Lineage Output Research Articles

The many faces of hematopoietic stem cell heterogeneity.

Not all hematopoietic stem cells (HSCs) are alike. They differ in their physical characteristics such as cell cycle status and cell surface marker phenotype, they respond to different extrinsic signals, and they have different lineage outputs following transplantation. The growing body of evidence that supports heterogeneity within HSCs, which constitute the most robust cell fraction at the foundation of the adult hematopoietic system, is currently of great interest and raises questions as to why HSC subtypes exist, how they are generated and whether HSC heterogeneity affects leukemogenesis or treatment options. This Review provides a developmental overview of HSC subtypes during embryonic, fetal and adult stages of hematopoiesis and discusses the possible origins and consequences of HSC heterogeneity.

Lysine Methyltransferase Kmt5a Restricts Myeloid-Biased Output of Lymphoid-Primed Multipotent Progenitors

Distinct, lineage-biased subsets of multipotent progenitor cells (MPP) dynamically respond to the demands of the hematopoietic system to replenish mature hematopoietic cells as needed. It currently remains unclear as to whether distinct epigenetic mechanisms regulate lineage-specific expansion and differentiation from MPPs. Focusing on lymphoid-primed multipotent progenitor cells (LMPP/MPP4), we performed a lentiviral shRNA screen of 15 epigenetic factors, selected based on differential expression between myeloid-restricted and lymphoid-restricted progenitors. Following a 48 hour infection with lentiviral shRNA constructs or a non-targeting control, the lineage potential of lymphoid-primed multipotent progenitors was interrogated by myeloid and lymphoid colony forming unit (CFU) assays. From this screen, knockdown of the lysine methyltransferase Kmt5a most dramatically altered lineage output from lymphoid-primed multipotent progenitors through an expansion of myeloid lineage colonies without altering lymphoid colony production. To confirm target specificity, two independent shRNA hairpins targeting distinct locations of the Kmt5a transcript demonstrated that knockdown of Kmt5a (97.1% and 99.5% versus non-targeting control shRNA) increased macrophage colony production by 1.94 and 1.95 fold, respectively (P < 0.01 and P < 0.05, n = 3). Preliminary single cell culture experiments support that the enhanced myeloid lineage output from lymphoid-primed multipotent progenitors occurs at the single-cell level through increased cloning efficiency of myeloid-biased cells. Our results suggest that Kmt5a functions to restrict myeloid lineage output from lymphoid-primed multipotent progenitors. Mechanistically, KMT5A is responsible for monomethylation of histone H4K20 and the methylation of non-histone proteins (ex. p53K376). Our ongoing work aims to distinguish between these histone and non-histone targets to determine the precise mechanisms restricting myeloid lineage output from lymphoid-primed multipotent progenitors. This work has direct implications for a better understanding of the molecular drivers of transient myeloid lineage reprogramming of lymphoid-primed multipotent progenitors during hematopoietic regeneration, age associated myeloid lineage skewing of hematopoiesis, and myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Junctional Adhesion Molecule 2 Represents a Novel Subset of Hematopoietic Stem Cells Poised for T Lymphopoiesis

