HSC Niche Research Articles

Spatial Mapping Reveals Distinct Erythroid Niches in Mice and Humans during Development and Stress

Like hematopoiesis, erythropoiesis requires erythroid niche nurturing and support. While extensive studies have revealed various components and functions of the HSC niches, the erythroid niche is less characterized. The most studied erythroid niche is the bone marrow erythroblastic island (EBI), which comprises a central macrophage surrounded by developing erythroblasts. However, most of the reported studies of EBI involve in vitro reconstitution of mixed cell populations that do not recapitulate in vivo niches. Here, we utilized multiple spatial mapping technologies, including spatial transcriptomics and whole-mount imaging studies, to characterize erythropoiesis in mouse and human hematopoietic tissues during development and under stress. We first utilized the Visium platform from 10x Genomics to study the spatial transcriptomic profiles of the E14.5 mouse fetal liver, which is the major organ for murine fetal definitive erythropoiesis. The Visium platform contains barcoded spots with a diameter of 55 µm, which is suboptimal for single-cell analyses but ideal for the investigation of cell-cell interactions within their microenvironment. We found that each hepatic lobe exhibited different cluster distributions, indicating distinct cell compositions among different anatomic loci. Further deconvolution of the clusters and co-analysis with corresponding single-cell RNA sequencing data revealed that there was a higher positive correlation between erythroid cells and C1q+ macrophages compared to other macrophages, suggesting that C1q+ macrophages are more likely to be EBI macrophages in mouse. C1q+ macrophages exhibited high expression of known genes involved in EBI macrophage function and erythropoiesis. Cyclic immunofluorescence (CyCIF) and whole-mount immunofluorescence analyses further demonstrated the co-localization of C1q+ macrophages with erythroid cells in the E14.5 fetal liver. This strong positive correlation between C1q+ macrophages and erythroid cells was also observed across different stages of fetal liver development, newborn bone marrow, and adult spleen under physiologic and stress conditions. These findings demonstrate that C1q+ macrophages as EBI macrophages are conserved across different hematopoietic organs in mice. We next applied the same technologies to human hematopoietic tissues during development and stress. In contrast to the mouse, we did not observe a strong positive correlation between erythroid cells and C1q+ macrophages or other macrophages in the human fetal liver. Instead, there is a strong association between erythroid progenitor and maturing erythroid cells. This strong association is also found in different developmental stages of the human fetal liver as well as the bone marrow, which becomes even stronger in regenerating human bone marrow after chemotherapy. We also recapitulated these findings in a human bone marrow organoid model. These results demonstrate the erythroid cell self-sustaining capacities in human erythropoiesis. Taken together, our findings reveal the distinct erythroid niches in mice and humans during development and stress, which provides a comprehensive understanding of erythropoiesis during evolution.

Heterogeneous Intercellular Communication of Hematopoietic Stem Cells in the Mouse Bone Marrow

Introduction.Hematopoietic stem cells (HSCs) are responsible for maintaining the homeostasis of the blood and immune systems. This process must rapidly adapt to bleeding, infection, injury, and disease. HSCs reside in specialized microenvironments in the bone marrow, called “niche”. Several bone marrow cell types have been proposed as the key components of the niche. It is well established that HSCs rely on molecular signals from the niche cells to survive, migrate, proliferate, and differentiate. Recent studies suggest that HSCs differentiate heterogeneously. Additionally, the bone marrow is a heterogeneous environment consisting of more than thirty cell types. This study will test the hypothesis that individual HSCs are engaged in heterogeneous intercellular signaling in the bone marrow. Materials and Methods.We utilized CellChat (Jin et al., 2021), a database for ligand-receptor pairs and associated pathways, to predict cell-cell signaling at the single cell level. We used single cell RNA sequencing data from mouse hematopoietic stem and progenitor cells, blood and immune cells, and non-hematopoietic cells from the bone marrow to determine putative cell-cell interactions. The predicted signaling interactions were then adjusted based on the cell-cell spatial interaction revealed by PhenoCycler measurement (Akoya Biosciences). Mouse bone marrow transplantation experiments were performed to test the functional significance of the identified signaling pathway interactions. Results.We found that individual HSCs send and receive different levels of molecular signals across various signaling pathways. In addition to the expected interactions with the known HSC niche cells, we also found new interactions between HSCs and their progeny cells including myeloid progenitors and immune cells. These cell-cell signaling interactions are consistent with the spatial interaction frequencies of the corresponding cell types as revealed by our imaging data from the PhenoCycler analysis. Further, we found that some signaling pathways are positively or negatively correlated across individual HSCs, indicating complex intercellular signaling networks. The most significant negative correlation was found between HSCs sending MHC-I signals and sending PARs signals (Figure). Moreover, this MHC-I/PARs signaling correlation network also involves signaling from the HSC niche, including those that are known to play roles in HSC homing such as ESAM and VCAM. Therefore, we tested the engraftment efficiency of HSCs that receive PARs and those that do not using the HSC mouse transplantation model. We found that PAR1 + HSCs engraft much less efficiently. Finally, we present the HSC Interaction Data Explorer (HIDE), a database that comprehensively depicts the interactions between HSCs and other bone marrow cells including the involved signaling pathways, ligands and receptors of the pathways, and the pathway correlations. Conclusion.Our study uncovered novel interactions of HSCs with myeloid progenitor cells and immune cells, which were validated by spatial interaction data. These interactions indicate feedback signaling in regulating blood cell regeneration. Our findings further revealed signaling pathway correlation networks across individual HSCs, and we showed their functional significance using transplantation experiments. Finally, we present HIDE as a resource that informs the interactions between HSCs and various bone marrow cells, the relevant signaling pathways, and the pathway correlations. Figure Legend. HSC signaling correlation networks. Each node shows a signaling pathway and direction (e.g., HSCs sending MHC-I). The edges represent the spearman correlation score.

