Native Hematopoiesis Research Articles

Dynamic Tracking of Native Precursors in Adult Mice.

Hematopoietic dysfunction has been associated with a reduction in the number of active precursors. However, precursor quantification at homeostasis and under diseased conditions is constrained by the scarcity of available methods. To address this issue, we optimized a method for quantifying a wide range of hematopoietic precursors. Assuming the random induction of a stable label in precursors following a binomial distribution, estimates depend on the inverse correlation between precursor numbers and the variance of precursor labeling among independent samples. Experimentally validated to cover the full dynamic range of hematopoietic precursors in mice (1 to 105), we utilized this approach to demonstrate that thousands of precursors, which emerge after modest expansion during fetal-to-adult transition, contribute to native and perturbed hematopoiesis. We further estimated the number of precursors in a mouse model of Fanconi Anemia, showcasing how repopulation deficits can be classified as autologous (cell proliferation) and non-autologous (lack of precursor). Our results support an accessible and reliable approach for precursor quantification, emphasizing the contemporary perspective that native hematopoiesis is highly polyclonal.

Temporal multimodal single-cell profiling of native hematopoiesis illuminates altered differentiation trajectories with age

Aging negatively affects hematopoiesis, with consequences for immunity and acquired blood cell disorders. Although impairments in hematopoietic stem cell (HSC) function contribute to this, the invivo dynamics of such changes remain obscure. Here, we integrate extensive longitudinal functional assessments of HSC-specific lineage tracing with single-cell transcriptome and epitope profiling. In contrast to recent suggestions from single-cell RNA sequencing alone, our data favor a defined structure of HSC/progenitor differentiation that deviates substantially from HSC-derived hematopoiesis following transplantation. Native age-dependent attrition in HSC differentiation manifests as drastically reduced lymphoid output through an early lymphoid-primed progenitor (MPP Ly-I). While invitro activation fails to rescue lymphoid differentiation from most aged HSCs, robust lymphopoiesis can be achieved by culturing elevated numbers of candidate HSCs. Therefore, our data position rare chronologically aged HSC clones, fully competent at producing lymphoid offspring, as a prime target for approaches aimed to improve lymphopoiesis in the elderly.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Hematopoietic Flt3+ Multipotent Progenitors Are Organized in Discrete Functionally and Hierarchically Distinct Populations

Blood production in the bone marrow (BM) is classically viewed as a constant hematopoietic flux originating from a small pool of self-renewing hematopoietic stem cells (HSCs). In this model, quiescent HSCs regularly differentiate and give rise to a complex set of transient multipotent progenitors (MPPs) that serve as compartments of amplification through division, while undergoing progressive lineage specification Recent studies exploiting sophisticated genetic marking for in vivo cell-fate tracking have challenged this paradigm and suggested that HSCs have a limited contribution to steady state blood production (Sun, Nature 2014, Busch, Nature 2015; Rodriguez-Fraticelli, Nature 2018). The interpretation of these results and the extent of the HSC contribution to native hematopoiesis remains a matter of debate (Pucella, Annu Rev Cell Dev Biol 2020; Rodriguez-Fraticelli, Curr. Opin. Hematol 2021). Beyond this debate, these studies have highlighted the underestimated contribution of the MPP compartment to the daily hematopoietic output in unperturbed conditions. This new understanding raises questions about the functional properties of the MPP compartment in steady state condition and its ability to independently sustain prolonged hematopoietic cell production. Here we revisited the early hematopoietic hierarchy present in the Lin- Kit+ Sca1+ (LSK) fraction that includes all HSC and MPP (MPP1-5) compartments. Highly-multiplexed CITE-Seq single cell analyses with over 60 antibodies identified a novel LSK CD62L- Flt3+ population (CD62L- MPP4) that clusters within the MPP compartments but displays a cell cycle gene signature similar to the one found in quiescent HSCs. Analysis of the Mki67 reporter mouse model and nucleotide analogue incorporation assays directly confirmed the existence of this unexpected cell cycle diversity in MPP4 during unperturbed hematopoiesis. Consistent with the molecular data, these analyses characterized a resting CD62L- and a proliferative CD62L+ MPP4 compartments. These two populations showed similar cloning efficiency in methylcellulose assays indicating that both populations behave as intermediary MPP compartments. When assessed in short-term culture, both populations were responsive to Flt3L stimulation but displayed specific kinetics of division. They also showed distinct short-term reconstitution ability upon transplantation in correlation with their cell cycle activity. Analysis of these populations in native condition using the H2B-GFP label-retaining system further confirmed the specific properties of the resting CD62L- MPP4 cells, distinct from the other proliferative MPP2, MPP3 and CD62L+ MPP4 compartments. Finally, these studies uncovered a novel early path of differentiation that involves the MPP5 cells (LSK Flt3+ CD48- CD150-) sequentially acquiring Flk2 and CD48 expression to generate the resting CD62L- MPP4 compartment, which in turn gives rise to cycling CD62L+ MPP4 cells. Altogether, these results challenge the accepted dichotomy between quiescent HSCs and dividing progenitors. While difference in lineage potential has been historically a major criterium to classify MPP populations, we established a new dimension at the MPP diversity, based on their proliferative activity at steady state. These results particularly highlight a new path of differentiation directly linking MPP5, a quiescent compartment with residual self-renewal capacity, to the resting CD62L- Flt3+ MPP cells. Based on these results, we proposed that slow-dividing Flt3+ MPPs act as a key transitional compartment that can buffer the hematopoietic cell production and therefore regulate hematopoietic homeostasis at steady state. We envision that this novel compartment could be a key intermediary for hematopoietic malignancies driven by the acquisition of Flt3 mutations and a cellular target for intervention.