Phenotypically well-characterized hematopoietic stem cells (HSCs) still represent a heterogeneous pool of primitive cells regarding to their functionality. In particular, different lineage potential of HSCs have been considered as one of key features of the HSC heterogeneity. The lineage output of HSCs is often coupled with cell cycle status or long-term reconstitution potential, however molecular mechanisms of the mutuality are unclear and other type of the regulation may exist. In addition, prospective isolation of such HSCs biased towards specific lineage(s) is still problematic, as many of categorizations highly rely on retrospective information, e.g. transplantation assay. Although several markers have been reported to be able to subdivide HSCs into subcategories, exploration of additional markers will allow us understanding further molecular mechanisms of HSC regulations including activation and lineage choice.Here, we show that cell surface expression of Junctional adhesion molecule 2 (Jam2) represents higher reconstitution capacity of HSCs and the T cell potential. Flow cytometry analyses revealed that a subset of CD150+CD48-KSL cells in mouse bone marrow (BM) were positive for Jam2 (Jam2+HSC, 36.6 ±13.0 %), while other Jam family member Jam1 (F11r) was expressed on all HSCs and Jam3 was not detected. To examine functional differences of Jam2+ and Jam2-HSCs, 30 cells were separately transplanted into lethally irradiated mice. Peripheral blood analyses revealed that Jam2+HSCs reconstituted more efficiently than Jam2-HSCs (77.5 ±15.9 and 51.7 ±29.3 %, respectively). In case of transplantation using 5 cells, the frequency of reconstituted mice was higher in Jam2+HSCs (7 in 11) compared to Jam2-HSCs (4 in 11), indicating that Jam2+ population is more enriched for functional HSCs. The expression of Jam2 on HSC is reversible, but not hierarchical, as both Jam2+ and Jam2-HSCs reconstituted opposite population in the BM.Lineage analyses revealed that Jam2+HSCs have a greater potential in lymphoid cell reconstitution, particularly T cells, whereas the chimerism in myeloid cells was not significantly different from Jam2-HSCs. This tendency of higher contribution to the T cell development was even more pronounced in the secondary transplantation experiments, where the contribution of Jam2+HSCs in T cells was close to 100 %. Of note, most of Jam2+HSCs were in a dormant state, suggesting that the T cell (or lymphoid) potential of Jam2+HSCs is independent of cell cycle progression.Jam2 has been reported to interact with Jam1, which mediates the Notch signaling (Kobayashi et al., Nature, 2014). Competitive co-culture of Jam2+ vs Jam2-HSCs on OP9-DL1 showed that Jam2+HSCs dominated the T cell production, whereas no difference was seen in B cell production upon OP9 co-culture. Since Jam2 positivity correlates to T cell potential, we asked if altered T lymphopoiesis environment affects the cell surface Jam2 expression. Comparison of C57BL/6, NOD, NOD-Scid and NOD-Scid Il2rγ KO (NSG) mice showed that HSCs of NSG mice have significantly higher frequency of Jam2+HSCs, suggesting that cell surface Jam2 expression might be regulated by specific cytokine(s) binding to IL2Rγ.Our findings suggest Jam2 is a new marker for a subset of HSCs that preferentially generate T cells. In addition, this work uncouples the lineage choice and cell cycle status, which proposes a novel model to the lineage-determining machineries. Since efficient and immediate generation of T cells in transplantation therapy is important to minimize infectious risks, understanding the molecular basis of the Jam-Notch cooperation would contribute to establish safer and more efficient treatment. DisclosuresNo relevant conflicts of interest to declare.

Chronic interleukin-1 exposure drives haematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal.

Haematopoietic stem cells (HSC) maintain lifelong blood production and increase blood cell numbers in response to chronic and acute injury. However, the mechanism(s) by which inflammatory insults are communicated to HSCs and their consequences for HSC activity remain largely unknown. Here, we demonstrate that interleukin-1 (IL-1), which functions as a key pro-inflammatory ‘emergency’ signal, directly accelerates cell division and myeloid differentiation of HSCs via precocious activation of a PU.1-dependent gene program. While this effect is essential for rapid myeloid recovery following acute injury to the bone marrow (BM), chronic IL-1 exposure restricts HSC lineage output, severely erodes HSC self-renewal capacity, and primes IL-1-exposed HSCs to fail massive replicative challenges like transplantation. Importantly, these damaging effects are transient and fully reversible upon IL-1 withdrawal. Our results identify a critical regulatory circuit that tailors HSC responses to acute needs, and likely underlies deregulated blood homeostasis in chronic inflammation conditions.

Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells.

Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis.

A somatic mutation of GFI1B identified in leukemia alters cell fate via a SPI1 (PU.1) centered genetic regulatory network

We identify a mutation (D262N) in the erythroid-affiliated transcriptional repressor GFI1B, in an acute myeloid leukemia (AML) patient with antecedent myelodysplastic syndrome (MDS). The GFI1B-D262N mutant functionally antagonizes the transcriptional activity of wild-type GFI1B. GFI1B-D262N promoted myelomonocytic versus erythroid output from primary human hematopoietic precursors and enhanced cell survival of both normal and MDS derived precursors. Re-analysis of AML transcriptome data identifies a distinct group of patients in whom expression of wild-type GFI1B and SPI1 (PU.1) have an inverse pattern. In delineating this GFI1B-SPI1 relationship we show that (i) SPI1 is a direct target of GFI1B, (ii) expression of GFI1B-D262N produces elevated expression of SPI1, and (iii) SPI1-knockdown restores balanced lineage output from GFI1B-D262N-expressing precursors. These results table the SPI1-GFI1B transcriptional network as an important regulatory axis in AML as well as in the development of erythroid versus myelomonocytic cell fate.

The Role of Symmetric Stem Cell Divisions in Tissue Homeostasis.

