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Bone marrow endothelial cells (BMECs) are an indispensable component for the hematopoietic stem cell (HSC) niche. In particular, Akt-activation endows ECs with the instructive capacity to support the self-renewal of HSCs to maintain homeostasis and promote the regeneration of the hematopoietic system following myeloablative stress, whereas MAPK signaling promotes HSC differentiation into lineage committed progenitors. Our group has recently demonstrated that in both in vitro and in vivo settings, aged BMECs can instruct an aged phenotype in young HSCs. Notably, we have generated preliminary data demonstrating that young BMECs maintain high levels of Akt signaling, whereas aged BMECs preferential signaling through MAPK. These data suggest that aged BMECs could be primed to negatively affect HSC function, which may lead to aged-related hematopoietic disorders, including the development of hematopoietic malignancies. Acute myeloid leukemia (AML) is the most common acute leukemia in adults, which incidence is predicted to rise with the expected increase in the elderly population. Disease recurrence is driven by a rare population of tumor cells, capable of self-renewal and expansion similarly to normal HSCs. These cells, referred as leukemia initiating cells (LIC), have the ability to persist during chemotherapy, and to repopulate disease. Notably, AML is known to alter the normal bone marrow vascular architecture, leading to increased vascular leakiness and to secrete a number of angiogenic factors, including VEGF-A, which has been suggested to support leukemic growth. As a consequence, the vascular niche might actively contribute to the maintenance and survival of LICs during the course of the disease and after induction of remission by chemotherapeutic agents. To this end we have generated data where we demonstrate that transplantation of AML LICs resulted in significant increases in the phosphorylation states of AKT and MAPK signaling. This data confirms that leukemic cells hijack the pro-HSC EC-specific Akt signaling pathway to enable their progression, but also over activate signaling pathways that have been demonstrated to support the differentiation of normal HSCs and support survival of AML. To formally test if endothelial MAPK signaling impacts the ability of BMECs to support functional hematopoiesis, we generated a mouse model where mice carrying a Rosa26 Stop/Floxed MEK1DD (mutant MAPKK1 that renders MAPK constitutively activate) were crossed to a tamoxifen inducible EC-specific VEcadherin Cre transgenic mouse to generate MAPKVCC mice. Analysis of these mice revealed that MAPKVCC mice manifested a profound decline in their HSC and progenitor potential resulting from loss of vascular integrity and enhanced inflammation within their BM microenvironment. We next isolated primary BMECs from MAPKVCC mice or littermate controls, and co-cultured them in an ex vivo platform with putative MLL-AF9 AML LICs (cKit+CD34+CD11b+). We observed that MAPK pathway activation conferred BMECs the ability to significantly expand putative LICs ex vivo. Moreover, transplantation of LICs first expanded on MAPKVCC BMECs resulted in notably shorter survival of recipient mice, compared to mice receiving control BMEC-expanded LICs. We further characterized MapkVCC BMEC metabolic profile by Seahorse XFe96 Extracellular Flux Analyzer. MapkVCC BMECs displayed a hyperglycolytic phenotype at steady state as well as increased glycolytic capacity under stress conditions, compared to control BMECs. Moreover, MapkVCC BMECs showed high respiratory capacity, thus phenocopying the metabolic profile of aged BMECs and tumor-associated ECs. Notably, MAPKVCC BMECs induced a metabolic switch in MLL-AF9 LICs ex vivo, towards increased glycolysis in both steady and stressed state and retained oxidative respiration potential under stress. These results suggest that age-associated aberrant MAPK signaling in the BM endothelial niche enhances the acquisition of the Warburg's effect in LICs, providing a selective microenvironment for the emergence of malignant clones at the expenses of normal hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem and progenitor cells (HSPCs) balance the physiological