Lineage-restricted Progenitors Research Articles

The molecular chronology of mammary epithelial cell fate switching.

The adult mammary gland is maintained by lineage-restricted progenitor cells through pregnancy, lactation, involution, and menopause. Injury resolution, transplantation-associated mammary gland reconstitution, and tumorigenesis are unique exceptions, wherein mammary basal cells gain the ability to reprogram to a luminal state. Here, we leverage newly developed cell-identity reporter mouse strains, and time-resolved single-cell epigenetic and transcriptomic analyses to decipher the molecular programs underlying basal-to-luminal fate switching in vivo . We demonstrate that basal cells rapidly reprogram toward plastic cycling intermediates that appear to hijack molecular programs we find in bipotent fetal mammary stem cells and puberty-associatiated cap cells. Loss of basal-cell specifiers early in dedifferentiation coincides with activation of Notch and BMP, among others. Pharmacologic blockade of each pathway disrupts basal-to-luminal transdifferentiation. Our studies provide a comprehensive map and resource for understanding the coordinated molecular changes enabling terminally differentiated epithelial cells to transition between cell lineages and highlights the stunning rapidity by which epigenetic reprogramming can occur in response to disruption of tissue structure.

Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.

Two developmental pathways generate functionally distinct populations of natural killer cells

Abstract Natural killer (NK) cells function by eliminating virus-infected cells or tumor cells during early defenses. However, the early development of NK cells and lineage relationships between NK cells and helper innate lymphoid cells (ILCs) remain elusive. Common precursors for ILCs (ILCPs) can differentiate into both helper ILCs and NK cells. Here, we identified a NK lineage-restricted progenitor population, early NK progenitor (ENKP), which does not develop from ILCPs, thus ENKP may represent the ILCP-independent pathway of NK cell development. Competitive chimera experiment shows ENKPs generate NK cells more efficiently than ILCPs, suggesting that ENKP-dependent pathway is the major pathway for NK cell development. scRNA-seq shows ENKP-derived NK cells express Ly49 receptors and higher levels of cytotoxic genes whereas ILCP-derived NK cells have very low expression of Ly49 receptors and express higher levels of genes implicated in tissue residency such as CD69 and CD200R. Furthermore, Ly49H+ NK cells which response to MCMV infection mostly develop from ENKPs but not ILCPs. Consistently, ENKP-derived NK cells but not ILCP-derived NK cells expanded dramatically after MCMV infection. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets.

HSCs Engraft in a Stochastic Pattern and Form Clonal Pools after Transplantation

Hematopoietic stem cells (HSCs) are considered the only cell type capable of self-renewing and generating all blood lineages. HSCs are further assumed to be rare, barely proliferating, and not contributing to short-term hematopoietic recovery after transplantation. Although widely accepted, individual definitions have been challenged. Tracking of murine HSCs showed continuous contribution during steady-state hematopoiesis. Similarly, single HSCs can reconstitute the entire murine host illustrating extensive proliferative capabilities without immediate exhaustion while maintaining long-term persistence. Studying these findings in humans is challenging. Knowledge about the engraftment kinetics and contribution of human HSCs during recovery is limited to clinical trials of gene therapy patients enrolled in clonal tracking studies using retroviral integration site analysis (ISA). However, the tracking of human HSCs remains under-investigated due to the limited number of specimens possible to obtain, especially during the early phases of reconstitution where blood cell counts are low. In addition, clone tracking is impacted by technical limitations. While ISA is reliable to monitor the potential outgrowth of dominant clones, low sensitivity and high error rates require significant data exclusion and sophisticated statistical tests to ensure data reliability (Adair et al. 2020, Molecular Therapy MCD). Lack of sensitivity and loss of low abundance clones can be overcome by increasing the frequency of sampling (high density) as well as repeated sampling, aspects hard to achieve in patients. To investigate the clonal dynamics in the early phases of hematopoietic reconstitution and determine the onset of HSC contribution, we performed high-density sampling for ISA in nonhuman primates (NHPs). In the first month of hematopoietic recovery, weekly blood samples were taken to enhance data density. We applied a new clonal tracking approach to reliably detect the contribution of multipotent long-term persisting HSCs during neutrophil recovery even in samples with low cellularity during early recovery. Animals were followed for 4.5 years to collect HSC clone signatures, and multipotent stem cell clones were retrospectively identified in early samples. Finally, observed changes in clonal diversity in vivo were used to inform a simulation of hematopoietic reconstitution to determine the temporal involvement of HSCs. Contribution of multipotent long-term persisting HSCs clones was detected in the very first blood samples taken 2 to 3 weeks post-transplant during neutrophil recovery. HSCs became the dominant source for mature blood cells in the peripheral blood as early as 50 days post-transplant. Based on these finding, simulations predicted that hematopoietic recovery is primarily HSC driven, differentiating HSC clones are the main source of blood production during recovery, and that long-term persisting HSC clones expand forming clonal pools. The ability of HSCs to symmetrically expand was confirmed ex vivo in colony-forming cells assays as well as in vivo using ISA and tracking of expressed barcodes. As predicted by the simulation, we observed a rapid decline of clonal diversity and individual HSCs symmetrically expand forming clonal pools. Here, we show evidence that long-term persisting multipotent HSCs actively contribute during early hematopoietic reconstitution after transplantation. Enhanced sampling showed that multipotent HSCs produce neutrophils during recovery, are the main source of mature blood cells as early as 50 days post-transplant, and symmetrically divide to form clonal pools long-term. Most importantly, observed changes in the clonal diversity during early recovery suggest a stochastic HSC engraftment and differentiation pattern rather than a bi-phasic reconstitution initially driven by short-term progenitors. Early blood production is driven by these HSC clones that are not predetermined but stochastically contribute to a limited number of lineages before they disappear - a population of cells previously incorrectly identified as lineage-restricted short-term engrafting progenitors. Consequently, we here propose a revised model of hematopoietic reconstitution and provide novel insights into HSC biology with direct implications for the design of HSC transplantation as well as gene therapy studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transcription Factor Profiling Reveals That CEBPA Promotes Ribosomal RNA Synthesis in Myeloid Progenitors

