Hematopoietic Stem Cells Heterogeneity Research Articles

Aging is associated with functional and molecular changes in distinct hematopoietic stem cell subsets

Age is a risk factor for hematologic malignancies. Attributes of the aging hematopoietic system include increased myelopoiesis, impaired adaptive immunity, and a functional decline of the hematopoietic stem cells (HSCs) that maintain hematopoiesis. Changes in the composition of diverse HSC subsets have been suggested to be responsible for age-related alterations, however, the underlying regulatory mechanisms are incompletely understood in the context of HSC heterogeneity. In this study, we investigated how distinct HSC subsets, separated by CD49b, functionally and molecularly change their behavior with age. We demonstrate that the lineage differentiation of both lymphoid-biased and myeloid-biased HSC subsets progressively shifts to a higher myeloid cellular output during aging. In parallel, we show that HSCs selectively undergo age-dependent gene expression and gene regulatory changes in a progressive manner, which is initiated already in the juvenile stage. Overall, our studies suggest that aging intrinsically alters both cellular and molecular properties of HSCs.

Hematopoietic stem cell heterogeneity and age-associated platelet bias are evolutionarily conserved.

Hematopoietic stem cells (HSCs) reconstitute multilineage human hematopoiesis after clinical bone marrow (BM) transplantation and are the cells of origin of some hematological malignancies. Although HSCs provide multilineage engraftment, individual murine HSCs are lineage biased and contribute unequally to blood cell lineages. Here, we performed high-throughput single-cell RNA sequencing in mice after xenograft with molecularly barcoded adult human BM HSCs. We demonstrated that human individual BM HSCs are also functionally and transcriptionally lineage biased. Specifically, we identified platelet-biased and multilineage human HSCs. Quantitative comparison of transcriptomes from single HSCs from young and aged BM showed that both the proportion of platelet-biased HSCs and their level of transcriptional platelet priming increase with age. Therefore, platelet-biased HSCs and their increased prevalence and transcriptional platelet priming during aging are conserved features of mammalian evolution.

Hematopoietic stem cell heterogeneity in non-human primates revealed by five-lineage output bias analysis.

Understanding hematopoietic stem cell (HSC) heterogeneity is crucial for treating malignant blood disorders. Compared with mice, we have limited knowledge of the heterogeneity of human HSCs. Fortunately, non-human primates (NHPs) have become the best animal models for studying human HSCs. Here, we employed a public dataset derived from NHP autologous bone marrow transplantation, and focused on a total of 820 HSC clones with reconstitution capacity of all available five lineages (granulocyte, monocyte, B cell, T cell, and natural killer cell) at two time points (11/12 and/or 42/43 months). Intriguingly, unsupervised clustering on these clones revealed six HSC subtypes, including a lymphoid/myeloid balanced (LM-balanced) subtype and five single-lineage-biased subtypes. We also observed that the subtypes of these HSC clones might change over time, and a given subtype could transition into any one of the other five subtypes, albeit with a certain degree of selectivity. Particularly, each of the six subtypes was more likely to turn into lymphoid-biased rather than myeloid-biased ones. Additionally, our five-lineage classification method exhibited strong correlation with traditional lymphoid/myeloid bias classification method. Specifically, our granulocyte- and monocyte-biased subtypes were predominantly attributed to α-HSCs, while LM-balanced, B cell-biased, and T cell-biased subtypes were primarily associated with β-HSCs. The γ-HSCs were composed of a small subset of B cell-biased and T cell-biased subtypes. In summary, our five-lineage classification identifies more finely tuned HSC subtypes based on lineage output bias. These findings enrich our understanding of HSC heterogeneity in NHPs and provide important insights for human research.

Deciphering the metabolic heterogeneity of hematopoietic stem cells with single-cell resolution

Metabolic status is crucial for stem cell functions; however, the metabolic heterogeneity of endogenous stem cells has never been directly assessed. Here, we develop a platform for high-throughput single-cell metabolomics (hi-scMet) of hematopoietic stem cells (HSCs). By combining flow cytometric isolation and nanoparticle-enhanced laser desorption/ionization mass spectrometry, we routinely detected >100 features from single cells. We mapped the single-cell metabolomes of all hematopoietic cell populations and HSC subpopulations with different division times, detecting 33 features whose levels exhibited trending changes during HSC proliferation. We found progressive activation of the oxidative pentose phosphate pathway (OxiPPP) from dormant to active HSCs. Genetic or pharmacological interference with OxiPPP increased reactive oxygen species level in HSCs, reducing HSC self-renewal upon oxidative stress. Together, our work uncovers the metabolic dynamics during HSC proliferation, reveals a role of OxiPPP for HSC activation, and illustrates the utility of hi-scMet in dissecting metabolic heterogeneity of immunophenotypically defined cell populations.

