Long-term Repopulating Hematopoietic Stem Cells Research Articles

Human Adult Long-Term Repopulating Hematopoietic Stem Cells Can be Purified By Surface cMPL Expression

Simple combinations of markers that reliably demarcate human long-term repopulating hematopoietic stem cells (LT-HSCs) would facilitate our understanding of their unique molecular state and the development of stem cell-based therapeutics. The complex CD34+CD38-CD45RA-CD90+CD49f+ phenotype is now routinely accepted to define a subset of cord blood CD34+ cells enriched in LT-HSCs (Notta, Science 2011). The same phenotype with the addition of rhodamine can enrich human LT-HSCs within mobilized peripheral blood (MPB) CD34+ cells, but lower stem cell enrichment is observed (Huntsman, Blood 2015). The hematopoietic cytokine thrombopoietin (TPO) and its receptor cMPL act as primary regulators of HSC function. In recent clinical studies, the synthetic TPO agonist eltrombopag was shown to improve long-term trilineage hematopoiesis in patients with immune aplastic anemia, providing evidence that activation of cMPL receptor stimulates residual multipotent LT-HSCs within the bone marrow (BM). Building on these observations, we hypothesized that cMPL might also be a relevant marker to delineate LT-HSC activity within human adult CD34+ cell populations and provide the basis for a novel LT-HSC purification scheme. To assess the utility of surface cMPL expression for purification of human adult LT-HSCs, we first performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) of human MPB CD34+ cells from a healthy volunteer. CITE-seq was conducted with antibodies designed to react with human cMPL receptor and previously described HSC epitopes (CD34, CD38, CD90, CD45RA, and CD49f). We identified 7 distinct clusters in dimension-reduction (UMAP) analysis. By confirming expression levels of HSPC gene signatures and HSC surface markers, such as CD38, we assigned each CD34+ cell cluster to a distinct HSPC subpopulation, including a single LT-HSC population (cluster 2) (Fig A). Notably, CD34+ cell subsets expressing higher levels of surface cMPL expression were highly enriched in transcriptionally defined HSC population (cluster 2) and consistently co-expressing the known human LT-HSC phenotypic markers, including lower levels of surface CD38 and CD45RA expression and higher levels of surface CD90 and CD49f expression (Fig B). To determine whether surface cMPL expression can enrich functional LT-HSCs, we next performed serial xenotransplantation assays. We used fluorescence-activated cell sorting to partition human MPB CD34+ cells into cMPL high (top 10%) and cMPL low (bottom 10%) populations, and transplanted these cells into immunodeficient NBSGW mice. Serial peripheral blood (PB) sampling of primary xenografted mice revealed a striking difference in patterns of hematopoietic reconstitution over time between the two groups. In mice transplanted with cMPL high cells, human cell chimerism increased gradually throughout the 16-week engraftment period, while a progressive decline in engraftment was observed in the cMPL low group. Notably, mean human cell engraftment within the PB, BM and spleen of mice transplanted with cMPL high cells was 209-fold (p < 0.01), 37-fold (p < 0.0001) and 283-fold (p < 0.001) higher than mean human cell chimerism in the cMPL low group at 16 weeks post-transplantation, respectively. These data suggest that cMPL high and cMPL low populations are distinctly enriched in LT-HSCs and hematopoietic progenitors, respectively. Next, to quantitatively compare the frequency of self-renewing LT-HSCs within the cMPL high and cMPL low HSPC subsets, human CD45+ cells obtained from the BM of primary mice were injected into secondary NBSGW mice at limiting dilution and BM engraftment was measured at 16 weeks post-transplantation (total period of engraftment: 32 weeks). After accounting for engraftment parameters (i.e., cell dose and engraftment levels) in both primary and secondary mice, extreme limiting dilution analysis computed self-renewing LT-HSC frequencies of 1 in 1,267 within the cMPL high CD34+ cell population, and 1 in 149,010 in the cMPL low CD34+ cell fraction, representing a 116-fold enrichment of LT-HSCs using cMPL as a single marker purification strategy. Collectively, these data suggest that cMPL expression can effectively delineate LT-HSCs in human adult CD34+ HSPCs and thus serve as an additional relevant marker for the development of human adult HSC targeting therapeutics.

ABL001 (asciminib) Efficiently Targets Transplantable BCR::ABL1 Lymphoid Blast Crisis in the Scl-Tta-p210-BCR::ABL1 Mouse Model