Srsf2P95H/+ co-operates with loss of TET2 to promote myeloid bias and initiate a chronic myelomonocytic leukemia-like disease in mice.

Recurrent mutations in RNA splicing proteins and epigenetic regulators contribute to the development of myelodysplastic syndrome (MDS) and related myeloid neoplasms. In chronic myelomonocytic leukemia (CMML), SRSF2 mutations occur in ~50% of patients and TET2 mutations in ~60%. Clonal analysis indicates that either mutation can arise as the founder lesion. Based on human cancer genetics we crossed an inducible Srsf2P95H/+ mutant model with Tet2fl/fl mice to mutate both concomitantly in hematopoietic stem cells. At 20-24 weeks post mutation induction, we observed subtle differences in the Srsf2/Tet2 mutants compared to either single mutant. Under conditions of native hematopoiesis with aging, we see a distinct myeloid bias and monocytosis in the Srsf2/Tet2 mutants. A subset of the compound Srsf2/Tet2 mutants display an increased granulocytic and distinctive monocytic proliferation (myelomonocytic hyperplasia), with increased immature promonocytes and monoblasts and binucleate promonocytes. Exome analysis of progressed disease demonstrated mutations in genes and pathways similar to those reported in human CMML. Upon transplantation, recipients developed leukocytosis, monocytosis, and splenomegaly. We reproduce Srsf2/Tet2 co-operativity in vivo, yielding a disease with core characteristics of CMML, unlike single Srsf2 or Tet2 mutation. This model represents a significant step toward building high fidelity and genetically tractable models of CMML.