SPI1-KLF1/LYL1 Axis Regulates Lineage Commitment during Human Endothelial-to-Hematopoietic Transition

PU.1 ( SPI1) is a crucial transcription factor in hematopoiesis, yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. Given that the complexity of in vivo hematopoiesis can mask subtle cellular transitions, we sought to investigate the function of SPI1 in both complex in vivo environments and more controlled in vitro models of EHT to elucidate its precise role. By comparing the human in vivo and in vitro EHT transcriptomes, we observed similarities in the transcriptional profiles of SPI1, suggesting its regulatory role in EHT. Knocking down SPI1 in in vitro-generated hemogenic endothelial cells (HECs) led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis, we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells, respectively - as downstream targets ofSPI1. Concomitant overexpression of KLF1 or LYL1 with SPI1 knockdown in in vitro-generated HECs partially rescues the reduction in HPC formation induced by SPI1 knockdown. Specifically, KLF1 overexpression restores myeloid lineage potential, while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1- LYL1 axis in the regulatory network in in vivo EHT. Taken together, our findings shed new light on the role of SPI1 in regulating lineage commitment during EHT, potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs).

Investigation of CD83 As a Novel Human Hematopoietic Stem Cell Inflammatory Activation Marker

Hematopoietic stem cells (HSC) are responsible for life-long blood production and must balance homeostasis with sudden demand situations such as infections. However, the heterogeneity of molecular programs engaged by acute inflammatory stimuli to activate emergency hematopoiesis at the level of HSC are poorly understood. In a recent study linking human HSC heterogeneity to aging, recovery from severe COVID-19 infection, and clonal hematopoiesis (CH), we discovered two HSC subsets through multiomic analysis, denoted HSC-I and -II, in a xenograft model of inflammation-recovery. While HSC-I had few inflammation-driven molecular changes, HSC-II showed sustained epigenetic and transcriptional alterations in response to dual tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS) challenges despite a 2.5-month recovery period. Importantly, a signature of long-term memory T cells (Akondy et al 2017) was enriched in HSC-II (Zeng, Nagree, et al, in submission). We interrogated the human HSC multiomic signatures uncovered from this human xenograft model to identify potential regulators associated with HSC response to and recovery from acute inflammation. We identified CD83 as a candidate that is differentially expressed between our HSC subsets. Similarly, we found CD83 expression to be enriched in a subset of adult-derived HSCs from patients where CH status was established and enrichment of an HSC-II signature observed (Jakobsen et al, submitted). CD83, first identified as a dendritic cell marker, is expressed on many immune cells, with linkage to controlling murine T regulatory (T-Reg) vs memory cell fate (Doebbeler et al, 2018), but has not previously been shown to be relevant for human HSC biology. Flow cytometry analysis of cord blood (CB) hematopoietic stem and progenitor cells (HSPC) showed CD83 to be low or absent in HSC (Figure A) and their downstream progeny at steady state. However, robust CD83 expression was observed in multiple HSPC subpopulations including long-term (LT-) and short-term (ST-) HSC within 16 hours of ex vivo culture, with highest expression in LT-HSC (Figure A). Importantly, CD83 surface expression on LT-HSC was enhanced following culture with TNFα (Figure A). Bystander effects were excluded by demonstrating similar findings after culture of purified HSC populations. Acute in vivo LPS treatment of mice xenografted with CB-derived HSC was sufficient to upregulate CD83 surface expression after 16 hours on LT-HSC/HSPC. To elucidate the potential function of CD83-expressing HSC/multipotent progenitors (MPP), CD19-CD34+CD38-CD45RA- cells were sorted from xenografts following acute inflammatory challenge for low and high CD83 expression and subjected to bulk RNA sequencing. We found enrichment of a gene expression signature specific to activated human HSCs (Garcia-Prat et al, 2021) within CD83-positive HSC/MPP (Figure B). Strikingly, enrichment of a signature of stimulated vs resting T cells (Galetti et al, 2020) and of effector T cells (Akondy et al 2017) was found in CD83-positive compared to CD83-negative HSC/MPP (Figure B). T cell effector function is in part dictated by metabolic reprogramming. We thus examined reactive oxygen species (ROS) levels in LT-HSC following ex vivo culture with TNFα and found lower ROS levels with CD83 expression. We therefore hypothesize that CD83 may be an ‘effector’ marker defining a distinct HSC subset during homeostatic or inflammatory activation that is subsequently fated to become memory-like HSC-II. We are undertaking studies to determine the mechanistic role of CD83 in HSC function and HSC-II formation via Cas9-sgRNA-mediated knockout or lentivirus-mediated overexpression of CD83. In summary, CD83 represents a long-sought marker of inflammatory activation at the HSC level. CD83 may have significant clinical potential as a biomarker of aberrant activation in gene therapy manufacturing protocols or for HSC transplantation. Future studies will shed light on whether CD83 can functionally regulate the molecular programs underlying human HSC heterogeneity.