AML-derived extracellular vesicles negatively regulate stem cell pool size: A step toward bone marrow failure

Purpose of the studyLong-term repopulating hematopoietic stem cells (LTR-HSCs) have been previously shown to reside in close proximity to osteoblasts, where they take shelter in the bone marrow (BM) microenvironment against cytotoxic and apoptotic stimuli. Nevertheless, the function of the HSC niche is believed to undergo an adaptive evolutionary modification during leukemogenesis. Recent studies have demonstrated that leukemic clones can impact BM homing through extracellular vesicle (EV) secretion. However, the exact mechanism driving BM conversion is still unclear. In the present study, the human osteoblast cell line (MG-63) were subjected to various concentration of sera-derived EVs of patients with acute myeloid leukemia (AML) and healthy volunteers to assess if they are associated strongly enough to alter the expression pattern of cross-talk molecules involved in niche interactions. MethodTo gain a brief insight into the EVs secretion criteria, we first conducted a comparative analysis of sera-derived EVs by dynamic light scattering (DLS), transmission electron microscopy (TEM), and Bradford assay. After incubating MG-63 cell lines with increasing concentrations of the EVs, Trypan-blue and microculture tetrazolium test (MTT) assays were used to evaluate the cell survival, logarithmic growth, and metabolic activity. Finally, the expression levels of OPN, ANGPT-1, and JAG-1 transcripts were evaluated through the qRT-PCR technique. ResultsHere, we report that AML-derived EVs can affect the viability, cell growth, and metabolic activity of the human osteoblasts cell line (MG-63) compared to those that received healthy-derived EVs. We also found that leukemic EVs tend to induce overexpression of OPN but reduce the expression of ANGPT-1 and JAG-1 genes in the osteoblast transcriptome, which may provide a potential context imposing selective suppression of HSC pool size. ConclusionThese findings extend the general concept of a novel mechanism in which leukemic EVs would make it possible to create a specialized pre-metastatic microenvironment in the interest of tumor expansion, allowing leukemic clones to overcome their HSCs counterparts.

Establishing an Easy-to-Use 3D BM Niche Model for the Co-Culture of Primary Bone Marrow Mesenchymal Stromal Cells (BM-MSC) with Hematopoietic Stem and Progenitor Cells (HSPC)

Background BM-MSC are a collection of primitive cell populations, a major stromal component of the BM niche and are essential to maintain tissue homeostasis. 2D culture protocols are popular, though not standardized, and effective for BM-MSC expansion, but display limited niche function in co-culture systems. 3D co-culture leverages the importance of cell signaling networks activated by cell-cell and cell-matrix interactions in native tissues. Here, we establish a superior and easy-to-use 3D culture protocol and compare to conventional 2D approaches. Methods, Results and Discussion First, we compared commercially available expansion media to the most widely cited investigator-defined media for optimal 2D expansion and 3D culture of ex vivo murine BM-MSC. Based on this, we developed a five-marker flow cytometry panel to accurately quantify BM-MSC culture purity. To test BM-MSC niche function in 2D vs 3D cultures, we generated 3D spheroid hanging drop (HD) cultures from 2D expanded BM-MSC. In HD spheroids, cells are in direct contact with each other as well as extracellular matrix components and can be used to co-culture multiple different cell types. To assess the niche-like function of 3D HD cultures compared to 2D adherent cultures, we assessed Nestin protein expression since Nestin is a marker of HSC niches within mouse BM in vivo. BM-MSC were cultured in 2D or HD and Nestin protein expression assessed by immunofluorescence imaging, showing a significant increase in percent of Nestin-positive cells in HD-cultured BM-MSC, compared to matched 2D controls, in agreement with previous reports in human BM-MSC 3D culture. To assess 3D in vitro BM-MSC niche (IVBMN) function, we assayed stemness markers and survival of FACS isolated primary HSPC (Lineage-Sca1+cKit+, LSK) from mouse BM. LSK cells were cultured as standard (alone) or seeded as co-cultures with BM-MSC within HD with specific cytokine support, in low oxygen (6.5%) conditions (IVBMN) (Figure 1). LSK cells cultured within IVBMN exhibited superior survival as well as retention of stemness markers, demonstrating the benefit of BM-MSC support. In vivo, the BM niche also includes BM niche resident macrophages (BM-Mɸ), which were recently shown to be a limiting factor for engraftment and HSC reconstitution in irradiated mice, and are part of long term and short term HSC niches. To further extend our IVBMN, BM-Mɸ [F4/80+ Ly6G- VCAM1+ CD169+] were collected by FACS and added to IVBMN cultures (IVBMN-Mɸ). Notably, the IVBMN-Mɸ co-culture model further enhanced survival and stemness of primary LSK cells, when compared to LSK cultured alone or within IVBMN. Based on these preliminary data, we demonstrate that our IVBMN-Mɸ culture system is a unique tool which mimics niche properties in vitro and is easy-to-use for HSPC manipulation. Conclusions Here we describe a standardized flow panel for reliable murine BM-MSC immunophenotyping, and characterize a simple but effective 3D BM-MSC co-culture system which allows niche-like function in vitro. Fig. 1. In vitro BM niche (IVBMN) experimental workflow: A) Ex vivo expanded primary BM-MSC (20x) (i) -/+ FACS isolated BM niche resident Mɸ (ii) in co-culture with primary BM HPSC (LSK) (iii). B) Cells were cultured in a 3D mixed culture system with specific cytokine support, in 6.5% low oxygen. C) IVBMN-Mɸ [MSC+ BM-Mɸ +LSK] co-culture promotes maintenance of primary viable LSK compared to IVBMN [MSC+LSK] or LSK standard culture conditions alone, after 5 days culture. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P57Kip2 regulates embryonic blood stem cells by controlling sympathoadrenal progenitor expansion