Successful maintenance of cellular lineages critically depends on the fate decision dynamics of stem cells (SCs) upon division. There are three possible strategies with respect to SC fate decision symmetry: (a) asymmetric mode, when each and every SC division produces one SC and one non-SC progeny; (b) symmetric mode, when 50% of all divisions produce two SCs and another 50%—two non-SC progeny; (c) mixed mode, when both the asymmetric and two types of symmetric SC divisions co-exist and are partitioned so that long-term net balance of the lineage output stays constant. Theoretically, either of these strategies can achieve lineage homeostasis. However, it remains unclear which strategy(s) are more advantageous and under what specific circumstances, and what minimal control mechanisms are required to operate them. Here we used stochastic modeling to analyze and quantify the ability of different types of divisions to maintain long-term lineage homeostasis, in the context of different control networks. Using the example of a two-component lineage, consisting of SCs and one type of non-SC progeny, we show that its tight homeostatic control is not necessarily associated with purely asymmetric divisions. Through stochastic analysis and simulations we show that asymmetric divisions can either stabilize or destabilize the lineage system, depending on the underlying control network. We further apply our computational model to biological observations in the context of a two-component lineage of mouse epidermis, where autonomous lineage control has been proposed and notable regional differences, in terms of symmetric division ratio, have been noted—higher in thickened epidermis of the paw skin as compared to ear and tail skin. By using our model we propose a possible explanation for the regional differences in epidermal lineage control strategies. We demonstrate how symmetric divisions can work to stabilize paw epidermis lineage, which experiences high level of micro-injuries and a lack of hair follicles as a back-up source of SCs.

Interleukin-1 Drives Precocious Myeloid Differentiation of Hematopoietic Stem Cells at the Expense of Self-Renewal

Hematopoietic stem cells (HSCs) maintain lifelong blood homeostasis. While many of the cell-intrinsic mechanisms regulating HSC function at steady state have been well characterized, the role of inflammatory cytokines and other environmental factors in tailoring blood production following physiological insults has become a topic of emerging interest. The cytokine interleukin-1 (IL-1) is a prototypical pro-inflammatory cytokine that plays a key role in host inflammatory responses to injury and infection, and is associated with elevated myeloid cell production. Importantly, IL-1 also drives a wide range of chronic inflammatory conditions such as diabetes, obesity, and arthritis that are often characterized by deregulated blood homeostasis. Here, we show at single-cell resolution using continuous tracking technology that IL-1 drives accelerated HSC cell division kinetics and myeloid differentiation via the rapid activation of a precocious PU.1-dependent myeloid gene program. Activation of this program requires direct IL-1R signaling and subsequent activation of IKK kinases, and instructively primes HSCs to adopt a myeloid fate. Strikingly, we demonstrate that IL-1 produced by myeloid cells and endothelial cells of the bone marrow (BM) niche exerts similar effects in vivo, and is required for efficient myeloid recovery following acute challenges such as transplantation or myeloablation. On the other hand, we find that chronic IL-1 exposure substantially remodels HSC blood output, resulting in myeloid overproduction and expansion of myeloid-biased multipotent progenitor (MPP) compartments at the expense of lymphoid and erythroid lineages. Critically, chronic IL-1 erodes HSC self-renewal, significantly impairing their regenerative capacity following transplantation. On the other hand, chronically exposed HSCs recover their function upon IL-1 withdrawal. Collectively, these findings identify IL-1 as a critical regulator of HSC fate and lineage specification via activation of a PU.1 circuit. They also demonstrate a role for IL-1 as a double-edged sword in HSC biology, promoting HSC regeneration in response to acute insults while severely disrupting HSC self-renewal and lineage output during chronic exposure, hence identifying IL-1 as an important and therapeutically targetable factor underwriting myeloid overproduction and other deregulations that contribute to the pathogenesis of a variety of chronic inflammatory diseases and blood disorders. DisclosuresNo relevant conflicts of interest to declare.

Clonal Features of Hematopoietic Stem and Progenitor Cells in Aging

It is traditionally thought that Hematopoietic Stem Cells (HSCs) maintain blood homeostasis through long-term self-renewal and multilineage differentiation. This concept, however, is challenged by two recent studies in which the fundamental features of unperturbed hematopoiesis are evaluated by different approaches of lineage tracing. Both the kinetic analysis of HSC output by the Rodewald group and our clonal analysis with transposon barcoding suggest a dominant role of non-transplantable short-term HSCs and progenitors, but not the long-term HSCs, in driving native blood cell production. In addition, our longitudinal analysis of peripheral blood demonstrates extensive clonal succession in granulocyte production. These findings collectively suggest a distinct mechanism of native hematopoiesis that differs significantly from what has been learned in transplantation experiments. At the same time, they bring to light new questions regarding the ultimate fate of the progenitor population and the exact contribution of HSCs under normal physiological conditions. To address these questions, we examined clonal features of HSCs and progenitors in aged mice. Our results show a progressive reduction in clonal complexity and a concurrent increase in clonal stability when blood granulocytes are analyzed up to a hundred ten weeks after transposon barcoding. As time elapses, clonal overlapping between granulocytes and B cells become much more extensive, suggesting an increased tendency toward multilineage differentiation during aging. Analysis of stem and progenitor cells in bone marrow of aged mice reveals prevalent lineage output by multipotent progenitors (MPPs), whereas a lower fraction of HSC clones are found to produce mature progeny. While this overall pattern of differentiation is reminiscent of what has been observed in young and middle-aged animals, a two-fold increase in HSC clonal output was observed in these old mice, indicating their increased contribution to blood cell production. A comparison of clonal compositions in blood and marrow cell populations demonstrates an MPP origin of stable peripheral blood clones, and a smaller fraction of these clones can even be traced back to HSCs. These observations hence suggest extensive self-renewal and asymmetric cell division of these two cell populations in aging. Taken together, our results indicate that the aged hematopoietic system is characterized by reduced clonal complexity, increased clonal persistence, and HSC activation. The higher propensity to self-renewal during aging may also explain the elevated risk of malignant transformation in the elderly population. DisclosuresCamargo:Cell Signaling Technologies: Consultancy; Vital Therapies: Consultancy.