Activation of lineage competence in hemogenic endothelium precedes the formation of hematopoietic stem cell heterogeneity.

Hematopoietic stem and progenitor cells (HSPCs) are considered as a heterogeneous population, but precisely when, where and how HSPC heterogeneity arises remain largely unclear. Here, using a combination of single-cell multi-omics, lineage tracing and functional assays, we show that embryonic HSPCs originate from heterogeneous hemogenic endothelial cells (HECs) during zebrafish embryogenesis. Integrated single-cell transcriptome and chromatin accessibility analysis demonstrates transcriptional heterogeneity and regulatory programs that prime lymphoid/myeloid fates at the HEC level. Importantly, spi2+ HECs give rise to lymphoid/myeloid-primed HSPCs (L/M-HSPCs) and display a stress-responsive function under acute inflammation. Moreover,we uncover that Spi2 is required for the formation of L/M-HSPCs through tightly controlling the endothelial-to-hematopoietic transition program. Finally, single-celltranscriptional comparison of zebrafish and human HECs andhuman induced pluripotent stem cell-based hematopoietic differentiationresults support the evolutionary conservation of L/M-HECs and a conserved role of SPI1 (spi2 homolog in mammals) in humans. These results unveil the lineage origin, biological function and molecular determinant of HSPC heterogeneity and lay the foundation for new strategies for induction of transplantable lineage-primed HSPCs in vitro.

Hematopoietic Stem Cells and Their Bone Marrow Niches.

Hematopoietic stem cells (HSCs) are maintained in the bone marrow microenvironment, also known as the niche, that regulates their proliferation, self-renewal, and differentiation. In this chapter, we will introduce the history of HSC niche research and review the interdependencies between HSCs and their niches. We will further highlight recent advances in our understanding of HSC heterogeneity with regard to HSC subpopulations and their interacting cellular and molecular bone marrow niche constituents.

Study of the Dynamics and Diversity of Hematopoietic Stem Cells during Aging and Senolytic Drug Treatment Using Single Cell Transcriptomics