Hematopoiesis involves orchestrated differentiation of hematopoietic stem cells (HSCs) and lineage-restricted progenitors to mature blood cells. Different populations in this hierarchy vary in function, size, proliferation, and protein synthesis rates. It is also known that different normal hematopoietic cell types differ in the rates of ribosomal RNA (rRNA) transcription, the key rate-limiting step in ribosome biogenesis that occurs in the nucleolus. Leukemic blast cells have long been known to have enlarged nucleoli, indicating aberrantly increased rRNA transcription and ribosome biogenesis. Ribosome biogenesis is an energy-intensive process, beginning with the transcription of multi-copy rDNA genes by RNA polymerase I (Pol I) to produce 47S precursor rRNA (pre-rRNA), which is further processed into mature 18S, 5.8S, and 28S rRNA and assembled with 5S rRNA and 80 different ribosomal proteins to form mature ribosomes (Fig A). Because rRNA transcription is often considered a housekeeping process, the variations in its cell-type and disease-specific rates have not been studied, leading to a lack of understanding of the mechanisms underlying its regulation. To identify cell-type-specific regulators of rRNA transcription in hematopoiesis, we compiled a list of 249 hematopoietic transcription factors (TFs) and epigenetic factors, and mapped 2200 publicly available ChIP-Seq datasets for these factors to rDNA, creating the first atlas of hematopoietic TF-rDNA binding. Through this atlas, we identified that CEBPA shows consistent and abundant binding to human and mouse rDNA at a conserved, previously unknown motif (Fig B). CEBPA is an essential myeloid lineage-specific TF whose knockout leads to complete loss of granulocyte-monocyte progenitors (GMPs) and all downstream myeloid lineage cells. CEBPA also shows hotspot mutations in 10% of AML patients. To characterize the role of CEBPA in rRNA transcription, we used a mouse HoxA9-ER cell line (which closely resembles GMPs), and biallelically fused FKBP degron into endogenous loci of the Cebpa gene, enabling rapid degradation of CEBPA protein using small molecule ligand dTAGV-1 (Fig C, D). To quantify effects on rRNA transcription, we developed a novel assay named '47S-FISH-Flow’ that involves flowcytometric quantification of fluorescent probes hybridized to nascent rRNA in the nucleolus (Fig E, F). We found that depleting CEBPA caused a rapid reduction in 47S rRNA level, plateauing at 35% reduction within hours (Fig G). Decrease in rRNA production was followed by reduction in nucleolar size, reduction in mature ribosome subunit abundance, and reduced growth (Fig H, I). To understand the molecular function of CEBPA on rDNA machinery, we performed time course ChIP-Seq on key rDNA transcriptional machinery, and found that CEBPA degradation specifically reduced occupancy of Pol I along with its initiation factor RRN3 on rDNA (Fig J). In summary, we have found a novel role for the extensively studied myeloid lineage-specific TF CEBPA in directly binding rDNA and regulating rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by lineage-specific hematopoietic TFs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Myostatin is a negative regulator of adult neurogenesis after spinal cord injury in zebrafish.