1006 – MOLECULAR TO TISSUE-SCALE ANALYSIS OF HEMATOPOIETIC STEM/PROGENITOR CELL DYNAMICS

Natural sciences have long been divided into quantitative and qualitative, with physics an example of the former, and biology an example of the latter. Importantly, a quantitative understanding is essential for accurate prediction of future states including the impact of perturbations. Hematopoiesis research has greatly benefited from the qualitative description of the hematopoietic differentiation tree for over 30 years now, with renewed impetus provided through the addition of quantitative measurements of population dynamics in 2015. However, it is universally recognised that stem/progenitor populations defined by small combinations of cell surface markers are heterogeneous. Their use therefore represents a useful abstraction but cannot capture the true complexity of hematopoiesis. Hematopoiesis has a long track record of embracing single-cell functional and molecular profiling technologies. However, a single-cell resolution quantitative model of native hematopoiesis has never been reported. We have employed persistent labelling of stem cells combined with time-series single cell RNA-Seq to build the first quantitative model of native blood stem and progenitor dynamics in the mouse bone marrow at single cell resolution. We capture compartment-wide flux and net proliferation rates in real-time, thus revealing the distinct ways in which different blood lineages maintain their cellular output. By exploring the molecular layer of single cell RNA-Seq, we pinpoint molecular changes associated with changes in cell behaviour, such as increased differentiation or growth rates. Finally, unlike immunophenotyping data, our models can readily be integrated with external data thus enabling a quantitative comparison of stem cell behaviour during homeostatic versus transplantation settings. We anticipate that this work will not only impact our ability to perform quantitative analysis of perturbed hematopoiesis using mouse models, but also stimulate discussions on how a similarly quantitative understanding of human hematopoiesis might be achieved. Natural sciences have long been divided into quantitative and qualitative, with physics an example of the former, and biology an example of the latter. Importantly, a quantitative understanding is essential for accurate prediction of future states including the impact of perturbations. Hematopoiesis research has greatly benefited from the qualitative description of the hematopoietic differentiation tree for over 30 years now, with renewed impetus provided through the addition of quantitative measurements of population dynamics in 2015. However, it is universally recognised that stem/progenitor populations defined by small combinations of cell surface markers are heterogeneous. Their use therefore represents a useful abstraction but cannot capture the true complexity of hematopoiesis. Hematopoiesis has a long track record of embracing single-cell functional and molecular profiling technologies. However, a single-cell resolution quantitative model of native hematopoiesis has never been reported. We have employed persistent labelling of stem cells combined with time-series single cell RNA-Seq to build the first quantitative model of native blood stem and progenitor dynamics in the mouse bone marrow at single cell resolution. We capture compartment-wide flux and net proliferation rates in real-time, thus revealing the distinct ways in which different blood lineages maintain their cellular output. By exploring the molecular layer of single cell RNA-Seq, we pinpoint molecular changes associated with changes in cell behaviour, such as increased differentiation or growth rates. Finally, unlike immunophenotyping data, our models can readily be integrated with external data thus enabling a quantitative comparison of stem cell behaviour during homeostatic versus transplantation settings. We anticipate that this work will not only impact our ability to perform quantitative analysis of perturbed hematopoiesis using mouse models, but also stimulate discussions on how a similarly quantitative understanding of human hematopoiesis might be achieved.

The biology of hematopoietic stem cells and its clinical implications.

Hematopoietic stem cells (HSCs) give rise to all types of blood cells and self-renew their own population. The regeneration potential of HSCs has already been successfully translated into clinical applications. However, recent studies on the biology of HSCs may further extend their clinical use in future. The roles of HSCs in native hematopoiesis and in transplantation settings may differ. Furthermore, the heterogenic pool of HSCs dynamically changes during aging. These changes also involve the complex interactions of HSCs with the bone marrow niche. Here, we review the opportunities and challenges of these findings to improve the clinical use of HSCs. We describe new methods of HSCs mobilization and conditioning for the transplantation of HSCs. Finally, we highlight the research findings that may lead to overcoming the current limitations of HSC transplantation and broaden the patient group that can benefit from the clinical potential of HSCs.

Combined Biological and Modeling Approach of Hematopoiesis: From Native to Stressed Erythropoiesis

We developed here a new mathematical modelling approach which reconciliates native and stress hematopoiesis, focused on hematopoietic stem and progenitor compartments. We first proposed an erythropoiesis model leaning on a minimum of 6 cell-amplification compartments, able to reproduce native erythropoiesis. A phenylhydrazine-induced hemolytic stress was next applied, and in vivo data were used to estimate model parameters through an optimized algorithm and integrated regulatory processes. A reduction of all stem cell compartments was observed, due to a drastic differentiation without proliferation during 7 days, followed by a huge proliferation in all compartments including long-term hematopoietic stem cells, before returning to normal values. The 6-compartment model applied to stress erythropoiesis was complexified by integrating regulatory processes. Thus, regulation of hematopoiesis appears dispensable during native hematopoiesis but mandatory for stress hematopoiesis. In conclusion this multi-step and time-dependent model of immature hematopoiesis opens new opportunities to understand development of normal or pathological hematopoiesis.

Increased baseline RASGRP1 signals enhance stem cell fitness during native hematopoiesis.