During aging, the heterogeneity and function of hematopoietic stem cells (HSCs) decline dramatically, contributing to a wide array of age-associated deficiencies and diseases such as anemia, leukemia, and immune system deterioration. Using a genetically engineered mouse model that enables inducible elimination of senescent cells, we have previously demonstrated that their clearance restored the repopulation capacity of aged HSCs. In this study, we use a new generation senolytic drug, 753b, to target senescent cells in old (100 weeks) mice to further dissect the link between senescence and HSCs aging. A short-term treatment (8 doses) achieved efficient depletion of senescent cells as seen by decreased expression of p16Cdkn2a in the liver tissue from 753b-treated group compared to vehicle control. To gain a high-resolution insight into the molecular physiology of young and aged HSCs before and after senolytic treatment, we analyzed 34,000 single-cell HSC transcriptomes using 10X Genomics Chromium platform. Unsupervised clustering analysis using Seurat algorithm on sorted Lin-Sca1+cKit+ (LSK) CD150+ cells from young, aged-vehicle, and aged-drug groups identified 19 distinct clusters. We further grouped these clusters into lineage non-primed (6 clusters), intermediate (5 clusters), and lineage-primed (8 clusters) subgroups (Fig. 1A) using a pipeline developed by Hérault et al. (BMC Biology, 2021). Consistent with previous observations, aged HSCs were characterized by a significant expansion of non-primed cell subpopulations and a reduction of primed cells. Remarkably, a short 8-dose course of 753b senolytic drug treatment largely reversed aging-related changes in HSCs distribution-proportion of non-primed HSCs decreased, while dividing cells in intermediate HSC clusters increased along with slight elevation within primed HSCs (Fig. 1A). Interestingly, cell cycle analysis showed cells within intermediate and primed stages present in a similar cycling pattern across young, aged, and drug treated groups. Yet, among aged HSCs more cells were locked in a less proliferative early non-primed state, with fewer cells transitioning into lineage-primed state. Excitingly, short term 753b treatment partially restored cell cycling in non-primed clusters in aged mice, suggesting possible rejuvenation of HSCs. We further compared differentially expressed genes between young and aged groups in non-primed clusters and found enrichment of pathways related to programmed cell death and cell differentiation. Senescence score generated based on this gene list by Seurat algorithm showed normalization of the aging-related gene signature upon 753b treatment, especially in non-primed HSCs (Fig. 1B). Our study explores the transcriptomic heterogeneity and dynamics of HSCs during aging and after senolytic drug treatment at a single cell level and highlights potential mechanisms of senolytic drug-mediated hematologic recovery. The senescence score we generated can be further evaluated as a biomarker of aging of HSCs. Figure 1. (A) UMAP showing the distribution of HSC cells among 19 clusters. Clusters were sub grouped into non-primed, intermediate, and primed groups. Cell proportions in each cluster were shown in bar graph from the indicated groups. (B) Senescence scores were calculated based on gene expression of top elevated genes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dysfunctional Clusterin-Positive Hematopoietic Stem Cells Expand with Aging

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes dysregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old), and aged (20-month-old) mice to gain insight into the dynamics of HSC aging. scRNA-seq data revealed that transcriptomic profiles of HSCs change with aging, although middle-aged and aged HSCs showed very similar profiles to each other. scRNA-seq data divided young and aged HSCs into 2 and 3 clusters, respectively. While both young and aged HSCs had clusters with myeloid or lymphoid gene signature, the cluster with myeloid signature dominated in aged HSCs, in which aged gene signature (Nupr1, Mt1, Mt2, Selp) was highly enriched. Clusterin (Clu) was also significantly up-regulated in this cluster. Clu encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis (Itakura et al., JCB 2020). We hypothesized that Clu would be useful as a marker of aged HSCs, and conducted further analysis using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory elements of the Clu gene. Bone marrow (BM) analysis of Clu-reporter mice revealed that Clu expression is confined to a part of HSCs in BM and significantly up-regulated within the HSC compartment with aging (10 and 70 % of HSCs are Clu-positive in young and middle-aged mice, respectively). In competitive repopulation assays, Clu-positive HSCs showed severely impaired repopulation capacity and preferentially differentiated into myeloid cells while Clu-negative HSCs largely retained repopulation capacity and showed balanced differentiation. We analyzed the transcriptomic profile of Clu-positive HSCs with aging. RNA-sequence analysis revealed that young and middle-aged Clu-positive HSCs were significantly enriched for aged HSC gene signature and myeloid-biased HSC signature compared to Clu-negative HSCs. We evaluated the transcriptional regulation mechanism of Clu by Assay for Transposase-Accessible Chromatin sequence (ATAC-seq) analysis. Chromatin accessibility at the Clu promoter was already open in young HSCs, and largely enhanced in Clu-positive HSCs but not in Clu-negative HSCs among middle-aged HSCs. The open chromatin region at the Clu promoter had binding motifs of AP-1 and STAT family transcription factors, suggesting that external signals or stresses activate Clu expression. These results suggest that Clu-positive cells represent myeloid-biased HSCs which expand with aging and that Clu serves as a faithful marker to trace alterations in HSC heterogeneity with aging.

Single Cell Transcriptomics to Understand HSC Heterogeneity and Its Evolution upon Aging.