Objectives: This study aimed at investigating the therapeutic efficacy of ABL001 in the ScltTA-p210-BCR::ABL1 transgenic mouse model. ABL001 (asciminib) is a novel allosteric inhibitor of ABL kinase demonstrating its particular efficacy against BCR::ABL1 mutations that emerge during treatment with traditional TKIs. However, the activity and tolerability of ABL001 in a primary mouse model of BCR::ABL1 disease remain unknown. In addition, resistance mutations against ABL001 were observed in the myristoyl pocket of BCR::ABL1, but their emergence has not been analyzed in BCR::ABL1+ murine hematopoietic cells. Importantly, leukemic stem cells (LSCs) expressing BCR::ABL1 have developed mechanisms to evade eradication by tyrosine kinase inhibitors, underscoring the necessity for alternative therapeutic approaches. Methods: 5-fluorouracil- (5-FU-) treated bone marrow (BM) cells harvested from FVB/N-tg(ScltTA)(tetO- p210-BCR::ABL1) double-transgenic (dtg) donor mice (CD45.1+) were transplanted into lethally irradiated recipient mice (CD45.2+). One week after transplantation, tetracycline (tet) was removed from the drinking water to induce Bcr-Abl1 expression. Following an additional week, mice were treated daily with ABL001 or vehicle control by oral gavage (30 mg/kg body weight). Therapeutic effects were assessed using flow cytometry (peripheral blood, spleen, and BM), histological examination, RNA analysis and DNA sequencing of the myristoyl pocket of BCR::ABL1. Secondary transplants using splenocytes were performed to analyze whether ABL001 treatment was able to suppress BCR::ABL1-positive repopulating malignant stem cells. Additionally, we assessed potential ABL001-resistant subclones after secondary transplantation by subjecting the mice to further ABL001 treatment, modeling clinically relevant mutations in BCR::ABL1-positive disease. Results: Within 3 weeks after tet withdrawal, the transplanted mice developed an aggressive B-lymphoblastic p210-BCR::ABL1-positive disease, mimicking lymphoid blast crisis, as described previously (Koschmieder et al., Blood, 2005). Interestingly, ABL001 treatment significantly reduced this BCR::ABL1-driven phenotype as indicated by suppression of malignant B cells and restoration of normal hematopoiesis. ABL001 also significantly improved survival compared to the vehicle control group (p=0.026). Histological analysis confirmed the alleviation of the BCR::ABL1-driven phenotype in spleen and BM. Furthermore, ABL001 treatment significantly reduced BCR::ABL1 transcripts to background levels, demonstrating its ability to suppress BCR::ABL1-induced disease in the BM. The efficacy of ABL001 in reducing the malignant clone correlated with normalization of spleen weight. Essentially, ABL001 treatment normalized the phenotypic long-term repopulating hematopoietic stem cell (LT-HSC) population in the BM, suggesting restoration of the stem cell pool and targeting malignant LT-HSCs. This was supported by secondary transplantation experiments, showing that several mice receiving ABL001-treated splenocytes failed to develop leukemia, while all mice who received vehicle-treated cells did. Moreover, none of the secondary transplanted mice who were treated with ABL001 developed leukemia. Interestingly, Sanger sequencing of the BCR::ABL1 myristoyl pocket region demonstrated wildtype sequences and the absence of resistance mutations. Conclusion: Our study demonstrates the efficacy of ABL001 in ameliorating p210-BCR::ABL1-induced lymphoid blast crisis by specifically targeting BCR::ABL1-positive malignant stem cells and restoring normal hematopoiesis in a transgenic mouse model. These promising findings highlight the potential of ABL001 as an alternative therapeutic strategy for first-line treatment of BCR::ABL1-driven leukemia, including B-lymphoblastic blast crisis. Furthermore, since TKI treatment alone has not proven successful in patients with lymphoid blast crisis, this now opens new avenues for the in vivo-testing of novel combination treatments of ABL001 with chemotherapy or other targeting agents.

Stromal STAT5-Mediated Trophic Activity Regulates Hematopoietic Multipotent Progenitor Niche Factors

Signal transducer and activator of transcription 5 (STAT5a and STAT5b isoforms) are latent transcription factors that are intrinsically critical for normal hematopoiesis but are also expressed in bone marrow stromal cells. However, despite known STAT5a (prolactin) and STAT5b (growth hormone) functions in breast and liver tissue respectively, hematopoiesis-supporting stromal functions have not been explored. Here, floxed STAT5ab double knockout (KO) was generated with a variety of hematopoietic and stromal Cre-expressing transgenic mouse strains to test whether STAT5a or STAT5b play a role in extrinsic regulation of hematopoiesis. Pan-hematopoietic deletion with Vav1-Cre was the positive control for loss of multipotent hematopoietic function but surprisingly Vav1-Cre also deleted STAT5ab in CD45neg cells, dysregulated stromal niche factor mRNA expression, and altered MSC osteo/chondro-lineage differentiation priming. Single cell transcriptome analysis of bone marrow from wild-type or Vav1-Cre KO mice long-term repopulating hematopoietic stem cells (LT-HSC) showed upregulated oxidative phosphorylation (P<10-10) and protein translation (P<10-39) genes in LT-HSCs and throughout myeloid-primed clusters, while in contrast common lymphoid progenitor-like cells were completely eliminated. Similarly, like Vav1+ cells and based on scRNAseq analysis, Nes+ cells were also detected in both CD45neg and CD45+ cell clusters. Deletion of STAT5ab with Nes-Cre reduced the number of LT-HSCs (30% reduction compared to Nes-Cre control mice, P=0.02, N=4) and caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45+ and CD45neg bone marrow cell populations, additional stroma-specific Cre strains (LepR, Osx, Ocn, Prrx1, Nes-CreERT2) as well as endothelial-specific (Cdh5) strains were generated lacking STAT5ab. Viable mice were assessed for peripheral blood hematology, competitive repopulation, as well as hematopoietic stem/progenitor subsets (LT-HSC, MPP1, MPP2, MPP3, MPP4, KLS) using well-accepted flow cytometry antibody panels. Deletion of STAT5 in stromal cells with LepR-Cre significantly reduced MPP4 (35% reduction, P=0.025, N=4). Overall, extrinsic stromal (but not endothelial) cells were deficient in supporting multipotent and lineage-committed hematopoietic progenitors. However, peripheral blood hematology was rescued through homeostatic production. Gene regulation profiles resembled that of cytokine-starved MPPs. Functional support for niche-supporting activity was also assessed using STAT5ab KO MSCs in vitro. With Lepr-Cre mediated STAT5ab deletion, bone marrow niche factor mRNAs were downregulated (Cxcl12, Angpt1, Igfbp4, Igf1, Il34) and support of multipotent hematopoietic cells was significantly impaired. ELISA assay showed that SDF-1a was reduced 3-fold (P<0.001, N=5) and IGF-1 was reduced 2-fold (P<0.01, N=5) compared to wild type MSCs. Inversely, mRNAs for Kitl and Grem1 were increased in the same stromal cells, consistent with loss of multipotency and mixed HSC vs. MPP stromal support activities. Furthermore, novel scRNAseq analysis on simultaneous bone marrow hematopoietic and stromal cells compared wild-type and KO mice. Differentially expressed gene sets with Maximum Mean Discrepancy (MMD) co-expression revealed a key central role for coordinated STAT5ab/Cish balance as a biomarker of the cellular progenitor activity state (metabolic, protein translation, survival) associated with multipotency, comparing KO to wild-type cells. Importantly, Cish was strongly co-expressed in cells primed for lineage-specific differentiation (MSC - osteo/chondrogenic-primed; HSC - myeloid-primed) with STAT5ab co-expressed genes being associated with multipotent potential. Therefore, novel STAT5ab/Cish-mediated gene regulation programs operate within the bone marrow microenvironment to mediate cytokine production and responsiveness. This work highlights the importance of examining heterotypic cell types within bone marrow to elucidate functional cross-talk underlying hematopoietic homeostasis and defines conserved STAT5ab-mediated gene signatures as potential biomarkers to test in human hematopoietic and immune cell disorders.