Intrinsic and extrinsic inhibition of neuronal regeneration obstruct spinal cord (SC) repair in mammals. In contrast, adult zebrafish achieve functional recovery after complete SC transection. While studies of innate SC regeneration have focused on axon regrowth as a primary repair mechanism, how local adult neurogenesis affects functional recovery is unknown. Here, we uncover dynamic expression of zebrafish myostatin b (mstnb) in a niche of dorsal SC progenitors after injury. mstnb mutants show impaired functional recovery, normal glial and axonal bridging across the lesion, and an increase in the profiles of newborn neurons. Molecularly, neuron differentiation genes are upregulated, while the neural stem cell maintenance gene fgf1b is downregulated in mstnb mutants. Finally, we show that human fibroblast growth factor 1 (FGF1) treatment rescues the molecular and cellular phenotypes of mstnb mutants. These studies uncover unanticipated neurogenic functions for mstnb and establish the importance of local adult neurogenesis for innate SC repair.

Cerebellum lineage allocation, morphogenesis and repair: impact of interplay amongst cells.

The cerebellum has a simple cytoarchitecture consisting of a folded cortex with three cell layers that surrounds a nuclear structure housing the output neurons. The excitatory neurons are generated from a unique progenitor zone, the rhombic lip, whereas the inhibitory neurons and astrocytes are generated from the ventricular zone. The growth phase of the cerebellum is driven by lineage-restricted progenitor populations derived from each zone. Research during the past decade has uncovered the importance of cell-to-cell communication between the lineages through largely unknown signaling mechanisms for regulating the scaling of cell numbers and cell plasticity during mouse development and following injury in the neonatal (P0-P14) cerebellum. This Review focuses on how the interplay between cell types is key to morphogenesis, production of robust neural circuits and replenishment of cells after injury, and ends with a discussion of the implications of the greater complexity of the human cerebellar progenitor zones for development and disease.

Competition between hematopoietic stem and progenitor cells controls hematopoietic stem cell compartment size

Cellular competition for limiting hematopoietic factors is a physiologically regulated but poorly understood process. Here, we studied this phenomenon by hampering hematopoietic progenitor access to Leptin receptor+ mesenchymal stem/progenitor cells (MSPCs) and endothelial cells (ECs). We show that HSC numbers increase by 2-fold when multipotent and lineage-restricted progenitors fail to respond to CXCL12 produced by MSPCs and ECs. HSCs are qualitatively normal, and HSC expansion only occurs when early hematopoietic progenitors but not differentiated hematopoietic cells lack CXCR4. Furthermore, the MSPC and EC transcriptomic heterogeneity is stable, suggesting that it is impervious to major changes in hematopoietic progenitor interactions. Instead, HSC expansion correlates with increased availability of membrane-bound stem cell factor (mSCF) on MSPCs and ECs presumably due to reduced consumption by cKit-expressing hematopoietic progenitors. These studies suggest that an intricate homeostatic balance between HSCs and proximal hematopoietic progenitors is regulated by cell competition for limited amounts of mSCF.

Divergence between Neuronal and Oligodendroglial Cell Fate, in Postnatal Brain Neural Stem Cells, Leads to Divergent Properties in Polymorphic In Vitro Assays.

Two main stem cell pools exist in the postnatal mammalian brain that, although they share some “stemness” properties, also exhibit significant differences. Multipotent neural stem cells survive within specialized microenvironments, called niches, and they are vulnerable to ageing. Oligodendroglial lineage-restricted progenitor cells are widely distributed in the brain parenchyma and are more resistant to the effects of ageing. Here, we create polymorphic neural stem cell cultures and allow cells to progress towards the neuronal and the oligodendroglial lineage. We show that the divergence of cell fate is accompanied by a divergence in the properties of progenitors, which reflects their adaptation to life in the niche or the parenchyma. Neurogenesis shows significant spatial restrictions and a dependence on laminin, a major niche component, while oligodendrogenesis shows none of these constraints. Furthermore, the blocking of integrin-β1 leads to opposing effects, reducing neurogenesis and enhancing oligodendrogenesis. Therefore, polymorphic neural stem cell assays can be used to investigate the divergence of postnatal brain stem cells and also to predict the in vivo effects of potential therapeutic molecules targeting stem and progenitor cells, as we do for the microneurotrophin BNN-20.