demands of maintaining peripheral leukocytes, erythrocytes, and platelets, while maintaining a potent stem cell reserve. These characteristics have made HSPC transplantation the only curative option for treating many hematological disorders. The regenerative potential of hematopoietic stem cells (HSCs) reside in their ability to home to a supportive niche, allowing for both HSC self-renewal and reconstitution of the hematopoietic system. The bone marrow (BM) microenvironment regulates HSC quiescence, self-renewal, and differentiation. The BM niche is composed of a number of cell types, including Lepr+ cells, Nestin+ cells, and endothelial cells. Collectively, the HSC niche modulates HSC fate decisions through the expression of paracrine factors, including Cxcl12 and Kitl. The BM microenvironment also plays a critical role in the reestablishment of hematopoiesis following myeloablative injury. Vegfr2-mediated vascular repair is critical for hematopoietic reconstitution following chemotherapeutic and radiation-mediated insult, while BM granulocyte production of Tnfa supports the regeneration of sinusoidal endothelium and subsequent hematopoietic recovery. While the importance of an HSC-supportive microenvironment during hematopoietic homeostasis and during regenerative conditions is coming into focus, poorly defined BM niche cells have limited the precise mechanistic insights necessary to elucidate new regenerative factors and strategies. Current methodology used to examine cellular subsets within the BM microenvironment rely on immunophenotypic fractionation, localization, and genetic lineage tracing. Ambiguous BM niche cellular immunophenotypes and gene expression have limited cellular resolution and confounded the interpretation of cre-mediated genetic deletion models. Herein, we aim to resolve the identities of distinct BM endothelial cell (BMEC) subpopulations to ultimately develop genetic tools to elucidate the paracrine requirements of the HSC-supportive endothelial niche. To this end, we sort-purified murine BMECs (VECAD+CD31+CD45-TER119-) for single cell RNA sequencing (scRNA-Seq). scRNA-Seq revealed the emergence of distinct BM arteriole, sinusoidal, and transitional endothelial populations, with arteriole BMECs significantly enriched for Kitl and Cxcl12. To confirm an arteriole enrichment in Kitl and Cxcl12 expression, we performed scRNA-Seq transcriptional analysis of sort-purified BM cells from KitlGFP and Cxcl12DsRed reporter mice. KitlGFP BM cells identified a distinct arteriole, but not sinusoidal, BMEC population. Cxcl12DsRed BM cells identified both arteriole and sinusoidal BMEC cell populations, but confirmed an increase in Cxcl12 expression in arterioles. We next examined candidate genes to generate BMEC subset-specific inducible cre mice. Analysis revealed that Vegfr3 (Flt4) expression was specific to sinusoidal BMECs, while Bmx1 appeared enriched in arterioles. We utilized a previously described Vegfr3YFP transgenic reporter mouse and found sinusoidal BMEC restricted expression. We then generated an inducible Vegfr3creERT2 line that directs efficient recombination to sinusoidal endothelium, with no detectable off-target activity. We next examined a previously described Bmx1creERT2 mouse line by generating Bmx1creERT2;ROSA26tdTomato reporter mice. In contrast to a recent report, Bmx1creERT2 activity was not spatially confined to the BM arteriole niche, but also labeled additional niche components, making it an unsuitable for arteriole-specific deletion. Moreover, previously reported constitutive Eporcre mice used to delete Kitl in sinusoids displays detectable cre activity in both arteriole and erythrocyte populations. More refined genetic models will need to be generated to test current and future candidate factors in the BM niche. Using our transcriptional data set, we have generated and validated a new inducible Vegfr3creERT2 mouse line that displays sinusoidal-restricted expression in the BM. Arteriole-specific creERT2 lines are currently being evaluated. These models will be used to systematically evaluate novel candidate arteriole- and sinusoidal-specific hematopoietic paracrine factors identified in our transcriptional analysis. Disclosures No relevant conflicts of interest to declare.