Oncogenic mutations in RAS genes, like KRASG12D or NRASG12D, trap Ras in the active state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced in the bone marrow via Mx1CRE. The RAS exchange factor RASGRP1 is frequently overexpressed in T-ALL patients. In T-ALL cell lines overexpression of RASGRP1 increases flux through the RASGTP/RasGDP cycle. Here we expanded RASGRP1 expression surveys in pediatric T-ALL and generated a RoLoRiG mouse model crossed to Mx1CRE to determine the consequences of induced RASGRP1 overexpression in primary hematopoietic cells. RASGRP1-overexpressing, GFP-positive cells outcompeted wild type cells and dominated the peripheral blood compartment over time. RASGRP1 overexpression bestows gain-of-function colony formation properties to bone marrow progenitors in medium containing limited growth factors. RASGRP1 overexpression enhances baseline mTOR-S6 signaling in the bone marrow, but not in vitro cytokine-induced signals. In agreement with these mechanistic findings, hRASGRP1-ires-EGFP enhances fitness of stem- and progenitor- cells, but only in the context of native hematopoiesis. RASGRP1 overexpression is distinct from KRASG12D or NRASG12D, does not cause acute leukemia on its own, and leukemia virus insertion frequencies predict that RASGRP1 overexpression can effectively cooperate with lesions in many other genes to cause acute T cell leukemia.

3011 – MUTANT ASXL1 INDUCES EXPANSION OF HEMATOPOIETIC STEM CELLS THROUGH ACTIVATION OF AKT/MTOR PATHWAY

Epigenetic regulators, ASXL1, DNMT3A, and TET2 are frequently mutated in clonal hematopoiesis (CH). Dnmt3a- or Tet2-deficient mice increase self-renewal of hematopoietic stem cells (HSCs), suggesting that mutations in DNMT3A and TET2 provoke clonal expansion of hematopoietic cells, leading to subsequent CH in humans. On the contrary, ASXL1-mutated mice show reduced numbers and functions of HSCs. Thus, how ASXL1 mutations drive CH are not understood. Here, we investigated the effects of ASXL1 mutations on physiological aging of HSCs using knockin (KI) mice expressing a C-terminally truncated form of ASXL1 (ASXL1-MT). We found that HSCs expressing ASXL1-MT exhibited a competitive disadvantage after transplantation. On the other hand, in a genetic mosaic mouse, they displayed a growth advantage to occupy the HSC compartment over time, recapitulating CH in humans. As a mechanism by which ASXL1-MT causes CH, we show that ASXL1-MT binds and stabilizes phosphorylated Akt to activate Akt/mTOR pathway in coordination with Bap1. Activated Akt/mTOR induces dysregulated cell cycle progression and proliferation of HSCs. Meanwhile, it also compromises HSC functions along with activated mitochondrial metabolism, ROS overproduction, and increased DNA damage. Treatment with an mTOR inhibitor rapamycin suppressed aberrant proliferation of HSCs and ameliorated dysregulated hematopoiesis in aged ASXL1-MT KI mice. Taken together, ASXL1-MT impairs function of HSCs, whereas it confers a clonal advantage on HSCs during aging specifically in native hematopoiesis. ASXL1 mutation-mediated expansion of HSCs associated with increased DNA damage can induce development of CH, resulting in malignant transformation with secondary mutations. Akt/mTOR signaling could be a therapeutic target to individuals with CH harboring ASXL1 mutations.