BMP signalling differentially regulates distinct haematopoietic stem cell types.

Adult haematopoiesis is the outcome of distinct haematopoietic stem cell (HSC) subtypes with self-renewable repopulating ability, but with different haematopoietic cell lineage outputs. The molecular basis for this heterogeneity is largely unknown. BMP signalling regulates HSCs as they are first generated in the aorta-gonad-mesonephros region, but at later developmental stages, its role in HSCs is controversial. Here we show that HSCs in murine fetal liver and the bone marrow are of two types that can be prospectively isolated—BMP activated and non-BMP activated. Clonal transplantation demonstrates that they have distinct haematopoietic lineage outputs. Moreover, the two HSC types differ in intrinsic genetic programs, thus supporting a role for the BMP signalling axis in the regulation of HSC heterogeneity and lineage output. Our findings provide insight into the molecular control mechanisms that define HSC types and have important implications for reprogramming cells to HSC fate and treatments targeting distinct HSC types.

Functional evidence implicating chromosome 7q22 haploinsufficiency in myelodysplastic syndrome pathogenesis.

Chromosome 7 deletions are highly prevalent in myelodysplastic syndrome (MDS) and likely contribute to aberrant growth through haploinsufficiency. We generated mice with a heterozygous germ line deletion of a 2-Mb interval of chromosome band 5A3 syntenic to a commonly deleted segment of human 7q22 and show that mutant hematopoietic cells exhibit cardinal features of MDS. Specifically, the long-term hematopoietic stem cell (HSC) compartment is expanded in 5A3(+/del) mice, and the distribution of myeloid progenitors is altered. 5A3(+/del) HSCs are defective for lymphoid repopulating potential and show a myeloid lineage output bias. These cell autonomous abnormalities are exacerbated by physiologic aging and upon serial transplantation. The 5A3 deletion partially rescues defective repopulation in Gata2 mutant mice. 5A3(+/del) hematopoietic cells exhibit decreased expression of oxidative phosphorylation genes, increased levels of reactive oxygen species, and perturbed oxygen consumption. These studies provide the first functional data linking 7q22 deletions to MDS pathogenesis.

Aberrant Hematopoietic Stem Cell Function in Mice Haploinsufficient for a 2 Mb Deletion of Chromosome 5A3 Syntenic to a Region of Human 7q22 Frequently Deleted in Myelodysplastic Syndromes