The molecular pathways and microenvironmental cues that choreograph the conversion of endothelial cells (ECs) into true engraftable hematopoietic stem cells (HSCs) remain undefined. This is due to lack of models to recreate the short-lived transition from EC to hemogenic cells and to HSCs. Extending on our previous work (Sandler V. et al, Reprogramming of human endothelium into hematopoietic cells requires vascular niche induction. Nature, 511:312-8. 2014), we have developed a novel, sequential, clinically-translatable in vitro model of the adult EC to hematopoietic transition (EHT). This model uses precise, conditional, on-off expression of transcription factors (FosB, Gfi1, Runx1, and Spi1 - FGRS) and an inductive vascular niche to reprogram adult mouse ECs into true HSCs (rEC-HSCs) with high efficiency. During the induction phase (days 0-8), FGRS are conditionally expressed in adult non-lymphatic ECs isolated from Runx1-IRES-GFP reporter mice and co-cultured with the supportive vascular niche cells. During the specification phase (days 8-20), FGRS-transduced VEcad+Runx1-CD45- ECs activate expression of endogenous Runx1, initiating the hematopoietic program and silencing EC fate. The VEcad+Runx1+CD45+ cells, then complete specification and full commitment to VEcad-Runx1+CD45+ hematopoietic stem and progenitor cells (rEC-HSPCs). Specified rEC-HSPCs are then expanded (days 20-28) on the vascular niche generating a large number of hematopoietic cells and, at this point, expression of exogenous FGRS is turned off. Transplantation of rEC-HSPCs (CD45.2) into lethally irradiated (CD45.1) recipient mice reconstitute both short-term and long-term hematopoiesis, and are capable of engrafting secondary and tertiary recipients (rEC-HSCs). Once engrafted, rEC-HSPCs give rise to functional myeloid and lymphoid cells with full complement of polarized T cell subsets. rEC-HSC-derived immune cells undergo T-cell receptor (TCR) rearrangement and reconstitute adaptive immune function in Rag1-/- mice. To prove the stem cell potential of rEC-HSCs, we performed clonal analyses on the VEcad+Runx1+CD45+ cells (days 8-20), in which single cells were plated in coculture with vascular niche. Notably, 7 out of 386 CD45.2+ clones gave rise to expanding colonies that were capable of 4 months primary multilineage engraftment into lethally irradiated CD45.1+ recipients. In addition, limiting dilution transplantation of isochronic VEcad-Runx1+CD45+ cells indicated that 1 out of 538 rEC-HSPCs are repopulating rEC-HSCs with primary and secondary engraftment potential. Thus, based on both clonal and limiting dilution transplantation studies, we demonstrate that rEC-HSCs represent true repopulating prototypical HSCs. In addition, since during the expansion phase there are >200 fold expansion of the SLAM-coded KLS population, these data support the notion that during the 20-28 days expansion phase there is robust proliferation of rEC-HSCs. Serial analyses of the primary, secondary and tertiary engrafted rEC-HSCs, showed no evidence of leukemias. Molecular analyses indicated that both in vitro and in vivo expanded rEC-HSCs had complete erasure of vascular signature and stable expression of hematopoietic profile. Moreover, employing Runx1-IRES-GFP reporter mice enabled deconvolution of stage-specific pathways involved in generation of engraftable rEC-HSCs. Inhibition of TGFβ signaling along with activation of BMP and CXCL12 pathways reinforced the induction phase. Active Notch and CXCL12 signaling throughout the specification and expansion phases supported self-renewal of transplantable rEC-HSCs. This stepwise reprogramming approach provides an interrogatable in vitro platform to elucidate the critical pathways involved in the transition of ECs into hemogenic-like and ultimately hematopoietic cells in vitro. Specifically, these data unequivocally support that our approach lead to generation of large number HSCs, enabling therapeutic application. Thus, our reprogramming platform that does not require transition through a pluripotent state, will facilitate devising strategies to reprogram ECs into abundant autologous long-term repopulating HSCs amenable to genetic modification for treatment of inherited and acquired hematological disorders. Disclosures Ginsberg: Angiocrine Bioscience: Employment. Butler:Angiocrine Bioscience: Research Funding. Rafii:Angiocrine Bioscience: Equity Ownership, Other: Non-paid consultant.