AbstractHematopoietic stem cells (HSCs) are of major clinical importance, and finding methods for their in vitro generation is a prime research focus. We show here that the cell cycle inhibitor p57Kip2/Cdkn1c limits the number of emerging HSCs by restricting the size of the sympathetic nervous system (SNS) and the amount of HSC-supportive catecholamines secreted by these cells. This regulation occurs at the SNS progenitor level and is in contrast to the cell-intrinsic function of p57Kip2 in maintaining adult HSCs, highlighting profound differences in cell cycle requirements of adult HSCs compared with their embryonic counterparts. Furthermore, this effect is specific to the aorta-gonad-mesonephros (AGM) region and shows that the AGM is the main contributor to early fetal liver colonization, as early fetal liver HSC numbers are equally affected. Using a range of antagonists in vivo, we show a requirement for intact β2-adrenergic signaling for SNS-dependent HSC expansion. To gain further molecular insights, we have generated a single-cell RNA-sequencing data set of all Ngfr+ sympathoadrenal cells around the dorsal aorta to dissect their differentiation pathway. Importantly, this not only defined the relevant p57Kip2-expressing SNS progenitor stage but also revealed that some neural crest cells, upon arrival at the aorta, are able to take an alternative differentiation pathway, giving rise to a subset of ventrally restricted mesenchymal cells that express important HSC-supportive factors. Neural crest cells thus appear to contribute to the AGM HSC niche via 2 different mechanisms: SNS-mediated catecholamine secretion and HSC-supportive mesenchymal cell production.

The Infection Process Study in Changing to Malignancy

Hematopoiesis is regulated by a diverse cellular of BM microenvironment which supports stepwise multi-potent stem cells differentiation as well as maturation of progenitors and precursors into mature blood cells. Some imbalances between virus inherent transforming abilities and the host immune system can lead into the various diseases development. In this case, hematopoietic dynamics are perturbed during inflammation that we want to know exactly about the HSC niche interaction in the situation which can occur as a result. Here we discuss about some infections have deleterious effects injuring hematopoietic stem cell , inefficient hematopoiesis and also destruction of the cells and then can go to the remodeling of bone marrow microenvironment and ultimately change to malignancy. So it is important to demonstrate a BM microenvironment critical role in the response to infection.

Engineered Tissue Models to Replicate Dynamic Interactions within the Hematopoietic Stem Cell Niche.

Hematopoietic stem cells are the progenitors of the blood and immune system and represent the most widely used regenerative therapy. However, their rarity and limited donor base necessitate the design of ex vivo systems that support HSC expansion without the loss of long-term stem cell activity. This review describes recent advances in biomaterials systems to replicate features of the hematopoietic niche. Inspired by the native bone marrow, these instructive biomaterials provide stimuli and cues from cocultured niche-associated cells to support HSC encapsulation and expansion. Engineered systems increasingly enable study of the dynamic nature of the matrix and biomolecular environment as well as the role of cell-cell signaling (e.g., autocrine feedback vs paracrine signaling between dissimilar cells). The inherent coupling of material properties, biotransport of cell-secreted factors, and cell-mediated remodeling motivate dynamic biomaterial systems as well as characterization and modeling tools capable of evaluating a temporally evolving tissue microenvironment. Recent advances in HSC identification and tracking, model-based experimental design, and single-cell culture platforms facilitate the study of the effect of constellations of matrix, cell, and soluble factor signals on HSC fate. While inspired by the HSC niche, these tools are amenable to the broader stem cell engineering community.

A Novel Megakaryocyte Subpopulation Poised to Exert the Function of HSC Niche as Possible Driver of Myelofibrosis.

Careful morphological investigations, coupled with experimental hematology studies in animal models and in in vitro human cultures, have identified that platelets are released in the circulation by mature megakaryocytes generated by hematopoietic stem cells by giving rise to lineage-restricted progenitor cells and then to morphologically recognizable megakaryocyte precursors, which undergo a process of terminal maturation. Advances in single cell profilings are revolutionizing the process of megakaryocytopoiesis as we have known it up to now. They identify that, in addition to megakaryocytes responsible for producing platelets, hematopoietic stem cells may generate megakaryocytes, which exert either immune functions in the lung or niche functions in organs that undergo tissue repair. Furthermore, it has been discovered that, in addition to hematopoietic stem cells, during ontogeny, and possibly in adult life, megakaryocytes may be generated by a subclass of specialized endothelial precursors. These concepts shed new light on the etiology of myelofibrosis, the most severe of the Philadelphia negative myeloproliferative neoplasms, and possibly other disorders. This perspective will summarize these novel concepts in thrombopoiesis and discuss how they provide a framework to reconciliate some of the puzzling data published so far on the etiology of myelofibrosis and their implications for the therapy of this disease.

Prostacyclin is an endosteal bone marrow niche component and its clinical analog iloprost protects hematopoietic stem cell potential during stress.