Peptidoglycan-Mediated Bone Marrow Autonomic Neuropathy Impairs Hematopoietic Stem/Progenitor Cells via a NOD1-Dependent Pathway in db/db Mice.

Impairment of bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) contributes to the progression of vascular complications in subjects with diabetes. Very small amounts of bacterial-derived pathogen-associated molecular patterns (PAMPs) establish the bone marrow cell pool. We hypothesize that alteration of the PAMP peptidoglycan (PGN) exacerbates HSPC dysfunction in diabetes. We observed increased PGN infiltration in the bone marrow of diabetic mice. Exogenous administration of PGN selectively reduced the number of long-term repopulating hematopoietic stem cells (LT-HSCs), accompanied by impaired vasoreparative functions in db/db mouse bone marrow. We further revealed that bone marrow denervation contributed to PGN-associated HSPC dysfunction. Inhibition of NOD1 ameliorated PGN-induced bone marrow autonomic neuropathy, which significantly rejuvenated the HSPC pools and functions in vivo. These data reveal for the first time that PGN, as a critical factor on the gut-bone marrow axis, promotes bone marrow denervation and HSPC modulation in the context of diabetes.

NOTCH-mediated exvivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia.

SummaryActivation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.

Priming Human Hematopoietic Stem and Progenitor Cells for Cas9/Sgrna and rAAV6-Mediated Homologous Recombination

Highly efficient engineered nuclease-mediated gene editing through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) gives rise to the opportunity of developing gene corrective therapies for hematological disorders. The β-hemoglobinopathies, such as sickle cell disease (SCD) and β-thalassemia, are inherited blood disorders that manifest because of mutations in the beta-globin gene (HBB) and afflict millions of people worldwide. The only curative treatment for these blood disorders is allogeneic-hematopoietic stem cell transplant (allo-HSCT) from a healthy donor, which comes with severe limitations, such as a shortage of immunologically matched donors, graft-versus-host disease, and graft rejection. With the lack of effective curative therapies, it is postulated that autologous HSCT of HBB gene corrected HSPCs would be a potential one-time curative treatment for patients afflicted with β-hemoglobinopathies without the risk of graft versus host diseases and graft rejection, as well as avoiding the need to find an immunologically matched donor. Our previous work demonstrated a methodology to efficiently target the beta-globin gene (HBB) in HSPCs by inducing a site-specific double strand break (DSB) with the RNA-guided (sgRNA) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease (Cas9/sgRNA) system while supplying an homologous HBB gene corrective donor template via recombinant adeno-associated virus serotype 6 (rAAV6). Here, we further build upon that work to identify the critical parameters to reproducibly achieve high levels of HR in HSPCs. By optimizing Cas9 ribonucleoprotein (RNP) concentration and electroporation conditions, we were able to induce DSBs in long-term repopulating hematopoietic stem cells (LT-HSCs), notably in serial transplants engrafting into immunodeficient non-obese diabetic (NOD)- severe combined immunodeficiency (SCID) Il2rg−/− (NSG) mice where we found ~49% of human cells had INDELS in the HBB gene, matching our input, implying the CRISPR/Cas9 system is effective at inducing double strand breaks at the HBB locus in long term repopulating stem cells. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a two-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis and subsequently higher HR levels. Perhaps the most interesting finding is that when HSPCs are cultured at low densities (100,000 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies. Building upon that work, we show that culturing HBB -targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in NSG mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies, but also as a foundation for editing HSPCs at other loci for both basic and translational research. DisclosuresPorteus:CRISPRTx: Equity Ownership.