Single-cell transcriptomic technologies enable the uncovering and characterization of cellular heterogeneity and pave the way for studies aiming at understanding the origin and consequences of it. The hematopoietic system is in essence a very well adapted model system to benefit from this technological advance because it is characterized by different cellular states. Each cellular state, and its interconnection, may be defined by a specific location in the global transcriptional landscape sustained by a complex regulatory network. This transcriptomic signature is not fixed and evolved over time to give rise to less efficient hematopoietic stem cells (HSC), leading to a well-documented hematopoietic aging. Here, we review the advance of single-cell transcriptomic approaches for the understanding of HSC heterogeneity to grasp HSC deregulations upon aging. We also discuss the new bioinformatics tools developed for the analysis of the resulting large and complex datasets. Finally, since hematopoiesis is driven by fine-tuned and complex networks that must be interconnected to each other, we highlight how mathematical modeling is beneficial for doing such interconnection between multilayered information and to predict how HSC behave while aging.

STAT1 is essential for HSC function and maintains MHCIIhi stem cells that resist myeloablation and neoplastic expansion

AbstractAdult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here, we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs that protect HSCs, including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated major histocompatibility complex class II (MHCII) expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis and was refractory to 5-fluorouracil–induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field.

CD49b identifies functionally and epigenetically distinct subsets of lineage-biased hematopoietic stem cells.

SummaryHematopoiesis is maintained by functionally diverse lineage-biased hematopoietic stem cells (HSCs). The functional significance of HSC heterogeneity and the regulatory mechanisms underlying lineage bias are not well understood. However, absolute purification of HSC subtypes with a pre-determined behavior remains challenging, highlighting the importance of continued efforts toward prospective isolation of homogeneous HSC subsets. In this study, we demonstrate that CD49b subdivides the most primitive HSC compartment into functionally distinct subtypes: CD49b− HSCs are highly enriched for myeloid-biased and the most durable cells, while CD49b+ HSCs are enriched for multipotent cells with lymphoid bias and reduced self-renewal ability. We further demonstrate considerable transcriptional similarities between CD49b− and CD49b+ HSCs but distinct differences in chromatin accessibility. Our studies highlight the diversity of HSC functional behaviors and provide insights into the molecular regulation of HSC heterogeneity through transcriptional and epigenetic mechanisms.

A CRISPR view of hematopoietic stem cells: Moving innovative bioengineering into the clinic.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome engineering has emerged as a powerful tool to modify precise genomic sequences with unparalleled accuracy and efficiency. Major advances in CRISPR technologies over the last 5 years have fueled the development of novel techniques in hematopoiesis research to interrogate the complexities of hematopoietic stem cell (HSC) biology. In particular, high throughput CRISPR based screens using various “flavors” of Cas coupled with sequencing and/or functional outputs are becoming increasingly efficient and accessible. In this review, we discuss recent achievements in CRISPR‐mediated genomic engineering and how these new tools have advanced the understanding of HSC heterogeneity and function throughout life. Additionally, we highlight how these techniques can be used to answer previously inaccessible questions and the challenges to implement them. Finally, we focus on their translational potential to both model and treat hematological diseases in the clinic.

High Throughput Multi‐omic Characterization of Clonal Hematopoietic Stem Cells During Progression of Myeloid Malignancies

Hematopoietic stem cells (HSCs) are responsible for giving rise to all other lineages of blood cells in the body. Over time, mutations in HSCs can promote the outgrowth of clonal populations that outcompete other HSCs, resulting in a phenomenon called Clonal Hematopoiesis of Indeterminate Potential (CHIP). Though not cancerous in and of itself, CHIP can progress to more serious hematologic disorders, such as the Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). The mechanisms of clonal expansion, by which certain mutant HSCs acquire a competitive advantage over other HSCs, currently remain largely undeciphered, as are the mechanisms by which clonal HSCs drive the initiation of MDS and contribute to the development of AML. Moreover, previous studies have shown that such mutant HSCs are resistant to conventional therapies and may act as reservoirs for disease relapse and progression. In the past, researchers were hindered by bulk cell analyses and the relative rarity of HSCs, but advances in single‐cell omics have now enabled us to explore the molecular heterogeneity of clonal HSCs and identify distinct clonal populations based on genotype and cell surface phenotype. We used single cell RNA sequencing to examine the transcriptional dynamics of purified HSCs from MDS and AML patients before and after treatment, as well as from age matched elderly controls. Interestingly, dimensionality reduction methods such as UMAP and tSNE revealed a reservoir of control HSCs that clustered with MDS HSCs. Upon comparison with other normal control HSCs, we found that genes associated with aging, mitochondrial function, and particular ion channels were strongly upregulated in these “MDS‐like” control HSCs, while genes involved in ribosomal and translation activity, along with certain surface markers, were substantially downregulated. Additionally, ribosomal transcripts were depleted in MDS HSCs from patients who did not respond to treatment. These results support the notion that the most immature HSCs, which impose the strictest constraints on translation, might clonally expand and initiate CHIP and/or MDS upon acquiring driver mutations, also serving as treatment resistant populations that underlie disease relapse. We also subjected purified MDS HSCs to simultaneous single cell targeted DNA sequencing and cell surface phenotyping, which allowed us to correlate the cell surface phenotype and genotype for specific clonal populations and identify cell surface markers that can be used to isolate HSCs with enhanced engraftment ability. Taken together, these results indicate that combined single cell genotyping and phenotyping can be used to track clonal populations across different stages of pathogenesis, providing further insight about the development of CHIP and its progression to MDS and AML.