Bone marrow (BM) endothelial cells (BMECs) form a network of blood vessels (BVs) that regulate both leukocyte trafficking and hematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance between these dual regulatory roles and if these events occur at the same vascular site. We define the BM architecture of functionally distinct BVs, their spatial localization and association with specific stromal precursors, which cooperatively regulate HSPC fate. BM stem and progenitor cell maintenance in a metabolically non-active state and leukocyte trafficking occur at separate sites and are differentially regulated by specific BVs with distinct permeability properties. BM arteries were found to be mostly encircled by aSMA+ pericytes whereas the ensuing small-diameter endosteal and trabecular arterioles were predominantly surrounded by stem cell-niche supporting stromal precursor cells. Live imaging and flow analysis revealed that endosteal arteriole BVs exhibited high flow rate, low permeability to external plasma from the peripheral blood, and high levels of adhesion- and tight-junction molecules. Primitive HSPCs located in peri-arteriole regions were found in a non-activated, low reactive oxygen species (ROS) state. Exposure of BM HSPCs to peripheral blood plasma, enhanced their metabolic activity, exhibited by enhanced intracellular ROS levels, and glucose uptake. The same was also evident for circulating HSPCs in the blood. Plasma-exposed HSPCs displayed enhanced motility alongside with reduced long-term repopulation potential. Live imaging showed that all immature and mature leukocyte bi-directional trafficking occurred exclusively at the more permeable sinusoids, located downstream to the endosteal arterioles. Of note, BM sinusoids contained a higher prevalence of ROShigh cells in their microenvironment, including HSPCs. Rapid AMD3100-induced HSPC mobilization preferentially affected sinusoidal but not arterial BVs permeability and CXCL12 chemokine release. Endothelial specific in vivo interference with CXCL12-CXCR4 interactions, via conditional CXCR4 genetic deletion, hampered BM barrier integrity resulting in enhanced HSPC egress. In line with these results we found that during conditions favoring BM stem and progenitor cells expansion, endothelial integrity was enhanced along with reduced HSPC bi-directional trafficking. Conversely, conditional endothelial specific induced genetic or pharmacologic disruption of barrier integrity augmented ROS levels in HSPCs, enhancing their bi-directional trafficking and differentiation while reducing their BM pool size and maintenance in a metabolically non-active state. Of note, humanized mice engrafted with pre-B ALL cells exhibited reduced BM barrier permeability most probably due to BM endothelium modification via FGF-2 secretion by the leukemic clone. Interestingly, human pre-B ALL cells displayed hypersensitivity to plasmatic exposure. We hypothesize that malignant cells modify BM endothelium to provide themselves with a supportive and protective microenvironment composed of undifferentiated BM stromal progenitors and tightly sealed endothelial barrier. In conclusion, our study identifies anatomically distinct BM BVs with different barrier functions serving as systemic leukocyte trafficking or HSPC BM maintenance sites with clinical therapeutic relevance. Disclosures Rafii: Angiocrine Bioscience: Consultancy, Equity Ownership.

The molecular mechanisms regulating the aging of hematopoietic stem cells (HSCs) are poorly understood. To date, most studies describing age-related alterations have focused on HSC-intrinsic alterations, showing that the absolute number of immunophenotypically defined HSCs increases with age but that aged HSCs exhibit decreased long-term reconstitution potential, self-renewing capacity, altered transciptomes, cell-cycling and responses to cellular stress and DNA damage. Furthermore, old HSCs exhibit a significant myeloid bias at the expense of lymphopoiesis, which is thought to predispose the aging hematopoietic to the development of myeloid neoplasms. While these studies show that cell-intrinsic changes contribute to the aging of the hematopoietic system, most have not adequately addressed the effects of the aging microenvironment. A large body of evidence has demonstrated functional interactions between the HSC and its niche, suggesting that local and systemic factors may regulate HSC function; however, the role of the bone marrow (BM) microenvironment in regulating HSC aging has not been fully elucidated. Understanding the relationship between the BM microenvironment and the HSC during aging may aid in efforts to prevent or reverse the age-related functional decline observed in the hematopoietic system. We have shown that Akt-activated endothelial cells (ECs) within the hematopoietic microenvironment, are indispensable for supporting HSC self-renewal during both steady-state and regenerative hematopoiesis and that EC-specific Mapk signaling drives