Revisiting hematopoiesis: applications of the bulk and single-cell transcriptomics dissecting transcriptional heterogeneity in hematopoietic stem cells.

Hematopoietic system (HS) is one of the most unique, adaptive and comprehensive developmental systems on which various other body systems relies on. It consists of a central pool of multipotent hematopoietic stem cells (HSCs) differentiating into lymphoid and myeloid lineage by series of gradual loss of stemness potential. Thus, this highly coordinated phenomenon of blood cell renewal ensures robust immunity and limits autoimmunity. Any disease, chronic infection or stress interrupts HS homeostasis and breaks HSCs' dormancy, thereby activating HSCs to meet the peripheral demand for different immune cells via their expansion and differentiation into more lineage-restricted progenitors, primarily within the bone marrow (BM) in adult life. Therefore, a greater understanding of the overall regulatory landscape of HSC homeostasis and their perturbations is critical for dissecting protective immunity versus autoimmunity. Recent advancements in next-generation sequencing (NGS) viz genomic, transcriptomic, epigenomic and proteogenomic methods at bulk as well as single-cell levels have increased our apprehension for HSC working model. In this review, we discussed the recent findings and computational methods used to unravel the new HSC model revised over the classical model.

Assessment of Breast Cancer Stem Cell Activity Using a Spheroid Formation Assay.

Evidence is emerging that cancer cells are arranged as a hierarchy that spans from stem cells to lineage-restricted progenitor cells. The recent development of spheroid cultures with several tissue type has provided new opportunities to assess cancer stem cell (CSC) activity by allowing them to propagate under conditions that resemble the microenvironment for growth of tumors. One tissue type widely used for stem cell investigations is mammary tissue, and the sphere formation assay has been used in both normal mammary tissue and in breast cancer. Here, we describe detailed experimental methodology for generating and propagating spheres from normal mammary tissue and primary breasttumors of mice, patient derived xenografts (PDXs) and breast cancercell lines. We further describe how these sphere cultures can be employed for coculture assays to assess the effect of tumor microenvironment (TME) on self-renewal ability of CSCs in breast cancer.

A Novel Megakaryocyte Subpopulation Poised to Exert the Function of HSC Niche as Possible Driver of Myelofibrosis.

Careful morphological investigations, coupled with experimental hematology studies in animal models and in in vitro human cultures, have identified that platelets are released in the circulation by mature megakaryocytes generated by hematopoietic stem cells by giving rise to lineage-restricted progenitor cells and then to morphologically recognizable megakaryocyte precursors, which undergo a process of terminal maturation. Advances in single cell profilings are revolutionizing the process of megakaryocytopoiesis as we have known it up to now. They identify that, in addition to megakaryocytes responsible for producing platelets, hematopoietic stem cells may generate megakaryocytes, which exert either immune functions in the lung or niche functions in organs that undergo tissue repair. Furthermore, it has been discovered that, in addition to hematopoietic stem cells, during ontogeny, and possibly in adult life, megakaryocytes may be generated by a subclass of specialized endothelial precursors. These concepts shed new light on the etiology of myelofibrosis, the most severe of the Philadelphia negative myeloproliferative neoplasms, and possibly other disorders. This perspective will summarize these novel concepts in thrombopoiesis and discuss how they provide a framework to reconciliate some of the puzzling data published so far on the etiology of myelofibrosis and their implications for the therapy of this disease.

CEBPA Directly Binds Ribosomal DNA and Promotes Ribosomal RNA Transcription in Myeloid Progenitors