Unraveling Heterogeneity of Aged Hematopoietic Stem Cells By Single-Cell RNA Sequence Analysis

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes deregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old) and aged (20-month-old) mice to gain insight into the dynamics of HSC aging.scRNA-seq analysis revealed three major clusters (A, B, C) and three minor clusters (D, E, F) in young HSCs. Of interest, aged HSCs also showed similar cluster formation. One of the minor clusters was characterized by gene signature associated with inflammatory response and significantly increased with age, while the remaining two minor clusters, which showed cell cycle gene signature, did not change in proportion. One of the major clusters, Cluster C, which showed the strongest expression of HSC-specific gene sets compared with other clusters, moderately increased with age.Our scRNA-seq analysis confirmed upregulation of age-related genes previously reported, such as Selp, Mt1, and Vwf, in a considerable portion of aged HSCs. von Willebrand factor (Vwf) encodes a blood glycoprotein produced by megakaryocytes and endothelial cells. Several groups have reported that Vwf-expressing HSCs show myeloid/platelet-biased differentiation (Joana C et al., Nature 2013; Sandra P et al., Developmental Cell 2018). Importantly, our scRNA-seq data identified that Clusterin (Clu) is rarely expressed in young HSCs and is dramatically upregulated in aged HSCs. Clu expression was predominantly increased in one of the major clusters, Cluster C. Clusterin encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis. We hypothesized that Clu would be useful as a marker of a unique subpopulation of aged HSCs, and thus conducted further analysis by using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory sequences of the BAC gene.Clu/GFP was preferentially expressed in HSCs and at lower frequencies in MPP1 than HSCs in Lineage -Sca-1 +c-Kit + (LSK) cell fraction. Clu-positive HSCs expressed high levels of CD150 and were detected in 10% and 60% of HSCs in 10-week-old young mice and 8-month-old middle-aged mice, respectively, indicating that Clu-positive HSCs increase with aging. We next assessed the function of Clu-positive HSCs in young mice. Clu-positive young HSCs established significantly lower chimerism than Clu-negative young HSCs and preferentially differentiated into myeloid cells in competitive transplantation assays. RNA-seq analysis of Clu-positive and Clu-negative HSCs from young mice confirmed that Clu-positive HSCs show the gene signature of myeloid-biased HSCs. Characterization of Clu-positive and negative subpopulations in aged HSCs is currently underway. These results suggest that Clu-positive HSCs represent myeloid-biased HSCs which expand with aging, thus Clu expression serves as a novel marker to monitor the alterations in HSC heterogeneity with aging. DisclosuresIwama: Nissan Chemical Corporation: Research Funding.

Definitive Hematopoietic Stem Cells Minimally Contribute to Embryonic Hematopoiesis