Indispensable role of Galectin-3 in promoting quiescence of hematopoietic stem cells

Hematopoietic stem cells (HSCs) in adult bone marrow (BM) are usually maintained in a state of quiescence. The cellular mechanism coordinating the balance between HSC quiescence and differentiation is not fully understood. Here, we report that galactose-binding lectin-3 (galectin-3; Gal-3) is upregulated by Tie2 or Mpl activation to maintain quiescence. Conditional overexpression of Gal-3 in mouse HSCs under the transcriptional control of Tie2 or Vav1 promoters (Gal-3 Tg) causes cell cycle retardation via induction of p21. Conversely, the cell cycle of long-term repopulating HSCs (LT-HSCs) in Gal-3-deficient (Gal-3-/-) mice is accelerated, resulting in their exhaustion. Mechanistically, Gal-3 regulates p21 transcription by forming a complex with Sp1, thus blocking cell cycle entry. These results demonstrate that Gal-3 is a negative regulator of cell-cycling in HSCs and plays a crucial role in adult hematopoiesis to prevent HSC exhaustion.

Endothelial Jak3 expression enhances pro-hematopoietic angiocrine function in mice

Jak3 is the only non-promiscuous member of the Jak family of secondary messengers. Studies to date have focused on understanding and targeting the cell-autonomous role of Jak3 in immunity, while functional Jak3 expression outside the hematopoietic system remains largely unreported. We show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow. The bone marrow niche is understood as a network of different cell types that regulate hematopoietic function. We show that the Jak3–/– bone marrow niche is deleterious for the maintenance of long-term repopulating hematopoietic stem cells (LT-HSCs) and that JAK3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. This work may serve to identify a novel function for a highly specific tyrosine kinase in the bone marrow vascular niche and to further characterize the LT-HSC function of sinusoidal endothelium.

Combined Single Cell Lineage and Transcriptome Sequencing Unveils Cell-Autonomous Regulators of Hematopoietic Stem Cell Fate

Blood regeneration upon transplantation relies on the activity of long-term repopulating hematopoietic stem cells (LT-HSCs). One of the major controversies in hematopoiesis relates to the apparently different properties that HSCs have in transplantation versus unperturbed settings. In unperturbed steady state hematopoiesis, the most potent HSCs appear to be mostly dormant, and only producing platelet-lineage cells. In turn, upon transplant, even a single transplanted HSC can actively divide and regenerate hundreds of millions of blood progenitors of all lineages. It would thus appear that HSCs have different fundamental properties in each study system. However, most transplantation studies have only tracked the lineage output of the transplanted HSC clones, and rarely the regeneration of the HSC compartment itself. In addition, clonal assays have not been performed at sufficient resolution to fully capture the diversity and clonal complexity of the regenerated HSC compartment. Here, we have used expressible barcodes, which can be sequenced in conventional single cell RNAseq assays, to simultaneously record the functional outcomes and transcriptional states of thousands of HSCs. Our analysis revealed multiple clonal HSC behaviors following transplantation that drastically differ in their differentiation activity, lineage-bias and self-renewal. Surprisingly, we witnessed a large fraction of clones that efficiently repopulate the HSC compartment but show limited contribution to differentiated progeny. Furthermore, these inactive clones have increased competitive multilineage serial repopulating capacity, implying that shortly after transplant a subset of clones reestablishes the native-like LT-HSC behaviors. Our results also argue that this clonal distribution of labor is controlled by cell autonomous, heritable properties (i.e. the epigenetic cell state). Then, using only our clonal readouts to segregate single HSC transcriptomes, we unveiled the transcriptional signatures that associated with unique HSC outcomes (platelet bias, clonal expansion, dormancy, etc.) and unraveled, for the first time, a gene signature for functional long-term serially repopulating clones. We interrogated the drivers of this cell state using an in vivo inducible CRISPR screening and identified 5 novel regulators that are required to regenerate the HSC compartment in a cell autonomous fashion. In conclusion, we demonstrate that functional LT-HSCs share more similar properties in native and transplantation hematopoiesis than previously expected. Consequently, we unveil a definition of the essential, common functional properties of HSCs and the molecular programs that control them. Figure 1 Disclosures No relevant conflicts of interest to declare.

2027 - SIMULTANEOUS LINEAGE AND TRANSCRIPTIONAL ANALYSIS OF SINGLE HSCS IDENTIFIES MECHANISMS OF FUNCTIONAL HETEROGENEITY

The hematopoietic system follows a hierarchical organization, with long-term repopulating hematopoietic stem cells (LT-HSCs) occupying the top tier. Application of systems-level single cell methods has revealed a remarkable functional and transcriptional heterogeneity within stem cells. To elucidate the molecular underpinnings of this functional heterogeneity, we developed a technique to barcode hematopoietic cells at the RNA level in order to simultaneously capture the lineage relationships and transcriptional states of HSCs with unprecedented robustness. Using a droplet-based single cell RNAseq platform, we analyzed the total fraction of engrafted hematopoietic stem cells together with a sufficiently significant representation of downstream progenitor cells to estimate HSC output. Inspection of the resulting stem cell state maps revealed a variety of clonal behaviors, including cell quiescence, asymmetric and symmetric divisions, and clonal competition. We also correlated these behaviors with some of the previously observed heterogeneity during transplantation, including lineage bias and clonal dominance. Importantly, comparison of single HSC expression profiles revealed significant differences in the transcriptional programs related with some of these behaviors, which illuminated the molecular machineries that operate at the stem cell level to define this heterogeneity. Finally, a loss-of-function in vivo screening for clonal quiescence/expansion regulators identified several novel candidates of this process. Thus, our work has harnessed the power of combined functional/transcriptional single cell readouts to discover novel mechanisms that orchestrate the in vivo complexity of stem cell activities.