Hematopoietic stem cells (HSCs) form a hierarchy of lineage restricted progenitor cells to produce mature hematopoietic cells that vary in function, size, proliferation, and protein synthesis rates. Different hematopoietic cells also vary in the rate of ribosomal RNA (rRNA) transcription, the key rate-limiting step in ribosome biogenesis that occurs in the nucleolus. Leukemic blast cells have long been identified by their prominent nucleoli, indicating high ribosome biogenesis rates (Fig A). Ribosome biogenesis is an extremely energy intensive process begins with transcription of multi-copy rDNA genes by RNA polymerase I (Pol I) to produce 47S precursor rRNA (pre-rRNA) which further processed into the generation of mature 18S, 5.8S, and 28S rRNA and assembled with 5S rRNA and 80 different ribosomal proteins to form mature ribosomes (Fig B). This process is highly dynamic and regulated at the level of rRNA transcription. Despite cell-type and disease-specific variations, rRNA transcription has long been considered a housekeeping process. Hence, cell or tissue type-specific regulation of rRNA transcription has rarely been explored.To identify cell-type-specific regulators of rRNA transcription in hematopoiesis, we mapped 2200 publicly available ChIP-Seq datasets representing 249 hematopoietic transcription factors (TFs) and epigenetic factors to create an atlas of hematopoietic TF-rDNA binding. We identified CEBPA that shows consistent and abundant binding to rDNA at a conserved, previously unknown motif in both species (Fig C). CEBPA is a myeloid lineage specific TF whose knockout leads to complete loss of all myeloid lineage cells. It is also frequently mutated (10%) in AML patients. So we picked CEBPA to further characterize its role in rRNA transcription.Since CEBPA deletion causes loss of granulocyte-monocyte progenitors (GMPs), we used the mouse HoxA9-ER cell line (which closely resembles GMPs). To study the immediate consequences of CEBPA loss, We generated a stable degron cell line by biallelically fusing FKBP degron into endogenous loci of Cebpa, enabling to rapidly degrade endogenous CEBPA protein on treatment with dTagV ligand (Fig D, E). To precisely quantify the rate of rRNA transcription, we developed a novel assay called ‘47S-FISH-Flow’ that involves hybridizing fluorescent oligos unique to 5' end of 47S pre-rRNA, which only marks newly synthesized nascent rRNA in the nucleolus, and quantify using flow cytometry (Fig F, G). We found that depleting CEBPA caused rapid decrease in 47S rRNA level and occupancy of Pol I on rDNA (Fig H, I).In summary, we found that myeloid lineage specific TF CEBPA abundantly binds to a conserved motif in rDNA and the depletion of CEBPA rapidly reduces nascent 47S rRNA, indicating that it directly promotes rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by lineage-specific hematopoietic TFs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single-Cell Tracking By Time Lapse Imaging Confirms Thrombopoietin Promotes Megakaryocytic-Erythroid Progenitor Self Renewal, but Does Not Instruct Lineage Commitment

The molecular mechanisms underlying lineage commitment of stem and progenitor cells have implications for deriving specific cell types in vitro for regenerative medicine purposes. Current approaches to derive transfusable amounts of erythrocytes and platelets fall short of producing physiologically relevant amounts of each cell type. Thrombopoietin (TPO), commonly used in culture systems to increase platelet yield from cultured megakaryocytes, increases megakaryocyte (Mk) numbers and promotes platelet production. With the goal of generating adequate quantities of transfusion products in mind, establishing differentiation cultures that start with highly purified progenitors with Mk and E lineage potential as well as expansion potential is a very attractive option. However, a pervasive misconception exists that supplementing MEP cultures with TPO will increase the frequency with which the MEP produce megakaryocyte-committed daughter cells, thereby increasing the pool of lineage-restricted progenitor cells with high expansion capacity that ultimately yield higher numbers of transfusable platelets. We investigated whether TPO plays a role in instructing Megakaryocytic-Erythroid Progenitor (MEP) [1] commitment to the Mk lineage utilizing our previously presented time-lapse imaged colony forming unit assay and cell tracking approach [2]. This allowed us to calculate the frequency with which MEP give rise to lineage restricted progenitors in control conditions (with SCF, IL3, IL6, EPO, and TPO) compared to conditions lacking TPO to test the hypothesis that absence of TPO may diminish the frequency with which MEP give rise to Mk-restricted progenitors.We found that in the absence of TPO, MEP colony forming efficiency significantly (p<0.05) dropped by 22.4%. Importantly, the distribution of colony types was unaffected by the absence of TPO (Figure 1). By time-lapse imaging we observed the frequency with which MEP commit to the E lineage versus the Mk lineage to be approximately 2:1 in both control and -TPO conditions, demonstrating that TPO does not affect MEP lineage commitmentTime-lapse imaging also confirmed that MEP cultured without TPO underwent significantly (p<0.05) fewer symmetric self-renewal divisions resulting in less expansion of MEP, as well as less asymmetric self-renewal divisions resulting in more rapid exhaustion of MEP (Figure 2). 80% of control MEP divisions resulted in self renewal for an average of 8 generations and expansion divisions dropped below 10% after the 6 th generation, whereas MEP cultured without TPO lost the same propensity to self-renew within 3 generations. This confirms that TPO supports MEP self-renewal and expansion, similarly to how TPO supports HSC expansion [3].Cell death rates were at least 5-fold higher in the absence of TPO compared to control culture conditions containing TPO, and this effect was observed in both E and Mk-destined cells, demonstrating that TPO supports survival of both E and Mk-destined progenitors (p<0.05; Figure 3). Of note, CD41 mean fluorescence intensity was 2-fold dimmer in Mk progeny in the -TPO condition compared to control condition after 6 days in culture, which could indicate a delay in megakaryocytic maturation resulting in slower up-regulation of CD41.Our results confirm the hypothesis that TPO allows for greater self-renewal of MEP, as well as the survival of both the E and Mk-destined progenitors, however TPO is not instructive in MEP lineage commitment as evidenced by no change in the frequency of colony types. These findings also support the use of TPO in cultures of less committed progenitors to promote expansion leading to higher production of transfusable cell types. Further uses of this approach can include measuring kinetics of cellular processes such as division rate and motility, which may correlate with cell state and permit predictions of the probability of lineage choices downstream of specific progenitor populations.