The myelodysplastic syndromes (MDS) are clonal hematologic neoplasms characterized by normal, increased or decreased bone marrow cellularity, ineffective production of one or more blood cell lineages, morphologic dysplasia, and a variable risk of progression to acute myeloid leukemia. Somatic monosomy 7 (-7) and deletions affecting the long arm of chromosome 7 [del(7q)] are highly prevalent cytogenetic abnormalities in de novo and therapy-related MDS that are associated with advanced age, prior exposure to alkylating agents, a high risk of leukemic transformation, and intrinsic drug resistance. Although recent genome-wide and targeted DNA sequencing efforts have uncovered mutations in genes involved in chromatin modification, transcription, RNA splicing, and signal transduction in MDS, extensive sequencing and expression analysis have not revealed recurring “second hit” mutations of any gene located on chromosome band 7q22, which is commonly deleted in patients with myeloid malignancies. Therefore, answering the long-standing question of how recurrent deletions of the long arm of chromosome 7 contribute to initiation, maintenance, and clinical outcome of MDS remains a fundamental challenge. Given the importance of understanding the biology of -7/del(7q) and the urgent need to develop new mechanism-based treatments for hematologic cancers with these deletions, we generated mice with a heterozygous germline deletion of a 2 Mb interval of chromosome band 5A3 syntenic to an interval of human chromosome band 7q22 commonly deleted in human patients (Blood 88:1930,1996). We find that 5A3 haploinsufficiency perturbs hematopoietic stem cell (HSC) development and function in vivo. 5A3+/del mice exhibit a reduced bone marrow cellularity, an expanded proportion of long-term HSCs, and an altered distribution of lineage-committed progenitors. 5A3+/del HSCs display impaired competitive reconstitution of lymphoid hematopoietic cells and a myeloid output bias, but also paradoxically show enhanced stem cell reconstitution upon aging and after serial transplantation. These defects are cell autonomous. Like WT HSCs, 5A3+/del HSCs exhaust their self-renewal potential by the tertiary round of transplantation. Taqman quantitative real-time PCR confirmed that all seven genes within the deleted interval that are expressed at detectable levels in HSC and multi-potent progenitors (MPP)(Mll5, Armc10, Psmc2, Dnajc2, Orc5l, Pmpcb and Napepld) showed ~50% reduction in transcripts in 5A3+/delHSC and MPP. Transcriptome sequencing (RNA-seq) analysis identified broad changes in lineage signature gene expression, as well as down-regulation of genes and pathways involved in oxidative phosphorylation (OXPHOS) in 5A3+/del HSCs. Whereas reactive oxygen species (ROS) are increased in aged 5A3+/del MPP, treatment of 5A3+/delmice with antioxidant N-Acetyl Cysteine failed to correct the HSC defects, suggesting that increased ROS is not the primary cause of these hematopoietic defects. Together, the abnormal properties of 5A3+/del hematopoietic cells support a mechanistic role of 7q22 deletions as contributing to cardinal features of MDS, which include impaired differentiation, myeloid lineage output bias, and a pronounced age-associated increase in disease incidence. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2292: Enforced expression of miR-125b promotes the in vivo expansion of human Lin- CB multi-lymphoid progenitors (MLP) and AML leukemia stem cells.

Abstract We recently demonstrated stem cell gene signatures predict clinical outcome in acute myeloid leukemia (AML) (Eppert et. al., Nature Medicine, 2011). Concomitant to this work, miRNA signatures for hematopoietic stem cells (HSC) and leukemia stem cells (LSC) were also generated. miRNA are small non-coding RNAs that regulate the translation and mRNA stability of protein coding genes with significant roles in the maintenance of human HSC (Lechman et. al., Cell Stem Cell, in press). To understand the functional role of miRNA in normal human blood development, we undertook an in vivo over-expression screen of 10 miRNA candidates over-represented in HSC and LSC. Lineage depleted human umbilical cord blood cells (Lin- CB) were transduced with lentivirus expressing either a candidate miRNA or control vector and xeno-transplanted into NSG mice. Three miRNA displayed a competitive growth advantage while 4 miRNA induced a growth disadvantage along with skewing of lineage output. A top LSC array candidate, miR-125b, showed the most pronounced phenotype with overt expansion of marked cells, enlarged spleens and increased lymphoid and erythroid output. Detailed analysis of miR-125b grafts revealed a greatly expanded MLP population, in comparison to HSC and MPP. Furthermore, upon enforced in vivo expression of miR-125b in 3 AML patient samples, we observed large increases in the CD34+CD117+ populations for all three AML samples, suggesting increased LSC numbers. Secondary LDA experiments revealed up to a 34 fold increase in LSC activity in comparison to control vector transduced AML cells. These data suggest that miR-125b normally functions in the limited self-renewal of lymphoid committed early progenitors and this function may be usurped during leukemogenesis to enhance LSC self-renewal. Citation Format: Eric R. Lechman, Karin G. Hermans, Stephanie Dobson, Kolja Eppert, Mark Minden, John E. Dick. Enforced expression of miR-125b promotes the in vivo expansion of human Lin- CB multi-lymphoid progenitors (MLP) and AML leukemia stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2292. doi:10.1158/1538-7445.AM2013-2292

Time Course Studies of the Progeny of Barcoded CD34+ Human Cord Blood Cells Generated in Transplanted NOD/SCID-IL2Rγ−/− Mice Unveil the Clonal Dynamics of Short Term, Intermediate Term and Long Term Repopulating Cells