Hematopoietic stem cells (HSCs) with superior reconstitution potential are reported to be enriched in the endosteal compared to central bone marrow (BM) region. To investigate whether specific factors at the endosteum may contribute to HSC potency, we screened for candidate HSC niche factors enriched in the endosteal compared to central BM regions. Together with key known HSC supporting factors Kitl and Cxcl12, we report that prostacyclin/prostaglandin I2 (PGI2 ) synthase (Ptgis) was one of the most highly enriched mRNAs (>10-fold) in endosteal compared to central BM. As PGI2 signals through receptors distinct from prostaglandin E2 (PGE2 ), we investigated functional roles for PGI2 at the endosteal niche using therapeutic PGI2 analogs, iloprost, and cicaprost. We found PGI2 analogs strongly reduced HSC differentiation in vitro. Ex vivo iloprost pulse treatment also significantly boosted long-term competitive repopulation (LT-CR) potential of HSCs upon transplantation. This was associated with increased tyrosine-phosphorylation of transducer and activator of transcription-3 (STAT3) signaling in HSCs but not altered cell cycling. In vivo, iloprost administration protected BM HSC potential from radiation or granulocyte colony-stimulating factor-induced exhaustion, and restored HSC homing potential with increased Kitl and Cxcl12 transcription in the BM. In conclusion, we propose that PGI2 is a novel HSC regulator enriched in the endosteum that promotes HSC regenerative potential following stress.

15-PGDH inhibition activates the splenic niche to promote hematopoietic regeneration.

The splenic microenvironment regulates hematopoietic stem and progenitor cell (HSPC) function, particularly during demand-adapted hematopoiesis; however, practical strategies to enhance splenic support of transplanted HSPCs have proved elusive. We have previously demonstrated that inhibiting 15-hydroxyprostaglandin dehydrogenase (15-PGDH), using the small molecule (+)SW033291 (PGDHi), increases BM prostaglandin E2 (PGE2) levels, expands HSPC numbers, and accelerates hematologic reconstitution after BM transplantation (BMT) in mice. Here we demonstrate that the splenic microenvironment, specifically 15-PGDH high-expressing macrophages, megakaryocytes (MKs), and mast cells (MCs), regulates steady-state hematopoiesis and potentiates recovery after BMT. Notably, PGDHi-induced neutrophil, platelet, and HSPC recovery were highly attenuated in splenectomized mice. PGDHi induced nonpathologic splenic extramedullary hematopoiesis at steady state, and pretransplant PGDHi enhanced the homing of transplanted cells to the spleen. 15-PGDH enzymatic activity localized specifically to macrophages, MK lineage cells, and MCs, identifying these cell types as likely coordinating the impact of PGDHi on splenic HSPCs. These findings suggest that 15-PGDH expression marks HSC niche cell types that regulate hematopoietic regeneration. Therefore, PGDHi provides a well-tolerated strategy to therapeutically target multiple HSC niches, promote hematopoietic regeneration, and improve clinical outcomes of BMT.

The Role of Microglia in Glioblastoma.

Glioblastoma (GB), the most aggressive malignant glioma, is made up of a large percentage of glioma-associated microglia/macrophages (GAM), suggesting that immune cells play an important role in the pathophysiology of GB. Under physiological conditions, microglia, the phagocytes of the central nervous system (CNS), are involved in various processes such as neurogenesis or axonal growth, and the progression of different conditions such as Alzheimer’s disease. Through immunohistochemical studies, markers that enhance GB invasiveness have been shown to be expressed in the peritumoral area of ​​the brain, such as Transforming Growth Factor α (TGF-α), Stromal Sell-Derived Factor 1 (SDF1/CXCL12), Sphingosine-1-Phosphate (S1P) and Neurotrophic Factor Derived from the Glial cell line (GDNF), contributing to the increase in tumor mass. Similarly, it has also been described 17 biomarkers that are present in hypoxic periarteriolar HSC niches in bone marrow and in hypoxic periarteriolar GSC niches in glioblastoma. Interestingly, microglia plays an important role in the microenvironment that supports GB progression, being one of the most important focal points in the study of therapeutic targets for the development of new drugs. In this review, we describe the altered signaling pathways in microglia in the context of GB. We also show how microglia interact with glioblastoma cells and the epigenetic mechanisms involved. Regarding the interactions between microglia and neurogenic niches, some authors indicate that glioblastoma stem cells (GSC) are similar to neural stem cells (NSC), common stem cells in the subventricular zone (SVZ), suggesting that this could be the origin of GB. Understanding the similarities between SVZ and the tumor microenvironment could be important to clarify some mechanisms involved in GB malignancy and to support the discovering of new therapeutic targets for the development of more effective glioblastoma treatments.