The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal

AbstractThe histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac), and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used 2 complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1-null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow 2 to 6 weeks after Hbo1 deletion. Hbo1-deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors. The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-, and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1, and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

Epigenetic and Single Cell Analyses Reveal Dual Fate Decision Points in Pre-Primed Hematopoietic Stem Cells during Erythro-Megakaryopoiesis

Recent single cell analyses have shown that hematopoietic differentiation is more complex than the traditional hierarchical model suggests, and that lineage commitment may occur earlier in the hierarchy than previously thought (Notta, 2016; Psaila, 2017). In particular, the relationship between bipotential megakaryocyte-erythroid progenitors (MEP) and common myeloid progenitors (CMP) is unclear in erythro-megakaryopoiesis. While it has been established that mouse MEP and CMP populations contain different proportions of megakaryocytic and erythroid lineage-committed cells, our aim was to determine when lineage-commitment occurs and whether a ‘default’ differentiation pathway exists.We established the transcriptional and epigenetic profiles of 14 flow cytometry-enriched primary murine hematopoietic populations, including hematopoietic stem/progenitor cells (HSPC) and lineage-committed populations. We performed transcriptome profiling (RNASeq), chromatin accessibility profiling (Assay for Transposable-Accessible Chromatin; ATACSeq), and DNA methylation profiling (MethylCSeq). We also performed ChIPSeq of H3K27ac on erythroblasts (EB) and immature megakaryocytes (Mk) to identify putative active enhancer elements.Principle component analysis (PCA) of the RNASeq data demonstrated variation between lineages. Primary cells and cell lines were separated by PC1 (20% of variance) while PC2 (14% of variance) separated HSPC and Mk-committed cells from EB, monocytes and granulocytes. Likewise, PCA of ATACSeq data also indicated that HSPC and Mk shared more similarity than other populations: PC1 (19% of variance) separated EB and non-EB populations while PC2 (12% of variance) separated HSPC and Mk-committed cells from EB, monocytes, granulocytes and lymphocytes (Figure 1). These data demonstrate that the transcriptional and chromatin accessibility profiles of Mk are more similar to HSPC than to other populations.We defined putative active enhancers (pAE) as the intersection of ATACSeq and H3K27ac ChIPSeq peaks in EB and Mk. We identified 2098 EB- and 6386 Mk-specific pAE, and 5989 shared pAE. To predict pAE in progenitors we overlaid EB and Mk pAE with successive ATACSeq peaks in lineage-restricted progenitors (CFU-E, CFU-Mk), followed by CMP and the Lin-Sca+Kit+ population (LSK). 97% and 89% of Mk pAE were detected in CMP and LSK, whereas only 84% and 64% of EB pAE were detected. To validate which EB and Mk pAE in LSK were active (as opposed to poised), we compared our LSK pAE with the indexing-first ChIP histone marks identified by Lara-Astiaso et al., (Science , 2014). We found that 31% of Mk-, but only 13% of EB pAE, were active in the LSK. These data indicate that the MK enhancer profile is established earlier than that of EB.Our comparison of the DNA methylation (CH3) profiles showed that, unlike the RNASeq, ATACSeq, and H3K27ac profiles, erythropoiesis was associated with an 87% decrease in CH3 from CMP and LSK sets; 15% of EB CH3 peaks were established de novo. In contrast, megakaryopoiesis was associated with a 33% increase in CH3 peaks from CMP and LSK sets; 50% of Mk CH3 peaks were established de novo. Many of these de novo methylation sites were associated with genes directing non-Mk lineages that are downregulated compared to CMP (e.g., Dach2, Sox5, Kit, Klf1, Tet1, Fog1).To determine when lineage-commitment occurs we performed single cell RNASeq (10X Genomics platform) on ~9000 cells from each of LSK, CMP, MEP, and GMP (granulocyte-macrophage progenitors) populations. The Seurat software (Sajita, 2015) was used to ‘cluster’ cells with similar expression profiles. MEP contained 5 erythroid clusters of different maturation stages (57% of cells), and 1 cluster with an Mk gene signature (5% of cells). CMP contained, among others, 8 Mk clusters (60% of cells) and 1 cluster (2% of cells) co-expressing Mk and EB genes. LSK contained 4 Mk clusters (26% of cells) but no populations expressing EB-associated genes.We conclude that the transcriptional and epigenetic Mk profile is detectable at the earliest stages of hematopoiesis. The requirement of de novo methylation and silencing of other lineage-associated factors prevents the classification of Mk as a default pathway. In contrast, the EB profile is established after most megakaryocyte progenitor cells have become committed and extensive transcriptional and epigenetic reprogramming is required to permit erythropoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MLL3 Deletion Enhances HSC Self-Renewal