3148 – SRSF2P95H AND TET2 LOSS CO-OPERATE TO PROMOTE CHRONIC MYELOMONOCYTIC LEUKAEMIA IN VIVO

Sequencing of >3000 MDS and related myeloid neoplasms has provided a detailed picture of the genetic landscape of these cancers. Most harbour mutations in two pathways: the RNA spliceosome (eg. SRSF2, SF3B1, U2AF1) and epigenetic regulators (eg. DMNT3, TET2). Co-mutation of SRSF2 and TET2 are profoundly enriched in MDS and MDS/MPN (p=2.63 × 10-14; q=1.21 × 10-12). In CMML and the related chronic neutrophilic leukaemia and atypical chronic myeloid leukaemia, there is a positive association between mutation of SRSF2 and TET2 (p=0.002). This association is strongest in CMML (p=0.004), where SRSF2 mutations occur in ∼50% of patients and TET2 mutations in ∼60%. SRSF2/TET2 co-mutation influences clinical presentation, with co-mutated CMML having higher leukocyte counts yet less pronounced monocytosis compared to those with wild-type SRSF2/TET2. Based on the human clinical genetics, we crossed the conditional Srsf2P95H/+ mutants with Tet2fl/fl mice to establish animals where we could mutate both concomitantly. At 52 weeks post gene mutation there is a hypocellular bone marrow with increased myeloid bias at the expense of B lymphoid and erythroid lineages in the Srsf2/Tet2 double mutants. Under conditions of native haematopoiesis with aging, a distinct myeloid bias and monocytosis develop in the Srsf2/Tet2 mutants which is not seen in either single mutant or controls. The compound Srsf2/Tet2 mutants display increased granulocytic and monocytic proliferation (myelo-monocytic hyperplasia) with increased immature promonocytes and monoblasts (∼10-15% total nucleated cells) and evidence of binucleate promonocytes. These are features of CMML (CMML-2). Upon secondary transplant, recipients developed leukocytosis, monocytosis and splenomegaly. This demonstrates that our model is able to reproduce SRSF2/TET2 co-operativity in vivo, yielding a disease with core characteristics of CMML, unlike single Srsf2 or Tet2 mutation.

The Source and Dynamics of Adult Hematopoiesis: Insights from Lineage Tracing.

The generation of all blood cell lineages (hematopoiesis) is sustained throughout the entire life span of adult mammals. Studies using cell transplantation identified the self-renewing, multipotent hematopoietic stem cells (HSCs) as the source of hematopoiesis in adoptive hosts and delineated a hierarchy of HSC-derived progenitors that ultimately yield mature blood cells. However, much less is known about adult hematopoiesis as it occurs in native hosts, i.e., without transplantation. Here we review recent advances in our understanding of native hematopoiesis, focusing in particular on the application of genetic lineage tracing in mice. The emerging evidence has established HSCs as the major source of native hematopoiesis, helped to define the kinetics of HSC differentiation, and begun exploring native hematopoiesis in stress conditions such as aging and inflammation. Major outstanding questions about native hematopoiesis still remain, such as its clonal composition, the nature of lineage commitment, and the dynamics of the process in humans.

Enumerating Functional HSC Clones in the Zebrafish Using Gestalt

Hematopoietic stem cells (HSCs) are functionally and genetically diverse. This diversity decreases as a function of age and with the development of myeloid neoplasia. Numerous systems have been developed to genetically "barcode" HSCs and quantify HSC clone numbers in the setting of native hematopoiesis, but no experimental and computational framework has been established to perform this analysis on a large number of biological replicates. Here we have employed the transgenic zebrafish system, Genome Editing of Synthetic Target Arrays for Lineage Tracing (GESTALT), to develop a framework scaled to permit the investigation of putative oncogenes or niche factors with moderate to small effect sizes. The GESTALT line carries a single copy of an array of 10 tandem CRISPR target sites that is highly variable after being repaired by non-homologous end joining. GESTALT embryos were injected with sgRNAs and Cas9 protein to generate genetic barcodes at the GESTALT locus. Fifty-six zebrafish were raised to adulthood and GESTALT variants were read from a sample of peripheral blood by amplicon sequencing. In a training set of 35 fish we developed a computational model to discriminate informative barcodes from uninformative GESTALT variants in each sample. This model identifies variants shared among samples and excludes them as uninformative. Experimentally-defined thresholds were used to minimize inter-sample correlation (mean Pearson Correlation Coefficient = 0.0021) while maximizing the number of retained informative barcodes in each sample. Bootstrapping and k-means clustering were used to classify samples with a high or low fraction of informative (FI) barcodes (FI cutoff = 0.65). 24 samples within the training set were classified in the High-FI group (mean FI = 0.93 ± 0.02, compared to 0.40 ± 0.04 in the Low-FI group). We then evaluated 3 methods of quantifying GESTALT HSC clones from all sequenced barcodes in the High-FI samples and found that counting barcodes with a VAF>0.02 produced the most normal distribution across the training set (Shapiro-Wilk test for normality, W = 0.95, p = 0.21). 4.3 ± 0.38 GESTALT HSC clones were detected in the training set. In an independent validation set of 21 samples, the number of GESTALT HSC clones was 3.4 ± 0.55, (p = 0.2). This study provides the largest reference dataset to date for HSC genetic barcoding experiments in the zebrafish and addresses the validity of barcode sequences, sample selection and clone enumeration in a systematic, objective way. The GESTALT barcoding system and our analysis framework compare favorably with other methods of studying HSC clonal diversity under conditions of native hematopoiesis in terms of scale, cost, labor and barcode validity. Future studies aiming to understand the mechanisms of HSC clonal evolution in the context of health, ageing and disease will benefit from this framework for enumerating functional HSC clones. Disclosures No relevant conflicts of interest to declare.