Integrin-α3 Is a Functional Marker of Ex Vivo Expanded Human Long-Term Hematopoietic Stem Cells

Transplantation of expanded hematopoietic stem cells (HSCs) and gene therapy based on HSC engineering have emerged as promising approaches for the treatment of hematological diseases. Nevertheless, the immunophenotype of cultured HSCs remains poorly defined. Here, we identify Integrin-α3 (ITGA3) as a marker of cultured human HSCs. Exploiting the pyrimidoindole derivative UM171 to expand cord blood (CB) cells, we show that ITGA3 expression is sufficient to separate the primitive EPCR+CD90+CD133+CD34+CD45RA- HSC population into two functionally distinct fractions presenting mostly short-term (ITGA3-) and both short-term and long-term (ITGA3+) repopulating potential. ITGA3+ cells exhibit robust multilineage differentiation potential, serial reconstitution ability in immunocompromised mice, and an HSC-specific transcriptomic signature. Moreover, ITGA3 expression is functionally required for the long-term engraftment of CB cells. Altogether, our results indicate that ITGA3 is a reliable marker of cultured human long-term repopulating HSCs (LT-HSCs) and represents an important tool to improve the accuracy of prospective HSC identification in culture.

Transcriptional and Functional Description of Stem Cell Heterogeneity in Native and Transplantation Hematopoiesis

The hematopoietic system follows a hierarchical organization, with multipotent long-term repopulating hematopoietic stem cells (LT-HSCs) occupying the top tier. This paradigm, developed mostly through cell transplantation assays, has recently been contested by a series of studies performed under native conditions, without transplantation. Application of systems-level single cell methods in this setting has revealed a heterogeneity of cell states within progenitors and stem cells, prompting a reevaluation of the theories of hematopoietic lineage fate decisions. We have previously described that hematopoietic stem cell fates are clonally heterogeneous under steady state and uncovered that a fraction of LT-HSCs contributes to a significant proportion of the megakaryocytic cell lineage under steady state, while rarely generating other types of progeny in unperturbed conditions. To elucidate the molecular underpinnings of this functional lineage-output heterogeneity, we developed a technique to barcode hematopoietic cells at the RNA level in order to simultaneously capture the lineage relationships and transcriptional states of HSCs. Using a droplet-based massive single cell RNAseq platform, we analyzed thousands of engrafted hematopoietic stem cells together with a sufficiently significant representation of downstream progenitor cells to measure HSC output. Inspection of the resulting “stem cell state-fate maps” revealed a variety of stem cell behaviors, including single cell quiescence, asymmetric and symmetric divisions, and clonal expansion. We also connected these behaviors with some of the previously observed heterogeneity in stem cell outcomes, including lineage bias, lineage output and clonal competition. Importantly, clustering of expression profiles revealed significant differences in the transcriptional programs related with some of these behaviors, which illuminate the molecular machineries that operate at the stem cell level to define this heterogeneity. Thus, our work has identified potential novel mediators for stem cell heterogeneity, which we are functionally analyzing in further detail to understand their molecular mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Daily Onset of Light and Darkness Differentially Controls Hematopoietic Stem Cell Differentiation and Maintenance

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

Flt3 ligand-eGFP-reporter expression characterizes functionally distinct subpopulations of CD150+ long-term repopulating murine hematopoietic stem cells.

The pool of hematopoietic stem cells (HSCs) in the bone marrow is a mixture of resting, proliferating, and differentiating cells. Long-term repopulating HSCs (LT-HSC) are routinely enriched as Lin- Sca1+ c-Kit+ CD34- Flt3- CD150+ CD48- cells. The Flt3 ligand (Flt3L) and its receptor Flt3 are important regulators of HSC maintenance, expansion and differentiation. Using Flt3L-eGFP reporter mice, we show that endogenous Flt3L-eGFP-reporter RNA expression correlates with eGFP-protein expression. This Flt3L-eGFP-reporter expression distinguishes two LT-HSC populations with differences in gene expressions and reconstituting potential. Thus, Flt3L-eGFP-reporterlow cells are identified as predominantly resting HSCs with long-term repopulating capacities. In contrast, Flt3L-eGFP-reporterhigh cells are in majority proliferating HSCs with only short-term repopulating capacities. Flt3L-eGFP-reporterlow cells express hypoxia, autophagy-inducing, and the LT-HSC-associated genes HoxB5 and Fgd5, while Flt3L-eGFP-reporterhigh HSCs upregulate genes involved in HSC differentiation. Flt3L-eGFP-reporterlow cells develop to Flt3L-eGFP-reporterhigh cells in vitro, although Flt3L-eGFP-reporterhigh cells remain Flt3L-eGFP-reporterhigh . CD150+ Flt3L-eGFP-reporterlow cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L-eGFP-reporterhigh cells do express EPCR but not CD41. Thus, FACS-enrichment of Flt3/ Flt3L-eGFP-reporter negative, Lin- CD150+ CD48- EPCR+ CD41+ HSCs allows a further 5-fold enrichment of functional LT-HSCs.