AbstractAbstract 28Hematopoietic stem cells (HSC) exhibit heterogeneity in self-renewal and differentiation activity, but the extent to which this is intrinsically determined and extrinsically regulated is still poorly understood. In the mouse, purities of HSCs can now be achieved to allow such questions to be addressed directly. Interestingly, tracking the outputs of large numbers of serial transplantable clones produced from single-cell transplants, or the clonal progenies of vector-marked/barcoded cells indicate the existence in mice of 2 subsets of HSCs with durable self-renewal ability. These 2 subsets are characterized by distinct lineage output programs that are maintained through the many HSC self-renewal divisions required to serially propagate a clone in vivo. To begin to ask whether similar subsets of human HSCs exist, we have created a diverse lentiviral library encoding an estimated >105 different barcode sequences and GFP, and then used this library to track the in vivo clonal outputs of transduced human CD34+ cord blood cells in xenografted mice. For this experiment, CD34+ cells isolated immunomagnetically to a purity of >80% were exposed to virus for 6 hours in the presence of growth factors and then immediately injected intravenously into 2 sublethally irradiated NOD/SCID-IL2Rγ−/− mice (1.2 × 105 cells per mouse; 30% GFP+ cells after 3 days in vitro). Different subsets of human cells were then isolated by FACS from immunostained bone marrow cells aspirated sequentially from the femurs of the mice at intervals from 4–27 weeks post-transplant and the identity, number and size of clones in each established by next generation sequencing of barcoded amplicons derived from each sample. To identify barcodes arising from PCR and sequencing errors and calibrate clone sizes, we included 3 controls of 20, 100 and 500 cells with a known barcode at each datapoint. The data from these controls allowed a threshold of 20 cells per clone to be established with >95% confidence. We then compared the representation of clones among all samples from each mouse to derive the number and size of all clones detected, assuming a mouse contains 2×108 bone marrow cells. This analysis revealed a total of 154 uniquely barcoded clones containing up to 2×108 human hematopoietic cells in the 2 mice (8–30 × 106 in one and 4–165 × 106 in the other at any single time point). Analysis of the representation of each clone over time showed successive waves of repopulation from different clones with lineage output profiles consistent with those obtained by transplanting separate fractions of CD34+ cord blood cells distinguished by their surface phenotypes. Specifically, we detected 50 clones (32% of all clones) that were not sustained at detectable levels beyond 9 weeks post-transplantation and were characterized by robust myeloid differentiation with variable B cell outputs at 4 weeks. Another 30 clones (19%) showed significant but also transient outputs of either or both the myeloid and B cell lineage, disappearing between week 9 and 16 post-transplant. Mature cell output was detected from a total of 74 clones (48%) at the 27 week time point, among which 36 (23%) were not evident during the first 4 months post-transplant. These late-appearing clones were mostly small (contributing up to 3 × 105 total hematopoietic cells at week 27) and made a significantly higher contribution to the total human myeloid population than to the total human B cell population. Notably, the 12 long term clones that showed robust mature cell output detectable in all 3 sites sampled at week 27 when the mice were sacrificed (left leg vs right leg vs pelvis) contained both myeloid and lymphoid cells but with large (>100-fold) variations in their representation in the 3 different sites. This latter finding suggests less trafficking of human cells between sites than expected from parabiotic mouse experiments or substantial differences in the differentiation control exerted in different locations. Additionally, from one of the mice, we obtained the first direct evidence of a large output of human T cells (>9 × 106) that was part of a long term multi-lineage clone detectable at 27 weeks post-transplant. This first use of a barcoding strategy to analyze the clonal dynamics of normal human CD34+ cells with in vivo repopulating activity demonstrates the power of this approach to analyze their lineage outputs and sets the stage for novel applications to expanded and transformed populations. Disclosures:No relevant conflicts of interest to declare.

The Lymphoblastic Leukemia Gene 1 (Lyl1) Regulates Lymphoid Specification and Maintenance of Early T Lineage Progenitors