the differentiation of HSCs into lineage-committed progeny. Here, we demonstrate that young BMECs maintain high levels of Akt signaling, whereas aged ECs exhibit preferential signaling through Mapk. Utilizing a novel HSC/EC co-culture system, we demonstrate that aged BMECs co-cultured with hematopoietic stem and progenitor cells (HSPCs) isolated from young mice inhibit the expansion of repopulating HSCs and are unable to expand aged HSPCs that give rise to long-term, multilineage engraftment. Of note, when we co-cultured aged HSPCs with young BMECs we found that we were able to maintain their functional capacity when assessed by competitive repopulation assays. These data suggest that BMECs play an important role in regulating HSC function. Based on these observations, we set out to test if endothelial Mapk inhibits the vascular niche from supporting functional hematopoiesis. We generated a mouse model in which Mapk was selectively overexpressed in ECs (Mapk VCC) and demonstrated that these mice exhibit a defect in phenotypic and functional HSCs, resembling phenotypes similar to aged HSCs. In particular, transplanted HSCs from Mapk VCC mice lead to diminished engraftment ability with an in increase in myeloid contribution at the expense of B and T cells. To directly test if the functional defects in the HSCs were due to the Mapk-activated vascular niche, we isolated BMECs from these mice and found that Mapk-activated ECs have a decreased ability to support the ex vivo expansion of functional HSCs, with less HSCs in quiescence and more differentiation into granulocytic myeloid cells. Transcriptome and proteomic analyses revealed that aged and Mapk-activated BMECs have similar defects in their pro-HSC angiocrine repertoire, suggesting a possible mechanism for their diminished capacity to instruct and maintain a balanced and healthy hematopoietic system. Furthermore, we utilized an endothelial-based cellular therapy approach to rejuvenate the BM microenvironment and demonstrated that transplantation of young BMECs can enhance hematopoietic recovery and restore HSC function following myeloablative injury in aged mice. Taken together, our in vivo animal model and EC/HSC co-culture system will allow us to screen for angiocrine factors that support the functional attributes of the HSC. Additionally, we have unlocked a potential therapeutic application for the transplantation of ECs following myeloablative treatment. Transplantation of BMECs may create a more permissive microenvironment that promotes an increase in the number of engrafted HSPCs following BM transplantation and accelerates the rate of hematopoietic recovery following radiation or chemotherapeutic regimens decreasing the morbidity/mortality associated with life threatening pancytopenias in the elderly. Disclosures No relevant conflicts of interest to declare.

Long-term, adult-engrafting hematopoietic stem cells (HSC) first emerge from hemogenic endothelial (HE) precursors in the context of embryonic arterial vessels such as the dorsal aorta of the AGM (Aorta-Gonad-Mesonephros region), a process which requires Notch1 receptor signaling. However, a possible subsequent role for Notch receptor activation during the unique period of substantial HSC expansion in embryonic development remains less well defined. Here, we show that endothelial cells derived from the murine embryonic AGM region (AGM-EC) or fetal liver (FL-EC) provide an in vitro substrate for generation/maturation of HSC from HE/HSC-precursor populations derived from early stage murine embryos, which lack adult-engraftment capacity prior to co-culture. Notably, these EC substrates, endogenously expressing Notch ligands of the Jagged and Delta families, also support subsequent numerical expansion of AGM-derived HSC, expressing Notch1 and Notch2 receptors, as determined by limit dilution transplantation analysis. Consistent with a requirement for Notch activation in this process, phenotypic HSC expansion during EC co-culture is blocked by gamma-secretase mediated Notch pathway inhibition. Furthermore, we show that in vitro Notch activation by immobilized ligand Delta1, together with cytokines and inhibition of the TGF-beta pathway, is sufficient to increase the number of AGM-derived HSC in the absence of EC stroma. Expansion of phenotypic hematopoietic stem/progenitor cells generated by culture on Delta1 is inhibited by antibody-mediated blockade of the combination of Notch1 and Notch2, but not by either Notch1 or Notch2 alone. Consistent with this, Notch receptor-specific activation by either immobilized Notch1 or Notch2 antibody is sufficient to support AGM-derived HSC in vitro in preliminary experiments. Altogether, these studies suggest a role for Notch pathway activation by either Notch1 or Notch2 in supporting embryonic-stage HSC expansion subsequent to initial Notch1-mediated HSC specification. Disclosures Moon: Fate Therapeutics: co-founder Other. Rafii:Angiocrine Biosciences: Founder Other.