Abstract 917: CD82/KAI1 maintains the dormancy of long-term hematopoietic stem cells through interaction with DARC-expressing macrophages

Abstract Hematopoiesis is regulated by crosstalk between long-term repopulating hematopoietic stem cells (LT-HSCs) and supporting niche cells in the bone marrow (BM). Here, we examine the role of CD82/KAI1 (CD82 hereafter) in niche-mediated LT-HSC maintenance. We found that CD82 is expressed predominantly on LT-HSCs and rarely on other hematopoietic stem-progenitor cells (HSPCs). In Cd82-/- mice, LT-HSCs were selectively lost as they exited from quiescence and differentiated. Mechanistically, CD82-based TGF-b1/Smad3 signaling leads to induction of CDK inhibitors and cell-cycle inhibition. The CD82 binding partner DARC/CD234 is expressed on macrophages and stabilizes CD82 on LT-HSCs, promoting their quiescence. When DARC+ BM macrophages were ablated, the level of surface CD82 on LT-HSCs decreased, leading to cell-cycle entry, proliferation, and differentiation. A similar interaction appears to be relevant for human HSPCs. Thus, CD82 is a functional surface marker of LT-HSCs that maintains quiescence through interaction with DARC-expressing macrophages in the BM stem cell niche. Finally, we hope that further development of our study will allow us to improve leukemia treatment by awakening not only “good” stem cells to keep beneficial stem cell functions, but also “bad” stem cells (e.g. cancer stem cells) in order to increase the sensitivity to treatment and avoid relapse. Citation Format: Jae-Il Choi, Jin Hur, Hwan Lee, Pniel Nham, Cheong-Whan Chae, Young-Eun Choi, Taewan Kim, Ga-Young Lee, Sung Hee Baek, Hyo-Soo Kim. CD82/KAI1 maintains the dormancy of long-term hematopoietic stem cells through interaction with DARC-expressing macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 917. doi:10.1158/1538-7445.AM2017-917

Stat5 deficiency decreases transcriptional heterogeneity and supports emergence of hematopoietic sub-populations.

Aging is associated with significant changes in hematopoiesis, including clonal dominance, anemia, myeloid malignancies, and reduced activation of signal transducer and activator of transcription 5 (Stat5). In previous studies, Stat5 deletion surprisingly amplified FLT3/ITD+ myeloid expansion or Myc-driven lymphoid expansion. Here we show that Stat5 deficiency has a strong impact upon transcriptional heterogeneity in single sorted c-Kit+Lin−Sca-1+ (KLS) cells or CD150+CD48− KLS long-term repopulating hematopoietic stem cells (LT-HSC). Single cell polymerase chain reaction (PCR) was performed on selected regulators of multi-lineage hematopoiesis. At least two dominant sub-populations were identified by increased expression of cell cycle regulatory and leukemia-associated genes. Furthermore, in the top expressing quartile of cells, the majority of genes were proportionally overrepresented. In wild-type KLS cells, Stat5 mRNA levels were also strongly correlated with several genes. Since heterogeneity decreases with age or inflammatory or oncogenic stress, these results provide a potential mechanistic linkage to Stat5 expression.

EPCR Guides Hematopoietic Stem Cells Homing to the Bone Marrow Independently of Niche Clearance

Bone marrow (BM) homing and lodgment of long-term repopulating hematopoietic stem cells (LT-HSCs) are active and essential first steps during embryonic development and in clinical stem cell transplantation. Rare, BM LT-HSCs endowed with the highest self-renewal and durable repopulation potential, functionally express the anticoagulant endothelial protein C receptor (EPCR) and PAR1. In addition to coagulation and inflammation, EPCR-PAR1 signaling independently controls a BM LT-HSC retention-release switch via regulation of nitric oxide (NO) production within LT-HSCs. EPCR+ LT-HSCs are maintained in thrombomodulin+ (TM) periarterial BM microenvironments via production of activated protein C (aPC), the major ligand for EPCR. Restriction of NO production by aPC-EPCR-PAR1 signaling, activates VLA4-mediated adhesion, anchoring EPCR+ LT-HSCs to the BM and protecting them from chemotherapy insult, sparing hematological failure and premature death (Gur-Cohen S. et al, Nat Med 2015).We report that transplanted EPCR+ LT-HSCs preferentially homed ‎to and were retained in the BM, while immature progenitors were equally distributed between the BM and spleen. Specificity of BM homing was further confirmed by EPCR neutralizing treatment that block aPC binding and attenuate EPCR+ LT-HSC BM homing. Furthermore, short term aPC in vitro pretreatment dramatically augmented EPCR+ LT-HSC BM homing, lodgment and long-term repopulation. PAR1 deficient stem cells were irresponsive to treatment with aPC and displayed reduced BM homing efficiency, all pointing to the aPC-EPCR-PAR1 axis as a crucial mediator of BM LT-HSC homing. Additionally, aPC pretreated EPCR+ LT-HSCs had a striking advantage to competitively home to the BM. Consistently, BM HSCs obtained from Procrlow mice, expressing markedly reduced surface EPCR, failed to compete with wild type stem cells in competitive repopulation assays. Importantly, the competitive homing results strongly imply that the BM available niches for newly arrived EPCR+ LT-HSCs are limited. Indeed, aPC pretreated EPCR+ LT-HSCs BM homing reached a plateau, as increasing the transplanted cell dose above 5x106 BM mononuclear cells, did not yield higher donor EPCR+ LT-HSC homing. These results reveal that there is a limited BM space for newly arrived transplanted EPCR+ stem cells to non-irradiated hosts. Importantly, we found that EPCR+ LT-HSCs can engraft the BM of non-conditioned mice with high efficiency, while remaining in a dormant, non-cycling state. Furthermore, the dormant homed EPCR+ LT-HSCs were later awakened and activated solely by treating the engrafted hosts with a low dose 5-FU chemotherapy, or with NO donor SNAP, revealing that preconditioning and clearance of occupied BM HSC niches are not required.To further address the preferential homing of EPCR+ LT-HSCs to the BM, we found that TM is exclusively expressed by unique BM arterioles, and not in the spleen. BM homed EPCR+ LT-HSCs were found adjacent to TM+ arterioles, imposing their retention. Homed BM EPCR+ LT-HSCs highly express full-length TM with intact lectin-like domain, and the BM TM+ endothelium was found to be enriched with a Glycocalyx layer, in particular with Heparan Sulfate Proteoglycan-2 (HSPG-2). HSGP-2 might specifically interact with the lectin-like domain of TM-expressingLT-HSCs, providing BM specific recognition and accelerated homing. Intriguingly, stabilizing TM function by in vitro pretreatment with platelet factor-4 (PF4) bypassed BM-derived cues and increased EPCR+/TM+ LT-HSC homing also to the spleen, suggesting a supportive role for PF4, highly secreted by BM megakaryocytes, in guiding EPCR+/TM+ LT-HSCs to the BM.Herein we define EPCR as a guidance molecule, navigating LT-HSC specifically to BM TM+ aPC-secreting blood vessels, allowing stem cell retention and protection from DNA damaging agents. The BM harbors a limited number of available stem cell niches for newly arrived transplanted EPCR+/TM+ LT-HSCs, and in vitro aPC pretreatment dramatically augments EPCR+/TM+ LT-HSC BM homing. Our findings provide new mechanistic insights and identify key players concerning LT-HSC homing specifically to the BM, leading to better repopulation following transplantation. This up-to-date approach and new knowledge may potentially lead to improved BM transplantation protocols and to prevent chemotherapy resistance of EPCR-expressing cancer stem cell mediated relapse. DisclosuresRuf:Iconic Therapeutics: Consultancy.