Abstract 548Long-term thymopoiesis crucially depends on the recruitment and expansion of bone marrow derived progenitor cells. Therefore, a tight regulation of the thymus-settling progenitor cells is required to maintain T cell lineage homeostasis. Lyl1, a transcription factor involved in homeostatic control of immature hematopoietic cells, remains expressed in early T-lineage progenitors (ETPs) until T cell lineage commitment.Here we demonstrate a critical requirement for Lyl1 in lymphoid priming of bone marrow (BM) progenitors and in the maintenance of ETPs. Lyl1 deficient hematopoiesis was unable to generate sufficient numbers of lymphoid-primed progenitor populations such as LMPPs, CLPs and in particular ETPs. We found a significant (p>0.001) 4-fold reduction of LMPPs in the BM and a 20-fold reduction (p<0.001) of ETPs in the thymus of Lyl1 KO mice compared to wildtype controls. Transplantation assays revealed a selective defect of Lyl1−/− LMPPs for thymic engraftment and T lineage development. Intra-thymic injections of Lyl1−/− LMPPs demonstrated, in absence of homing requirements, a cell autonomous defect of Lyl1−/− progenitors in their ability to undergo the ETP to DN2 transition and to expand in response to Notch. Injection of Lyl1+/+ LMPPs resulted in a 5-fold higher recovery of total donor cells compared to injections of Lyl1−/− LMPPs (p<0.001). 40% of the donor cells derived after intra-thymic injection of Lyl1+/+ LMPPs were committed to the T cell lineage (DN3, DP, SP), whereas T lineage commitment after injection of Lyl1−/− cells was only seen in 20% of the recovered cells (p<0.01). By absolute numbers, wildtype LMPPs had generated more DN3 (8-fold), more DP (21-fold) and more SP (9-fold) T cell lineage committed thymocytes, and fewer Lyl1+/+ cells remained in the c-kit positive “ETP/LMPP-like” stage (-0.5-fold).In contrast, reintroduction of Lyl1 cDNA into Lyl1−/− BM progenitors using retroviral vectors restored the thymic progenitor pool and enhanced T cell lineage output. At 12 weeks after transplantation of MIG-Lyl1 transduced cells we observed a significant expansion of transfected (GFPpos) cells in the peripheral blood solely attributable to expansion of normal, mature and poly-clonal T cells (p<0.001). The thymuses of MIG-Lyl1 transplanted recipients showed significantly greater overall cellularity (p<0.01) attributable to a significantly higher proportion of GFPpos thymocytes (p<0.01) compared to the MIG-GFP transplanted control group.To gain a more detailed understanding of the underlying molecular mechanisms of Lyl1-mediated T-lymphoid specification, we performed global gene expression profiling of wildtype and Lyl1−/− LMPPs as well as whole genome ChIP-seqencing in HPC-7 cells after pull-down with anti-Lyl1 antibodies. Here, we identified the lymphoid-promoting factor Gfi1 as a critical transcriptional target of Lyl1-mediated T lymphopoiesis. We found that Gfi1 expression was decreased in Lyl1−/− LMPPs and ETPs by 2- and 7.5- fold respectively. ChIP assays using ckitpos BM cells from Lyl1+/+ and Lyl1−/− mice revealed a strong enrichment of Lyl1 at a known enhancer region of the Gfi1 locus located 35kb upstream of Gfi1 (p<0.001). Binding of Lyl1 activated the Gfi1 35kb enhancer element in transactivation assays (p<0.001). Finally, intra-thymic injection of Lyl1−/− progenitors after retroviral transduction with Gfi1 cDNA allowed lymphoid development and enhanced the T cell lineage output compared to GFP-transduced controls (p=0.08).Collectively, our data provide evidence that pro-T cell expansion in the thymus is regulated through intrinsic control of thymus progenitor cells that employ a transcriptional program already established in hematopoietic stem and progenitor cells. We identify Lyl1 as a critical component of this regulatory network, which is vital for the maintenance of T cell lineage homeostasis. Finally, identification of important downstream mediators of Lyl1 function not only illuminates the molecular mechanisms underlying early T-cell development, but also suggests previously unrecognized pathways likely to play a role in Lyl1-mediated development of leukemia and lymphoma. Disclosures:No relevant conflicts of interest to declare.

Is normal hematopoiesis maintained solely by long-term multipotent stem cells?

AbstractThe understanding of the hierarchical organization of the human hematopoietic system is of major biologic and clinical significance. The validity of the conventional model in which hematopoiesis is solely maintained by a pool of multipotent long-term hematopoietic stem cells (LT-HSCs) has been recently challenged by several mouse studies. These new data point to the existence of a heterogeneous stem cell population that consists of distinct subsets of LT-HSCs, which include stem cells biased toward lineage-specific differentiation programs. This review attempts to discuss the balanced versus biased patterns of lineage output of human LT-HSCs gathered in 3 different gene therapy trials on the basis of vector integration site analysis by deep sequencing. The distribution of integration sites observed tends to support the validity of the revised model.

The erythroid/myeloid lineage fate paradigm takes a new player

This issue of The EMBO Journal features a new paradigm for cell lineage commitment. By studying early haematopoiesis in zebrafish, Roger Patient and colleagues characterize the Tif1 gamma-ligase as a context-specific regulator of Gata1/PU1-driven fate decisions.

Uncovering Haematopoietic System Dynamics and Single Multipotent Progenitors Activity In Vivo In Humans by Retroviral Tagging.