Mixed lineage leukemia 3 (MLL3 ) is one of the most commonly mutated genes in human cancer, and it is believed to function as a tumor suppressor. It is one of many genes affected as a result of mono-allelic 7q deletions that occur commonly in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). MLL3 encodes a histone methyltransferase that catalyzes the monomethylation of Histone 3 lysine 4 (H3K4), a known feature of enhancer elements. MLL3 also nucleates a COMPASS-like complex that removes repressive H3K27me3 marks, recruits the histone acetyltransferase p300/CBP to enhancers and promotes transcript elongation at promoters. For these reasons, MLL3 binding at cis-regulatory elements is thought to potentiate enhancer activity and transcription. Previous studies have shown that Mll3 deficiency causes a mild myeloproliferative disorder in mice (PLoS ONE 11:e0162515), and shRNA knockdown of Mll3 has been shown to cooperate with loss of Nf1 and p53 to promote AML (Cancer Cell 25:652). In the latter study, Mll3 silencing was associated with a hematopoietic stem cell (HSC)-like gene expression signature within the AML cells. These studies implicate MLL3 as a hematopoietic tumor suppressor, though its precise function in HSC self-renewal and lineage commitment has not been described.To better understand how Mll3 regulates HSC self-renewal, we developed a novel loss-of-function mouse allele in which a 5-base pair deletion and frameshift mutation was created near the N-terminus of the protein. Homozygous Mll3-/- mice are viable through most of gestation, but they die just prior to birth. Heterozygous Mll3+/- mice are born at normal Mendelian ratios, their development is normal after birth and they are fertile. To test whether Mll3 regulates hematopoiesis, we determined the frequencies and absolute numbers of HSCs, lineage restricted hematopoietic progenitors (HPCs), and granulocyte-monocyte progenitors (GMPs) in control, Mll3+/- and Mll3-/- fetal livers (E18.5) and control and Mll3+/- adult bone marrow. Heterozygous and homozygous Mll3 deletion caused a modest increase in HSC numbers and a modest decrease in HPC frequency in fetal livers. Mll3 heterozygosity resulted in a significant expansion of the HSC population in adult bone marrow. To test whether these increases in HSC numbers reflected enhanced self-renewal, we performed serial transplantation assays with fetal and adult HSCs. While primary transplants showed only a modest increase in repopulating activity in Mll3+/- and Mll3-/- HSCs, this effect was greatly enhanced in secondary transplants. Thus, Mll3 deficiency enhances HSC self-renewal capacity. Despite this phenotype, RNAseq showed no differences in the transcriptomes of wild type and Mll3+/- HSCs or HPCs. This suggests that Mll3 loss may reduce the probability of lineage commitment during successive HSC divisions without altering the commitment pathways themselves. Ongoing experiments will test this hypothesis. Enhanced self-renewal, particularly under conditions of hematopoietic stress, may convey a selective advantage to Mll3 -heterozygous HSCs and enhance progression to MDS. DisclosuresNo relevant conflicts of interest to declare.