Definitive hematopoietic stem cells (HSCs) emerge in the embryo and sustain major adult hematopoietic lineages. Although their functional potential is detected by transplant, nascent HSC contribution during development is both unknown and difficult to address due to the overlapping emergence of HSC-independent progenitors-cells that lack the multipotency and/or longevity of HSCs but express many of the same markers. Using sorted hematopoietic stem and progenitor cells from zebrafish embryos, we performed single cell RNA sequencing to decipher HSC and HSC-independent progenitor heterogeneity during the time frame of their emergence and initial maturation. After batch correction and dimensional reduction, we identified seven distinct populations that are inferred from RNA velocity analysis to originate from pre-hemogenic endothelium and develop into three main differentiation trajectories. We also determined that HSCs can be distinguished from HSC-independent progenitors based on the temporal regulation and differential activity of the draculin (drl) promoter that was previously shown to mark adult-contributing HSCs. From these studies, we found that the drl promoter is active in HSCs and HSC-independent progenitors at 1-day post-fertilization (dpf) but becomes highly expressed primarily in HSCs by 2 dpf. We applied a drl:cre-ER T2 tamoxifen-inducible Cre-loxP lineage-tracing approach to selectively lineage trace HSCs starting at 2 dpf and track their myeloid and lymphoid contribution during larval development and adulthood. We determined that HSC-independent progenitors primarily contribute to developmental lymphomyelopoiesis with minimal HSC contribution until after 7 dpf. Consistent with this result, we demonstrated that although HSCs robustly regenerated after hematopoietic injury using a novel inducible larval HSC injury model, their depletion had almost no impact on lymphoid and myeloid cell numbers up to 7 dpf. These findings suggest that HSCs are not entirely dormant during development and that there exists an uncoupling of HSC self-renewal and differentiation in development. In conclusion, we determine that it is the HSC-independent progenitors, and not HSCs, that sustain embryonic and early larval lymphomyelopoiesis. Acquiring a greater understanding regarding developmental differences in progenitor and HSC specification and maturation will inform and improve the generation of functional HSCs from renewable pluripotent stem cells. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Stem Cell Mobilization:Current Collection Approaches, Stem Cell Heterogeneity, anda Proposed New Method for Stem Cell Transplant Conditioning.

Hematopoietic stem cells naturally traffic out of their bone marrow niches into the peripheral blood. This natural trafficking process can be enhanced with numerous pharmacologic agents - a process termed "mobilization" - and the mobilized stem cells can be collected for transplantation. We review the current state of mobilization with an update on recent clinical trials and new biologic mechanisms regulating stem cell trafficking. We propose that hematopoietic mobilization can be used to answer questions regarding hematopoietic stem cell heterogeneity, can be used for non-toxic conditioning of patients receiving stem cell transplants, and can enhance gene editing and gene therapy strategies to cure genetic diseases.

Modeling of human T cell development in vitro as a read-out for hematopoietic stem cell multipotency.

Hematopoietic stem cells (HSCs) reside in distinct sites throughout fetal and adult life and give rise to all cells of the hematopoietic system. Because of their multipotency, HSCs are capable of curing a wide variety of blood disorders through hematopoietic stem cell transplantation (HSCT). However, due to HSC heterogeneity, site-specific ontogeny and current limitations in generating and expanding HSCs in vitro, their broad use in clinical practice remains challenging. To assess HSC multipotency, evaluation of their capacity to generate T lymphocytes has been regarded as a valid read-out. Several in vitro models of T cell development have been established which are able to induce T-lineage differentiation from different hematopoietic precursors, although with variable efficiency. Here, we review the potential of human HSCs from various sources to generate T-lineage cells using these different models in order to address the use of both HSCs and T cell precursors in the clinic.

CD63 acts as a functional marker in maintaining hematopoietic stem cell quiescence through supporting TGFβ signaling in mice.

Hematopoietic stem cell (HSC) fate is tightly controlled by various regulators, whereas the underlying mechanism has not been fully uncovered due to the high heterogeneity of these populations. In this study, we identify tetraspanin CD63 as a novel functional marker of HSCs in mice. We show that CD63 is unevenly expressed on the cell surface in HSC populations. Importantly, HSCs with high CD63 expression (CD63hi) are more quiescent and have more robust self-renewal and myeloid differentiation abilities than those with negative/low CD63 expression (CD63-/lo). On the other hand, using CD63 knockout mice, we find that loss of CD63 leads to reduced HSC numbers in the bone marrow. In addition, CD63-deficient HSCs exhibit impaired quiescence and long-term repopulating capacity, accompanied by increased sensitivity to irradiation and 5-fluorouracil treatment. Further investigations demonstrate that CD63 is required to sustain TGFβ signaling activity through its interaction with TGFβ receptors I and II, thereby playing an important role in regulating the quiescence of HSCs. Collectively, our data not only reveal a previously unrecognized role of CD63 but also provide us with new insights into HSC heterogeneity.