Inverse PAR1 Activity of Hematopoietic Stem Cells and BM Stromal Cells Mediates G-CSF-Induced Mobilization By Regulation of Nitric Oxide Generation

Hematopoietic stem and progenitor cell (HSPC) egress from the bone marrow (BM) to the circulation is tightly regulated and is accelerated during stress conditions, a process utilized for BM harvest. Recently, we demonstrated that mouse long term repopulating hematopoietic stem cell (LT-HSC) BM retention and their rapid release to the blood circulation are governed by a switch in nitric oxide (NO) generation via distinct coagulation-related protease activated receptor 1 (PAR1) cascades (Gur-Cohen S. et al., NM, 2016).Herein we report that surface PAR1 expression can be exploited and serve as a positive predictive marker for the efficiency of human CD34+ HSPC mobilization among healthy donors in clinical G-CSF-induced mobilization for matched allogeneic transplantations. We found that PAR1 expression on circulating leukocytes before G-CSF administration was positively correlated with higher yields of mobilized leukocytes after stimulation (P<0.001). In addition, the percentage of PAR1-expressing CD34+ HSPCs in the blood and their absolute numbers before initiation of mobilization positively correlated with peripheral blood leukocyte counts (P<0.001) and yield of mobilized CD34+ HSPCs (P=0.002). Importantly, tracing the consequences of the follow-up BM transplantation, we found accelerated neutrophil recovery in patients transplanted with mobilized cells expressing higher surface PAR1 levels at baseline (P=0.074), shortening the time for neutrophil recovery (neutrophil count>0.5 x 109/L) from an average of 14.6 days to an average of 11 days post-transplant. In addition, a trend of accelerated platelet production was documented to be related with higher PAR1 expression by circulating leukocytes prior to G-CSF stimulation. Consequently, poor mobilizer donors were characterized by extremely low surface PAR1 expression on circulating CD34+ cells prior to G-CSF stimulations. Herein we present a case report of a thrombophilic donor carrying the MTHFR mutation, expressing exceedingly low PAR1 levels at baseline, with the outcome of inadequate numbers of mobilized CD34+ HSPC in the blood following G-CSF treatments.To further gain insight into the role played by PAR1 signaling in the regulation of G-CSF-induced HSPC mobilization, we used mice as a functional preclinical small animal model. We found that antagonizing PAR1 signaling attenuated both steady state release and G-CSF-induced HSPC mobilization. Furthermore, co-administration of G-CSF with PAR1 antagonist attenuated secretion of BM stromal CXCL12 and abrogated upregulation of surface CXCR4 and PAR1 expression by BM HSPCs, all leading to significantly reduced HSPC migration, differentiation and mobilization. In support, PAR1-/- mice failed to efficiently mobilize HSPCs in response to G-CSF compared to wild type counterparts. Enforced HSPC recruitment by G-CSF treatments dramatically accelerated PAR1-dependent NO production by eNOS, known to promote TACE-mediated EPCR shedding and rapid LT-HSC mobilization. Concomitantly, circulating steady state and G-CSF-mobilized stem cells lack surface EPCR expression. Intriguingly, while EPCR expression by primitive BM stem cells was transiently reduced after G-CSF treatments, antagonizing PAR1 signaling along with G-CSF stimuli blocked NO generation and synchronically expanded BM EPCR+ LT-HSC and their supportive stromal progenitor cells (MSPCs), as confirmed by increased repopulation in transplanted mice. Finally, we report an inverse PAR1 expression and regulation by BM HSPC and stromal MSPCs in mediating G-CSF-induced mobilization. G-CSF induced elevation of PAR1 expression on BM HSPCs, providing the driving force for their enhanced NO mediated migration, proliferation, differentiation and recruitment to the circulation. Contrary, the levels of PAR1 expression were reduced on MSPCs in response to G-CSF treatment, and following NO generation by eNOS activity and CXCL12 secretion, resulted in reduced mesenchymal differentiation leading to accumulated numbers of immature mesenchymal (CFU-F) and osteoblast (CFU-OB) progenitor cells.Taken together, our study identifies and highlights inverse PAR1 signaling and NO generation as essential regulator of G-CSF induced HSPC mobilization and MSPC development opening new avenues to advance therapeutics for enhancing clinical G-CSF induced stem cell mobilization and transplantation protocols. DisclosuresRuf:Iconic Therapeutics: Consultancy.