1004 - HOW DO SRSF2P95H MUTATIONS LEAD TO MYELODYSPLASTIC/MYELOPROLIFERATIVE SYNDROME (MDS/MPN)?

SRSF2 P95 mutations are prevalent in myelodysplastic (MDS) and myelodysplastic/myeloproliferative (MDS/MPN) syndromes. They occur early in the course of the disease, are positively selected during disease progression and are associated with poor outcomes. Understanding how founder mutations such as SRSF2 modifies haematopoiesis and ultimately contributes to the development of myeloid bias and MDS/MPN will be critical to understanding the initiation and maintenance of these cancers. We have generated a conditional murine Srsf2P95H mutation model, where the temporal and cell type specific expression of the P95H mutation can occur. As in humans, the Srsf2 P95H mutation is expressed heterozygously from its endogenous locus. Using multiple Cre lines, we find that Srsf2P95H must be expressed in the haematopoietic stem cell (HSC) containing populations to promote a myelo-monocytic bias and expansion during native haematopoiesis. These early phenotypic alterations correspond with transcriptional and RNA splicing changes in lymphoid, myeloid and stem cell related genes. Upon ageing, Srsf2P95H animals develop a progressive, transplantable disease characterised by monocytosis, dysplastic neutrophils and macrocytosis with reduced red blood cells and loss of bone marrow (BM) B lymphopoiesis and erythropoiesis, all features of human MDS/MPN. Long-term (LT) HSC numbers tend to be lower and the repopulation potential of mutant cells decreased unless transplanted with age and microenvironment matched wild type cells. Srsf2P95H cells then reveal a competitiveness consistent with the presence of SRSF2 mutations in age-related clonal haematopoiesis. Diseased mice do not develop leukaemia, but exome sequencing of the bone marrow shows the acquisition of additional mutations currently found in humans with SRSF2 mutation. These results demonstrate a valid and reproducible Srsf2P95H/+ mouse knock-in model that will help elucidate the effect of human SRSF2 mutation on initiation and maintenance of MDS/MPN.

TET2 binding to enhancers facilitates transcription factor recruitment in hematopoietic cells.

The epigenetic regulator TET2 is frequently mutated in hematological diseases. Mutations have been shown to arise in hematopoietic stem cells early in disease development and lead to altered DNA methylation landscapes and an increased risk of hematopoietic malignancy. Here, we show by genome-wide mapping of TET2 binding sites in different cell types that TET2 localizes to regions of open chromatin and cell-type–specific enhancers. We find that deletion of Tet2 in native hematopoiesis as well as fully transformed acute myeloid leukemia (AML) results in changes in transcription factor (TF) activity within these regions, and we provide evidence that loss of TET2 leads to attenuation of chromatin binding of members of the basic helix–loop–helix (bHLH) TF family. Together, these findings demonstrate that TET2 activity shapes the local chromatin environment at enhancers to facilitate TF binding and provides an example of how epigenetic dysregulation can affect gene expression patterns and drive disease development.

Murine HSCs contribute actively to native hematopoiesis but with reduced differentiation capacity upon aging.

A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age.