AbstractAbstract 2611The long-standing model of human haematopoiesis postulates that myeloid and lymphoid lineages branch separately at very early stages, producing myeloid or erythroid cells and T or B cells, respectively. Conversely, a revised scheme of haematopoietic hierarchy was recently proposed, in which myeloid cells represent a prototype of blood cells, while erythroid, T and B cells are specialized cell types. The validity of these models has been mainly tested in vivo in the mouse, and in vitro through clonal assays on human haemopoietic stem cells (HSC). However, a clear definitive elucidation of the real nature of human haemopoiesis should ideally involve the ability to track the dynamics, survival and differentiation potential of haemopoietic progenitor clones for a long period of time directly in vivo in humans. Upon retroviral gene transfer, transduced cells are univocally tagged by vector insertions allowing them to be distinguished and tracked in vivo by integration profiling. We previously showed that gene therapy (GT) for adenosine deaminase (ADA) deficient SCID based on infusion of transduced CD34+ cells and reduced intensity conditioning, resulted in full multilineage engraftment, in the absence of aberrant expansions. Therefore, long-term studies in these patients provide a unique human model to study in depth haemopoietic clonal dynamics by retroviral tagging. For this reason, we performed a comprehensive multilineage longitudinal insertion profile of bone marrow (BM) (CD34+, CD15+, CD19+, Glycophorin+) and peripheral blood (PB) (CD15+, CD19+, CD4+, CD8+ cells, naïve and memory T cell subpopulations) cells in 4 patients 3–6 years after GT, retrieving to date 1055 and 1999 insertions from BM and PB cell lineages respectively. We could shape the insertional landscape of each lineage through a tri-factorial analysis based on the number of integrations retrieved, the percentage of vector positive cells and the number of insertion shared with other lineages. We were able to uncover the effects of selective advantages of gene-corrected cells in periphery and the frequency of identical integrants in different haematopoietic compartments. BM cells displayed the highest proportion of shared integrants (up to 58.1%), reflecting the real-time repopulating activity of gene-corrected progenitors. On the other hand, PB samples carried in general a higher frequency of vector positive cells, with the exception of PB CD15+ cells showing insertional landscapes very similar to the one of BM lineages. Interestingly, the detection of exclusively shared myeloid-T\\B or myeloid-erythroid integrants may be supportive of a myeloid-based haemopoiesis model. We also uncovered “core integrants”, shared between CD34+ cells and both lymphoid and myeloid lineages, stably tagging active long-term multipotent progenitors overtime. Strikingly, one of these progenitor clones carried an insertion inside one of the two fragile sites of MLLT3 gene, involved by translocation events in mixed lineage leukemia. We were able to track this and another integrant (downstream the LRRC30 gene) by specific PCRs, confirming the multilineage contribution to haematopoiesis of the relative progenitor clones and their fluctuating lineage outputs over 4 years, without showing aberrant expansions. We also retrieved 170 and 174 integrations from 4 T cell subtypes (Naive, TEMRA, Central and Effector memory) in two patients under PBL-GT and HSC-GT respectively. We found evidences that single naive T cell clones may survive in patients for up to 10 years after last infusion while maintaining their differentiation capacity into different T cell subpopulations. Interestingly, a cluster of 4 insertions (one of them shared among all T cell subtypes) was found in proximity of the interferon regulatory factor 2 binding protein 2 (IRF2BP2) gene in naive T cells from PBL-GT patient, thus suggesting an influence of transcriptional activity of this region on selective advantage of gene-corrected lymphocytes. In conclusion, through retroviral tagging, we can uniquely track single transduced haemopoietic cells directly in vivo in humans. The application of mathematical models to our insertion datasets is allowing to uncover new information on the fate and activity of haematopoietic progenitors and their differentiated progeny years after transplantation in GT patients. Disclosures:No relevant conflicts of interest to declare.

Lineage-specific combinatorial action of enhancers regulates mouse erythroid Gata1 expression

AbstractPrecise spatiotemporal control of Gata1 expression is required in both early hematopoietic progenitors to determine erythroid/megakaryocyte versus granulocyte/monocyte lineage output and in the subsequent differentiation of erythroid cells and megakaryocytes. An enhancer element upstream of the mouse Gata1 IE (1st exon erythroid) promoter, mHS−3.5, can direct both erythroid and megakaryocytic expression. However, loss of this element ablates only megakaryocytes, implying that an additional element has erythroid specificity. Here, we identify a double DNaseI hypersensitive site, mHS−25/6, as having erythroid but not megakaryocytic activity in primary cells. It binds an activating transcription factor complex in erythroid cells where it also makes physical contact with the Gata1 promoter. Deletion of mHS−25/6 or mHS−3.5 in embryonic stem cells has only a modest effect on in vitro erythroid differentiation, whereas loss of both elements ablates both primitive and definitive erythropoiesis with an almost complete loss of Gata1 expression. Surprisingly, Gata2 expression was also concomitantly low, suggesting a more complex interaction between these 2 factors than currently envisaged. Thus, whereas mHS−3.5 alone is sufficient for megakaryocytic development, mHS−3.5 and mHS−25/6 collectively regulate erythroid Gata1 expression, demonstrating lineage-specific differences in Gata1 cis-element use important for development of these 2 cell types.

Heterogeneity of Embryonic and Adult Stem Cells

New studies suggest that stem cells of embryonic, neural, and hematopoietic origin are heterogeneous, with cells moving between two or more metastable states. These cell states show a bias in their differentiation potential and correlate with specific patterns of transcription factor expression and chromatin modifications.