Fetal and Neonatal Hematopoietic Progenitors Are Exquisitely Sensitive to MLL-ENL-Driven Leukemia Initiation

Translocations involving the MLL1/KMT2A gene are observed in both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), but the frequency at which these mutations occurs is different at different stages of life. For example, MLL rearrangements are extremely common in infant leukemia (~70% of cases), but they are much less common in adolescent or adult AML (~5% of de novo adult AML). While the function of MLL fusion proteins has been extensively studied, it remains unclear why MLL-rearrangements are so common in infant leukemias as compared to adolescent or adult leukemias. One possibility is that fetal hematopoietic progenitors are more readily transformed by MLL fusion proteins than adult progenitors. Recent work in our lab has shown that a mutation commonly associated with adult AML, the FLT3-Internal Tandem Duplication, selectively activates leukemogenic transcriptional programs in adult as compared to fetal/neonatal progenitors (eLife 5:e18882). We have developed a system to test whether an analogous effect holds for MLL translocations - i.e. do they preferentially initiate leukemogenesis in fetal progenitors. We made use of a previously described tet-O doxycycline inducible mouse allele (Cell Reports 9:1246) to both spatially and temporally control expression of MLL-ENL, a common MLL fusion product (Fig. 1A). We found that MLL-ENL induction at fetal (E10.5) or neonatal (P0) stages caused AML to develop in most mice, but MLL-ENL induction at juvenile (P21) or adult (8 weeks) stages did not (Fig. 1B). Remarkably, AML developed with the greatest penetrance (100%) when MLL-ENL was induced at P0 as compared to both earlier and later developmental stages. In neonates, MLL-ENL profoundly depleted hematopoietic stem cells (HSCs) and, to a lesser extent, lineage-restricted hematopoietic progenitors (HPCs), and it caused expansion of myeloid progenitor populations. These effects began to dissipate by 10 weeks after birth. Changes in HSC and HPC frequency were not at all evident when MLL-ENL was induced at an adult stage, but by a year after MLL-ENL induction, the surviving mice had a significantly expanded HSC pool. MLL-ENL induced widespread changes in gene expression in neonatal HPCs. In contrast, very few changes in gene expression were observed in MLL-ENL expressing adult HPCs. Known targets of MLL-ENL, such as HoxA9, were more highly induced in neonatal HPCs than in adult HPCs, though expression was observed in older mice. Altogether, our data demonstrate a dynamic relationship between developmental stage and the transcriptional response to MLL-ENL, and they may help account for the high percentage of MLL rearrangements observed in infant leukemia. Furthermore, our findings have opened new lines of investigation into the relationship between the epigenetic landscapes of developing HSCs/HPCs and the programs that drive leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

SummaryAge-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

Heterogeneous subpopulations of adventitial progenitor cells regulate vascular homeostasis and pathological vascular remodelling.

Cardiovascular diseases are characterized by chronic vascular dysfunction and provoke pathological remodelling events, such as neointima formation, atherosclerotic lesion development, and adventitial fibrosis. While lineage-tracing studies have shown that phenotypically modulated smooth muscle cells (SMCs) are the major cellular component of neointimal lesions, the cellular origins and microenvironmental signalling mechanisms that underlie remodelling along the adventitial vascular layer are not fully understood. However, a growing body of evidence supports a unique population of adventitial lineage-restricted progenitor cells expressing the stem cell marker, stem cell antigen-1 (Sca1; AdvSca1 cells) as important effectors of adventitial remodelling and suggests that they are at least partially responsible for subsequent pathological changes that occur in the media and intima. AdvSca1 cells are being studied in murine models of atherosclerosis, perivascular fibrosis, and neointima formation in response to acute vascular injury. Depending on the experimental conditions, AdvSca1 cells exhibit the capacity to differentiate into SMCs, endothelial cells, chondrocytes, adipocytes, and pro-remodelling cells, such as myofibroblasts and macrophages. These data indicate that AdvSca1 cells may be a targetable cell population to influence the outcomes of pathologic vascular remodelling. Important questions remain regarding the origins of AdvSca1 cells and the essential signalling mechanisms and microenvironmental factors that regulate both maintenance of their stem-like, progenitor phenotype and their differentiation into lineage-specified cell types. Adding complexity to the story, recent data indicate that the collective population of adventitial progenitor cells is likely composed of several smaller, lineage-restricted subpopulations, which are not fully defined by their transcriptomic profile and differentiation capabilities. The aim of this review is to outline the heterogeneity of Sca1+ adventitial progenitor cells, summarize their role in vascular homeostasis and remodelling, and comment on their translational relevance in humans.