Complete Spatial Mapping of Steady-State and Stress Erythropoiesis

The anatomy of differentiation in the bone marrow (BM) is poorly understood due to lack of markers to image stepwise HSPC differentiation. We analyzed 250+ cell surface molecules in all hematopoietic progenitors and identified 56 differentially expressed markers in at least one HSPC that can be "mixed and matched" to prospectively image any HSPC of interest in the bone marrow. We used this data to develop a pipeline to map stepwise erythropoiesis in vivo. We found that all erythroid progenitors can be defined as Ly6C-CD27-ESAM-CD117+ cells and then Pre-MegE (earliest erythroid progenitor Cell Stem Cell. 2007 1(4):428-42) are CD150+CD71-. These give rise to CD71+CD150+ Pre-CFU-E that differentiate into CD71+CD150- CFU-E that then generate early erythroblasts. All BFU-E activity was restricted to Pre-MegE and Pre- CFU-E (70 and 30% of all BFU-E) whereas all CFU-E colonies were spread between Pre-MegE (44%), pre-CFU-E (10%) and CFU-E (46%). We also confirmed previously published data showing that CD71 and Ter119 can be used to image stepwise terminal erythropoiesis; CD71+Ter119dim early erythroblasts, CD71+Ter119bright late erythroblasts, CD71dimTer119bright reticulocytes and CD71-Ter119bright erythrocytes. Importantly, all populations were detected at identical frequencies using FACS or confocal imaging indicating that our imaging strategy detects all erythroid cells (Pre-CFU-E: 0.022 vs 0.027 %; CFUE: 0.32 vs 0.30%; Early-Ery: 0.62 vs 0.66%; Late-Ery: 32.05 vs 32.12%; Reticulocyte: 5.98 vs. 3.36%; Erythrocytes: 12.49 vs. 13.47%). We mapped the 3D location of every erythroid lineage cell in mouse sternum and interrogated the spatial relationships between the different maturation steps and with candidate niches. We compared the interactions found in vivo with those found in random simulations. Specifically, we used CD45 and Ter119 to obtain the spatial coordinates of every hematopoietic cell. Then we randomly placed each type of erythroid lineage cell at identical frequencies as those found in vivo to generate random simulations. We found erythroid progenitors show no specific association with HSC, indicating that Pre-Meg-E or more primitive progenitors leave the HSC niche after differentiation. Both Pre-Meg-E and Pre-CFU-E are found as single cells through the central BM space and do not specifically associate with other progenitors, or components of the microenvironment. In contrast almost all CFU-E locate to strings (28 strings per sternum) containing 8 CFU-E that are selectively recruited to sinusoids (mean CFU-E to sinusoid distance=2.2µm). As soon as CFU-E detach from sinusoids they downregulate CD117 and upregulate CD71 giving rise to a cluster of early erythroblasts that buds from the vessel. These progressively upregulate Ter119 to generate large clusters of late erythroblasts that in turn differentiate into clusters of reticulocytes and erythrocytes. To examine the clonal architecture of erythropoiesis we used Ubc-creERT2:confetti mice where a tamoxifen pulse leads to irreversible expression of GFP, CFP, YFP or RFP. Four weeks later we found that the CFU-E strings are oligoclonal with each clone contributing 2-6 CFU-E to the string. The budding erythroblasts clusters are similarly organized. These indicate that different CFU-E are serially recruited to the same sinusoidal spot where they self-renew 1-2 times and then undergo terminal differentiation. We then tracked how this architecture changed in response to stress (hemorrhage). Two days after bleeding we found that Pre-Meg-E and Pre-CFU-E numbers and locations were unaltered. The number of CFU-E strings remained constant (30 CFUE strings/sternum) but all strings contained more CFU-E (2-fold) suggesting increased self-renewal. Unexpectedly, fate mapping showed that the size of CFU-E clones did not increase when compared to steady-state. These results indicate that all CFU-E expand in respond to stress and that this is mediated via increased recruitment and differentiation of upstream progenitors. In summary we have found 56 differentially expressed markers that can be combined to detect most HSPC; validated a 5-color stain to image and fate map all steps of red blood cell maturation in situ; demonstrated that terminal erythropoiesis emerges from strings of sinusoidal CFU-E, and revealed the clonal architecture of normal and stress erythropoiesis. Disclosures No relevant conflicts of interest to declare.

In Situ Fate Mapping of Native and Stress Myelopoiesis Reveals a Unique Niche for Mono- and Dendritic Cell -Poiesis

In contrast to virtually all other tissues in the body the anatomy of differentiation in the bone marrow remains unknown. This is due to the lack of strategies to examine blood cell production in situ, which are required to better understand differentiation, lineage commitment decisions, and to define how spatial organizing cues inform tissue function. We developed approaches to image and fate map -using confetti mice- myelopoiesis in situ and generated 3D atlases of granulocyte and monocyte/dendritic cell differentiation during homeostasis and after emergency myelopoiesis induced by infection with Listeria monocytogenes. Figure 1 shows stepwise differentiation during myelopoiesis. We have found that -in imaging studies- CD11b-Ly6C-CD117+CD115+ cells are MDP; Lin-CD117+CD16/32+CD115- cells are GMP; CD11b-Ly6C+CD117+CD115+ are MOP; CD11b-CD117+CD115-Ly6C+ are GP; CD11b+CD115+Ly6Chi and CD11b+CD115+Ly6Clo cells are Ly6Chi and Ly6Clo monocytes; and MHCIIhi reticular cells are dendritic cells (DC). We used these markers to map every myeloid cell in the sternum and assessed the relationships between myeloid progenitors, their offspring and candidate niches in situ with single cell resolution. To test whether the interactions observed were specific we obtained the X, Y and Z coordinates for every hematopoietic cell in the sternum (detected using αCD45 and αTer119). We then used these coordinates to randomly place each type of myeloid cell, at the same frequencies found in vivo, through the BM to generate random distributions for each myeloid cell type. We found that myeloid progenitors do not localize with HSC indicating that they leave the HSC niche during differentiation. In the steady-state GP, MOP, and MDP are found as single cells that do not associate with each other indicating that granulo-, mono-, and dendritic cell-poiesis take place in different location. Myeloid progenitors are specifically recruited to sinusoids but are depleted near endosteum and arterioles (e.g. mean MDP distance to sinusoids, arterioles, and endosteum observed 5, 134, and 105μm vs 9, 86, and 69µm in the random simulation). GP form clusters with preneutrophils and immature neutrophils, in situ fate mapping demonstrated that these clusters are oligoclonal and that additional GP are serially recruited to the cluster as the old ones differentiate. Ly6Clo monocytes and dendritic cells are selectively enriched near MDP (2.0 DC and 4.4 Ly6Clo monocytes observed within 50µm of an MDP vs 0.9 DC and 1.8 Ly6Clo monocytes in the random simulation p=0.02 and p<0.0001). Fate mapping experiments demonstrated that the monocytes around MOP and monocytes and dendritic cells around the MDP are oligoclonal but are not the derived from the MOP/MDP they associate with. These indicate that Ly6Clo monocytes and DC are produced elsewhere but are then selectively recruited to regions enriched in MDP. The results above suggest that different sinusoids might be responsible for supporting different myeloid lineages. We found that dendritic cells localize to a unique subset (8% of all vessels) of colony stimulating factor 1 (CSF1, the major regulator of monopoiesis) -expressing sinusoids. Csf1 deletion in the vasculature disrupted MDP interaction with sinusoids, leading to reduced MDP numbers and differentiation ability, with subsequent loss of Ly6Clo monocytes and dendritic cells. L. monocytogenes infection dramatically changed the architecture of myelopoiesis and caused massive expansion of myeloid progenitors leading to the formation of monoclonal GP clusters and oligoclonal MOP clusters whereas MDP are still found as single cells associated with dendritic cells. Even after this massive insult granulopoiesis and mono/DC poiesis remained spatially segregated to different sinusoids. Csf1 deletion in the vasculature prevented generation of MDP and dendritic cells in response to infection. In summary we have developed strategies to image and fate map myelopoiesis in situ; revealed spatial segregation of -and distinct clonal architectures for- granulopoiesis and mono/DCpoiesis; and identified rare CSF1+ sinusoids that maintain mono/DCpoiesis in the steady-state and after infection. These data indicate that there is a specific spatial organization of definitive hematopoiesis and that local cues produced by distinct blood vessels are responsible for this organization. Figure Disclosures No relevant conflicts of interest to declare.

