health/National Institute of Deafness and Other Communication Disorders Grant PO1 DC0011