Beta-Globin Gene Targeting in Human Hematopoietic Stem Cells Using Cas9 Ribonucleoprotein and rAAV6

The b-hemoglobinopathies including sickle cell disease (SCD) and b-thalassemia (b-thal) affect millions of people worldwide. SCD and b-thal are caused by mutations in the b-globin gene (HBB) resulting in either abnormal sickling or severely reduced protein production, respectively. A curative strategy for the b-hemoglobinopathies would be ex vivo HBB gene correction in patient-derived hematopoietic stem and progenitor cells (HSPCs) followed by autologous hematopoietic stem cell transplantation (auto-HSCT). We report the first CRISPR/Cas9 gene-editing platform for achieving homologous recombination (HR) at the HBB gene in long-term repopulating HSCs derived from mobilized peripheral blood. We combine electroporation of Cas9 protein complexed with chemically modified sgRNAs and delivery of a HR donor by recombinant adeno-associated viral vectors, serotype 6 (rAAV6). Notably, by including a reporter gene in the HR donor, we are able to identify and purify a population of HSPCs with >90% of cells having targeted integration at the HBB gene. These cells can be identified because HR-mediated integration causes the reporter gene to be expressed at log-fold higher levels than the non-integrated reporter. When transplanted into immunodeficient mice, the purified population gives rise to engraftment of HBB-edited human cells in primary and secondary recipients, confirming the presence of long-term repopulating hematopoietic stem cells (LT-HSCs). Importantly, we show efficient correction of the SCD-causing E6V mutation in SCD patient-derived CD34+ HSPCs by either editing the nucleotide mutation or knocking in an anti-sickling b-globin cDNA. Edited SCD CD34+ cells were shown to express adult b-globin (HbA) mRNA after HSPCs were differentiated into erythrocytes in vitro, confirming intact transcriptional regulation of the edited HBB allele. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting HSCs for HR at the HBB locus to advance the development of next generation therapies for b-hemoglobinopathies. DisclosuresPorteus:CRISPR Therapeutics: Consultancy, Equity Ownership.

39. FACS-Based Enrichment of a Highly Purified HBB-Targeted Hematopoietic Stem and Progenitor Cell Population Using rAAV6 and CRISPR/Cas9

Precise engineered nuclease-mediated gene correction via homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the power to transform curative therapies for monogenic diseases of the immune system. Sickle cell disease (SCD) is one of the most common monogenic diseases, affecting millions of people worldwide. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with SCD; however, immunocompatibility issues, graft-versus-host disease, and graft rejection are major roadblocks for efficacious therapy. In theory, the ideal curative strategy for SCD and most monogenic immune diseases is ex vivo gene correction of patient-derived HSPCs followed by autologous HSCT of a highly purified targeted population to avoid possible complications of competition between unedited and gene-targeted HSPCs in vivo. By supplying a homologous GFP-expressing HBB donor via recombinant adeno-associated virus serotype 6 (rAAV6) in combination with a double strand break created by the CRISPR/Cas9 system, we achieved HR frequencies of 15-49% of HSPCs, and more importantly, identified a population of HBB-targeted HSPCs (HSPCHBB) through a log-fold MFI increase in chromosomal transgene expression. Fluorescence activated cell sorting (FACS) of this population revealed consistent GFP expression in >95% of cells over two weeks in vitro in multiple CD34+ donors isolated from either bone marrow, cord blood or peripheral blood. Single-cell FACS of HSPCHBB into methylcellulose led to both myeloid and lymphoid colony formation, and on-target PCR analysis revealed >95% of these HSPCHBB clones had either a mono or biallelic-targeting event. Notably, a fraction of HSPCHBB displayed the CD34+/CD38-/CD90+/CD45RA-immunophenotype, suggesting successful targeting of long-term repopulating hematopoietic stem cells (LT-HSCs). Furthermore, HSPCHBB displayed long-term engraftment in the bone marrow of immunodeficient NSG mice at 12 weeks post-transplant, where we identified that ~70% of human cells were GFP+ and also produced both myeloid (CD33+) and lymphoid (CD19+) cell types, implying that the HSPCHBB population contains true LT-HSCs that can repopulate a functional immune system. Altogether, these proof-of-concept studies showed that by combining a homologous rAAV6 donor, the CRISPR/Cas9 system, and FACS, it is feasible to generate and enrich a highly purified population of gene-corrected HSPCs that include LT-HSC potentials.