VEGF-C protects the integrity of the bone marrow perivascular niche in mice

AbstractHematopoietic stem cells (HSCs) reside in the bone marrow (BM) stem cell niche, which provides a vital source of HSC regulatory signals. Radiation and chemotherapy disrupt the HSC niche, including its sinusoidal vessels and perivascular cells, contributing to delayed hematopoietic recovery. Thus, identification of factors that can protect the HSC niche during an injury could offer a significant therapeutic opportunity to improve hematopoietic regeneration. In this study, we identified a critical function for vascular endothelial growth factor-C (VEGF-C), that of maintaining the integrity of the BM perivascular niche and improving BM niche recovery after irradiation-induced injury. Both global and conditional deletion of Vegfc in endothelial or leptin receptor–positive (LepR+) cells led to a disruption of the BM perivascular niche. Furthermore, deletion of Vegfc from the microenvironment delayed hematopoietic recovery after transplantation by decreasing endothelial proliferation and LepR+ cell regeneration. Exogenous administration of VEGF-C via an adenoassociated viral vector improved hematopoietic recovery after irradiation by accelerating endothelial and LepR+ cell regeneration and by increasing the expression of hematopoietic regenerative factors. Our results suggest that preservation of the integrity of the perivascular niche via VEGF-C signaling could be exploited therapeutically to enhance hematopoietic regeneration.

Abstract LB-225: Twisting stemness in AML using nanoparticle as a delivery system for siRNA targeting Twist

Abstract Twist-1, a basic helix-loop-helix transcription factor, regulates embryogenesis and epithelial-mesenchymal transition (EMT). Twist-1 is essential for the differentiation and specification of mesoderm-derived tissues and hematopoietic stem cells. In hematopoiesis, Twist-1 plays an important role in maintaining the bone marrow niche to support normal hematopoiesis. In human tumorigenesis, Twist-1 is overexpressed and promotes EMT, a key process in the metastases and drug resistance. In addition, recent evidence indicates that Twist-1-Bmi1 axis confers acute myeloid leukemia (AML), cell self-renewal and apoptosis resistance which highlights the importance of the Twist1-Bmi1 axis in the regulation of self-renewal in both normal hematopoietic and leukemia stem cells (LSCs). The hypothesis of the current study states that dysregulation of Twist-1 is implicated in the pathogenesis of various hematological malignancies, AML. This study demonstrates the efficacy of nanoparticle delivery using hyaluronic-acid conjugated mesoporous silica nanoparticles (MSN-HA) as an intracellular delivery mechanism for siRNA designed to knock down Twist-1 (siTwist-1). The nanoparticle is designed for tumor stem cell specificity as its HA component exclusively targets LSCs through their native ligand, CD44. In addition, the nanoparticles' electrostatically bonded polyethylene (PEI) coat protects the siRNA from nuclease degradation. Using siRNA nanotechnology, previous studies demonstrated the ability to target and silence cytoplasmic Twist-1 mRNA and re-sensitize cancer cells to conventional chemotherapeutic agents in solid tumor models. In this study, we demonstrate the efficacy of this nanoparticle siRNA technology on the knockdown of Twist-1 in the AML cell line, KG1a, and the consequent cascading effect of upregulating the tumor suppressor genes and downregulating stemness genes. Our data indicate that Twist-1 promotes leukemia cell growth and colony-forming units (CFU) through the Twist/c-MPL axis, and knockdown of Twist1 inhibits tumor growth and reduce the number of CFUs. Our Results also showed that Twist-1 interacts with Runt domain of Runx-1, and c-MPL, reported to be negatively regulated by RUNx-1. Thus, nanotechnology which combines CD44 targeting and Twist Knockdown, has shown promising results for LSCs targeting, mitigating metastasis in AML. Further investigations of the role of Twist HSC and bone marrow niche will provide more insight into strategic management and treatment for leukemic stem cells. Citation Format: Ebtesam Nafie, Angelina Flores, Idoroenyi Amanam, Ruining Wang, Jeffrey Zink, Lucy Ghoda, Guido Marcucci, Carlotta Glackin. Twisting stemness in AML using nanoparticle as a delivery system for siRNA targeting Twist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-225.