Somatic Mutations Reveal Lineage Relationships and Age-Related Mutagenesis in Human Hematopoiesis

SummaryMutation accumulation during life can contribute to hematopoietic dysfunction; however, the underlying dynamics are unknown. Somatic mutations in blood progenitors can provide insight into the rate and processes underlying this accumulation, as well as the developmental lineage tree and stem cell division numbers. Here, we catalog mutations in the genomes of human-bone-marrow-derived and umbilical-cord-blood-derived hematopoietic stem and progenitor cells (HSPCs). We find that mutations accumulate gradually during life with approximately 14 base substitutions per year. The majority of mutations were acquired after birth and could be explained by the constant activity of various endogenous mutagenic processes, which also explains the mutation load in acute myeloid leukemia (AML). Using these mutations, we construct a developmental lineage tree of human hematopoiesis, revealing a polyclonal architecture and providing evidence that developmental clones exhibit multipotency. Our approach highlights features of human native hematopoiesis and its implications for leukemogenesis.

What do the lineage tracing studies tell us? Consideration for hematopoietic stem cell origin, dynamics, and leukemia-initiating cells.

The recent advance of technologies enables us to trace the cell fate in vivo by marking the cells that express the gene of interest or by barcoding them at a single cell level. Various tamoxifen-inducible Cre-recombinase mice combined with Rosa-floxed lines are utilized. In this review, with the results revealed by lineage tracing assays, we re-visit the long-standing debate for the origin of hematopoietic stem cells in the mouse embryo, and introduce the view of native hematopoiesis, and possible leukemic-initiating cells emerged during fetal stages.

30 Whole-genome sequencing of normal stem cells provides novel insights into human native hematopoiesis and leukaemia aetiology

IntroductionMature blood and immune cells are produced by the process of hematopoiesis, which is orchestrated by self-renewing hematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs) in the bone marrow. As people age, clonal expansion of mutated stem cells within the blood more commonly occur, which is associated with increased risk of developing haematological malignancies. However, the processes underlying mutation accumulation in normal stem cells and the clonal composition of the blood tissue within the human bone marrow compartment remain unknown.Material and methodsWe determined genome-wide mutation patterns in HSCs and downstream MPPs by sequencing clonally expanded primary cells derived from bone marrow of multiple human donors of different ages.Results and discussionsWe found that base substitutions accumulate gradually during life at a rate of approximately 16 novel mutations per year, while insertions and deletions occur more sporadically and at low numbers. The number and types of mutations were similar between HSCs and MPPs, suggesting that differentiation and quiescence status does not affect somatic mutation load. The majority of the base substitutions in blood progenitors could be attributed to a novel mutational signature, which also explains the majority of base substitutions in acute myeloid leukaemia (AML). Additionally, using base substitutions, we reconstructed the developmental hematopoietic phylogenetic tree and demonstrate that branching takes place early during development.ConclusionOur data demonstrate that developmental clones have differential contribution to the HSC and MPP compartments, and while these sub-clones exhibit multipotency, branches contributing predominantly to HSCs have an enriched presence in the megakaryocyte lineage. Together, our approach highlights novel features of human hematopoiesis and its implications for leukemogenesis.

Clonal analysis of lineage fate in native haematopoiesis.

SUMMARYHematopoiesis, the process of mature blood and immune cell production, is functionally organized as a hierarchy, with self-renewing hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells sitting at the very top1,2. Multiple models have been proposed as to what the earliest lineage choices are in these primitive hematopoietic compartments, the cellular intermediates, and the resulting lineage trees that emerge from them3–10. Given that the bulk of studies addressing lineage outcomes have been performed in the context of hematopoietic transplantation, current lineage branching models are more likely to represent roadmaps of lineage potential rather than native fate. Here, we utilize transposon (Tn) tagging to clonally trace the fates of progenitors and stem cells in unperturbed hematopoiesis. Our results describe a distinct clonal roadmap in which the megakaryocyte (Mk) lineage arises largely independently of other hematopoietic fates. Our data, combined with single cell RNAseq, identify a functional hierarchy of uni- and oligolineage producing clones within the MPP population. Finally, our results demonstrate that traditionally defined long-term HSCs (LT-HSCs) are a significant source of Mk-restricted progenitors, suggesting that the Mk-lineage is the predominant native fate of LT-HSCs. Our study provides evidence for a substantially revised roadmap for unperturbed hematopoiesis, and highlights unique properties of MPPs and HSCs in situ.