Adult hematopoietic stem cells (HSCs) are defined by their ability to undergo self-renewal and maintain the capacity to generate all of the mature hematopoietic cell types within the blood and immune system. These unique qualities make the HSC clinically useful in bone marrow (BM) transplantation settings for a wide variety of hematological diseases. It has been demonstrated that maintenance of the HSC is dependent upon the cell-intrinsic properties of the HSC itself, as well as the extrinsic properties of the BM microenvironment. Within the hematopoietic microenvironment, we have shown that endothelial cells (ECs) are indispensable in supporting HSC self-renewal and differentiation into lineage-committed progeny during regenerative hematopoiesis. Furthermore, we have demonstrated that Akt signaling endows ECs with the capacity to instructively support HSC self-renewal through the expression of pro-hematopoietic angiocrine factors during homeostasis and hematopoietic regeneration following myelosuppressive stress. However, despite advances in the understanding of HSC biology, the exact mechanisms that regulate the balance between self-renewal and lineage-specific differentiation are still unknown. In order to expand our understanding of the body’s vascular network in regulating HSCs and hematopoietic regeneration, we have now focused on identifying the downstream signaling pathways of Akt within ECs that are responsible for the production of the pro-hematopoietic angiocrine factors. Because of the strong supporting data demonstrating that NF-kB signaling regulates hematopoietic function, we have focused on the Akt/NF-kB signaling axis in the vascular niche and demonstrated that inhibition of NF-kB within ECs results in a significant expansion of functional HSCs. Inhibition of the NF-kB pathway by expression of an IkBa super suppressor (IkBa-SS) via lentiviral transduction in primary ECs resulted in the expansion of phenotypic HSCs, while blocking differentiation of progenitor cells in vitro with an increase in the functional potential of the expanded HSCs. Utilizing a transgenic mouse model (Tie2.IkBa-SS) in which the NF-kB signaling pathway is inhibited specifically in ECs, we found that there was a significant increase in phenotypic and functional HSCs in vivo. Endothelial-specific inhibition of NF-kB signaling resulted in an increase in HSC quiescence and serial administration of low-dose chemotherapeutic agents resulted in an increase in self-renewal activity, suggesting that suppressing NF-kB signaling in ECs controls hematopoiesis by preventing premature exhaustion of the HSC pool. Following hematopoietic insult, Tie2.IkBa-SS mice undergo a rapid recovery of hematopoiesis and the hematopoietic system is largely protected following myelosuppression when compared to controls. Gene profiling of freshly isolated BM ECs from Tie2.IkBa-SS mice suggests that the enhancement of functional hematopoiesis is, in part, due to BM ECs upregulating pro-HSC angiocrine factors, as well as suppressing the production of cytokines and growth factors responsible for eliciting inflammatory responses, forcing the differentiation of HSCs. Furthermore, transplantation of BM ECs isolated from Tie2.IkB-SS mice significantly enhanced overall hematopoietic recovery following an LD50 dose of myeloablation, suggesting that transplantation of ECs could have tremendous therapeutic potential in mitigation the side effects of myeloablative injury by decreasing the morbidity and mortality associated with hematopoietic insults. In conclusion, our data demonstrates that the IkBa-dependent NF-kB pathway in ECs can regulate the production of pro-hematopoietic angiocrine factors that promote the maintenance and expansion of the HSC pool. Additionally, we have potentially unlocked a novel therapeutic application for the transplantation of genetically modified BM ECs following myeloablative treatment. Therapeutic transplantation of BM ECs may create a more permissive microenvironment that promotes an increase in the number of engrafted HSCs following BM transplantation, accelerating the rate of hematopoietic recovery following radiation or chemotherapeutic regimens and decreasing the morbidity and mortality associated with life threatening pancytopenias. Disclosures No relevant conflicts of interest to declare.