3045 – MAPPING MYELOID DIFFERENTIATION IDENTIFIES A CSF1+ VASCULAR NICHE FOR MYELOPOIESIS

Mounting evidence indicates that lineage decisions are controlled by local structures that support differentiation in distinct regions of the bone marrow. Our ability to manipulate hematopoiesis to treat disease has been hampered by our lack of understanding of these anatomical cues. Here we described the development of pipelines to simultaneously image myeloid progenitors together with their offspring, HSC, and endosteal and vascular niches. We found that myeloid progenitors move away from HSC during differentiation. Myeloid progenitors do not localize with each other, instead they localize to different sinusoids where they give rise to clusters of immature cells. Granulocyte, monocyte, and dendritic cell production takes place in different sinusoids suggesting the existence of specialized microenvironments that organize myelopoieis. Indeed, MDP selectively cluster with Ly6Clo monocytes and conventional dendritic cells; and localize to a unique subset of CSF1-expressing sinusoids (8% of all sinusoids), Csf1 deletion in the vasculature disrupted the MDP clusters and their interaction with sinusoids, leading to reduced MDP numbers and differentiation ability, with subsequent loss of Ly6Clo monocytes and dendritic cells. These indicate that there is a specific spatial organization of definitive hematopoiesis and that local cues produced by distinct vascular niches are responsible for this organization.

3043 – A PIPELINE TO IMAGE HEMATOPOIETIC DIFFERENTIATION REVEALS THE ARCHITECTURE OF ERYTHROPOIESIS IN THE BONE MARROW

The anatomy of differentiation in the BM is poorly understood due to a lack of markers to image stepwise HSPC differentiation. We immunophenotyped all types of multipotent and lineage committed HSPC and identified 56 cell surface markers that can be "mixed and matched" to prospectively image HSPC. We used this data to develop a pipeline to map erythropoiesis in whole-mounted sternum. Pre-Meg-E are ESAM-Ly6C-CD117+CD150+CD71-, Pre-CFU-E are Ly6C-CD117+CD150+CD71+ and CFU-E are Ly6C-CD117+CD150-CD71+ cells. We found that all three types of erythroid progenitors show no specific association with LT-HSC/MPP2/MkP, indicating that Pre-Meg-E or more primitive progenitors leave the HSC niche after differentiation. Both Pre-Meg-E and Pre-CFU-E are found as single cells through the central BM space and do not specifically associate with other progenitors, or components of the microenvironment. In contrast almost all CFU-E locate to strings (28 strings per sternum) containing 6±0.28 CD117brightCD71dim CFU-E that are selectively recruited to sinusoids (mean CFU-E to sinusoid distance=2.2µm). As soon as CFU-E detach from sinusoids they show sharp reductions in CD117 and upregulation of CD71 giving rise to a cluster of early erythroblasts that buds from the vessel. These progressively upregulate Ter119 to generate large clusters of late erythroblasts that in turn differentiate into clusters of reticulocytes and erythrocytes. To understand how the architecture of erythropoiesis changes in response to stress we performed phlebotomy. Two days later we found Pre-Meg-E and Pre-CFU-E numbers and locations was unaltered. The number of CFU-E strings remained constant (30 CFUE strings/sternum) but all strings contained more CFU-E (2-fold) that then differentiated as above. Our imaging pipeline can be quickly adapted to image all other HSPC during normal, stress, and diseased hematopoiesis.

Connecting secretome to hematopoietic stem cell phenotype shifts in an engineered bone marrow niche.

Hematopoietic stem cells (HSCs) primarily reside in the bone marrow, where they receive external cues from their local microenvironment. The complex milieu of biophysical cues, cellular components and cell-secreted factors regulates the process by which HSC produce the blood and immune system. We previously showed direct coculture of primary murine hematopoietic stem and progenitor cells with a population of marrow-derived mesenchymal stromal and progenitor cells (MSPCs) in a methacrylamide-functionalized gelatin (GelMA) hydrogel improves hematopoietic progenitor maintenance. However, the mechanism by which MSPCs influenced HSC fate decisions remained unknown. Herein, we report the use of proteomic analysis to correlate HSC phenotype to a broad candidate pool of 200 soluble factors produced by combined mesenchymal and hematopoietic progeny. Partial least squares regression (PLSR), along with an iterative filter method, identified TGFβ-1, MMP-3, c-RP and TROY as positively correlated with HSC maintenance. Experimentally, we then observe exogenous stimulation of HSC monocultures in GelMA hydrogels with these combined cytokines increases the ratio of hematopoietic progenitors to committed progeny after a 7-day culture 7.52±3.65-fold compared to non-stimulated monocultures. Findings suggest a cocktail of the downselected cytokines amplifies hematopoietic maintenance potential of HSCs beyond that of MSPC-secreted factors alone. This work integrates empirical and computation methods to identify cytokine combinations to improve HSC maintenance within an engineered HSC niche, suggesting a route toward identifying feeder-free culture platforms for HSC expansion. Insight Hematopoietic stem cells within an artificial niche receive maintenance cues in the form of soluble factors from hematopoietic and mesenchymal progeny. Applying a proteomic regression analysis, we identify a reduced set of soluble factors correlated to maintenance of a hematopoietic phenotype during culture in a biomaterial model of the bone marrow niche. We identify a minimum factor cocktail that promotes hematopoietic maintenance potential in a gelatin-based culture, regardless of the presence of mesenchymal feeder cells. By combining empirical and computational methods, we report an experimentally feasible number of factors from a large dataset, enabling exogenous integration of soluble factors into an engineered hematopoietic stem cell for enhanced maintenance potential of a quiescent stem cell population.

The Autonomic Nervous System Pulls the Strings to Coordinate Circadian HSC Functions.

As for many other adult stem cells, the behavior of hematopoietic stem and progenitor cells (HSPCs) is subjected to circadian regulatory patterns. Multiple HSPC functions, such as proliferation, differentiation or trafficking exhibit time-dependent patterns that require a tight coordination to ensure daily blood cell production. The autonomic nervous system, together with circulating hormones, relay circadian signals from the central clock—the suprachiasmatic nucleus in the brain—to synchronize HSC niche physiology according to light/darkness cycles. Research over the last 20 years has revealed how specific neural signals modulate certain aspects of circadian HSC biology. However, only recently some studies have started to decipher the cellular and molecular mechanisms that orchestrate this complex regulation in a time-dependent fashion. Here we firstly review some of the recent key findings illustrating how different neural signals (catecholaminergic or cholinergic) regulate circadian HSC egress, homing, maintenance, proliferation, and differentiation. In particular, we highlight the critical role of different neurotransmitter receptors in the bone marrow microenvironment to channel these neural signals and regulate antagonistic processes according to circadian cues and organismal demands. Then, we discuss the potential biological meaning of HSC circadian regulation and its possible utility for clinical purposes. Finally, we offer our perspective on emerging concepts in HSC chronobiology.

Local Irradiation Induces Systemic Inflammatory Response and Alteration of the Hematopoietic Stem Cell Niche

Radiotherapy is used in the treatment of ~50% of tumors. We and others have reported long-term suppression of hematopoietic stem and progenitor cells (HSPCs) in the setting of total body irradiation; however, it has been shown that even relatively small irradiation volumes can result in systemic adverse events, such as myeloablation and secondary malignancies. The mechanisms underlying these effects are unclear. We hypothesize that localized radiation may activate a systemic inflammatory response that can acutely alter HSPCs and bone marrow microenvironment (BMME) components, including marrow stromal cells (MSCs), thereby contributing to late effects. We therefore established a murine model of targeted irradiation (TR) using a small animal radiation research platform (SARRP). Methods: We administered local irradiation to a single tibia of 6-8 week old C57BL/6 male mice using a single dose of 15 Gy. Subsequently, we analyzed peripheral blood, BM, BM extracellular fluid (BMEF), collagenase-1 digested bone associated cells of both the irradiated (TR) and non-irradiated, contralateral (CONT) tibiae at 2, 6, 48 hours, 1 and 3 weeks post-TR, performing phenotypic (flow cytometry) and cytokine analyses. For all studies, n = 10-13 mice/time point. Results: In the TR tibia at 2 hours, although total cell numbers were unchanged, there was a significant upregulation of inflammatory cytokines (interleukin 1β (IL1b), IL18), chemokines (CXCL2, CXCL10, CCL2, CCL3) and macrophage colony stimulating factor (M-CSF). Of note, most of these changes normalized by 48 hours (M-CSF at 1 week). Changes in mediator expression were followed, at 6 hours post-TR, by significant increases in macrophage (macs) numbers, including CD206 phagocytic macs, neutrophils (PMNs) and cytotoxic lymphocytes, including CD8+ cells expressing CXCR3+, the receptor for CXCL9 and CXCL10. Interestingly, similar to the TR tibia, CXCL2 expression was also increased significantly in the CONT at 2 hours, followed (6 hours) by significant increases in macs and CD8+ cells, suggesting a systemic or abscopal effect. With respect to the effects of radiation on HSPCs, by 6 hours, most of the stem and progenitor cell (HSPC) populations in the TR marrow were significantly decreased; the decrease in long-term-HSCs was delayed until 48 hours post-TR. All populations remained severely depleted until 3 weeks post RT, demonstrating a rapid and sustained effect of TR on all HSPCs within the irradiation volume. In comparison, in the CONT tibia at 6 hours, CD41+ HSCs were expanded; this is consistent with previous demonstrations that CD41+ LT-HSCs expand with inflammatory signals and suggests that TR-induced signals induced a systemic impact on the non-irradiated HSPCs. By 1 week post-radiation, short term-HSCs were significantly decreased in the CONT marrow, likely due to mobilization since CFU-Cs were correspondingly significantly increased in the circulation. Finally, MSCs, previously shown to support HSCs, were found to be significantly increased in the TR tibia starting at 6 hours and peaking at 48 hours post-radiation. Surprisingly, MSCs were also expanded in the CONT marrow at 48 hours; this expansion was likely associated with the increased CXCL12 levels seen in both TR and CONT marrow, although the CXCL12 levels were higher in the irradiated tibia. Taken together, these changes indicate TR-induced global disruption of the HSC niche. Furthermore, in addition to the transient effects of localized irradiation, we observed a second wave of inflammatory signals, including a significant increase in CCL3, at 1 week post-TR and increased IL1b in the CONT marrow at 3 weeks, changes that may have contributed to the sustained loss of HSPC populations. Conclusions: We present the effects of local irradiation on global hematopoiesis, showing that, in addition to the anticipated acute local changes in the irradiated bone marrow, TR-induced persistent and, more importantly, systemic inflammation. We believe that using this murine model will allow us to dissect the contribution of direct (local) and indirect (systemic) responses to radiation on treatment effects, such as marrow failure and secondary malignancies. Disclosures No relevant conflicts of interest to declare.