Niche Factors Research Articles

IGF2 enhanced the osteo-/dentinogenic and neurogenic differentiation potentials of stem cells from apical papilla.

In dental tissue engineering, niche is important for maintaining stem cell function and regenerating the dental tissues. However, there is limited knowledge for the growth factors in niche to maintain the function of stem cells. In this study, we investigated the effect of IGF2, a growth factor in stem cells from apical papilla (SCAPs) niche, on differentiation and proliferation potentials of SCAPs. Recombinant human IGF2 protein (rhIGF2) was used. Cell counting kit-8 assay, Carboxyfluorescein succinimidyl ester assay, alkaline phosphatase (ALP) activity, Alizarin Red staining, quantitative calcium analysis, immunofluorescence staining and real-time RT-PCR were performed to investigate the cell proliferation and differentiation potentials of SCAPs. And proteomic analysis was used to identify the differential secreted proteins. By ALP activity assay, we found that 5ng/mL rhIGF2 might be the optimal concentration for treatment. Then, Alizarin Red staining, quantitative calcium analysis and osteogenesis-related gene expression results showed that 5ng/mL rhIGF2 could enhance the osteo-/dentinogenic differentiation potentials in SCAPs. Immunofluorescence staining and real-time RT-PCR results showed that neurogenic markers were significantly induced by 5ng/mL rhIGF2 in SCAPs. Then, CCK-8 assay and CFSE assay results showed that 5ng/mL rhIGF2 could enhance the cell proliferation in SCAPs. Furthermore, proteomic analysis showed that IGF2 could induce some secreted proteins which function related to the osteogenesis, neurogenesis and cell proliferation. Our results identified that IGF2 might be the potential mediator in niche to promote SCAP function and dental tissue regeneration.

2011 - ENGINEERING A HEMATOPOIETIC STEM CELL NICHE BY REVITALIZING MESENCHYMAL STEM CELLS

Hematopoietic stem cells (HSCs) are maintained by bone marrow (BM) niches in vivo, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors by Nestin-GFP+ mesenchymal-derived stem cells (MSCs) is downregulated upon culture, suggesting that transcriptional rewiring may contribute to this reduced HSC maintenance potential. Using an RNA sequencing screen, we identified 5 transcription factors (Klf7, Ostf1, Xbp1, Irf3, Irf7) that restored HSC niche function in cultured BM-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed higher repopulation capacity and protected lethally irradiated recipient mice. Competitive reconstitution assays revealed 7-fold expansion of functional HSCs by rMSCs. rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. We have also identified Mef2c as an important transcription factor downstream of KOXII. Our results suggest that all five KOXII genes are necessary to fully restore the niche activity in MSCs ex vivo. Some of these genes may act singly, perhaps by preventing MSC differentiation or driving expansion of hematopoietic growth factors. Our results thus establish a new platform that provides critical insight in the regulatory network of the HSC niche and forms the basis toward the engineering of supportive niches for curative cell therapies.

PROSTACYCLIN IS A NOVEL HEMATOPOIETIC STEM CELL REGULATOR ENRICHED IN THE ENDOSTEAL BONE MARROW NICHE

Potent functional HSCs are enriched at the endosteal bone marrow (BM), which comprises ∼10% of total BM. To identify novel niche factors that regulate HSCs, we performed a gene expression microarray seeking genes overexpressed in the endosteal relative to central BM. Prostacyclin/prostaglandin I2 (PGI2) synthase (Ptgis) was one of the highest enriched genes in the endosteal versus central BM (27-fold). PTGIS is the sole enzyme for biosynthesis of PGI2. PGI2 has no reported roles in HSC regulation in the BM and was chosen for further investigation. We found PGI2 analogue iloprost treatment of sorted BM lineage- Kit+ Sca1- (LKS+) cells in vitro for 7 days potently reduced proliferation and differentiation compared to vehicle control. BM cells pulse treated ex vivo with iloprost for 1 hour also resulted in 14-fold increased multilineage reconstitution potential compared to vehicle controls in competitive transplant assay. Iloprost administration in vivo partially rescued BM HSC reconstitution potential in mice sub-lethally (6.5 Gy) irradiated or administered pro-inflammatory granulocyte colony stimulating factor (G-CSF) for 3 days. These data suggest that PGI2 protects HSC from stress. Mechanistically, we found iloprost pulse treatment in vitro was associated with increased pSTAT3 and cell cycle progression in HSCs. Additionally, we found ∼4-fold greater proportion of untreated LKS+ HSPC homing to the BM of recipients co-administered iloprost + G-CSF compared to vehicle + G-CSF at 4 hours post-transplant. We found that G-CSF administration also decreased Scf and Cxcl12 mRNA expression in BM, which were partially rescued with iloprost co-administration. These data suggest that PGI2 protects HSC during stress by acting directly on HSCs and indirectly to preserve BM niche functions. In summary, we identified PGI2 as a novel HSC niche factor abundant in the endosteal BM.

Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function

Intestinal organoids have emerged as a powerful in vitro tool for studying intestinal biology due to their resemblance to in vivo tissue at the structural and functional levels. However, their sphere-like geometry prevents access to the apical side of the epithelium, making them unsuitable for standard functional assays designed for flat cell monolayers. Here, we describe a simple method for the formation of epithelial monolayers that recapitulates the in vivo-like cell type composition and organization and that is suitable for functional tissue barrier assays. In our approach, epithelial monolayer spreading is driven by the substrate stiffness, while tissue barrier function is achieved by the basolateral delivery of medium enriched with stem cell niche and myofibroblast-derived factors. These monolayers contain major intestinal epithelial cell types organized into proliferating crypt-like domains and differentiated villus-like regions, closely resembling the in vivo cell distribution. As a unique characteristic, these epithelial monolayers form functional epithelial barriers with an accessible apical surface and physiologically relevant transepithelial electrical resistance values. Our technology offers an up-to-date and novel culture method for intestinal epithelium, providing an in vivo-like cell composition and distribution in a tissue culture format compatible with high-throughput drug absorption or microbe-epithelium interaction studies.

Abstract 108: Efficiency improvement niche factors in drug susceptibility test using two dimension organoids derived from colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading cause of cancer-related death of world wide. About one in five patients with CRC has metastatic disease, and it is the major cause of death. Metastasis of the primary tumor is directly related to the patient survival. Systemic chemotherapy is the standard approach to treat the metastatic CRC (mCRC), and there are several protocols currently used for the mCRC, and the median survival time has increased by the systemic chemotherapy such as irinotecan, oxaliplatin, cetuximab and bevacizumab. However, the effect of the chemotherapy depends on the character of the individual tumor; we still have many patients who could not receive any benefit from the systemic chemotherapy. Therefore, we examined the individual drug sensitivity using organoids derived from each patient to predict the clinical efficacy of the chemotherapeutic drugs. We established a novel culture method for the patient’s tumor, named “two-dimension organoids (2DO)” using surgically resected specimens harboring phenotypic heterogeneity as an ideal patient’s tumor model. The gene expression of these organoids was similar to the each parental tumor, and they constructed a ductal structure of the tumor in the xenograft model. The previous study using 2DO allowed us to predict the clinical efficacy of chemotherapeutic drugs. However, all patients who underwent surgical resection do not need the chemotherapy. We examined the culture method and evaluated additional niche factors to establish 2DO efficiently from small-size samples, such as biopsy samples. Inflammation is a well-known risk factor for the development of colorectal cancer and several cytokines play the important roles in inflammatory reactions. Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine which is predominantly produced by macrophages as well as tumor cells. It was reported to promote tumor progression in some cancers where TNF-alpha activates the NF-κB pathway, which represents the act in the oncogenesis and cancer cell proliferation. The relationship between TNF-alpha and 2DO growth was examined in surgically resected specimens and endoscopic biopsies (n=15, each). The growth rates of 2DOs with TNF-alpha were significantly faster than the others without TNF-alpha (P=0.039). Using these 2DO cultured with TNF-alfa, the correlations for the chemotherapeutic drugs were observed between the 2DO and clinical outcomes of chemotherapy. In conclusion, TNF-alpha supports the growth of 2DO even from the small-size samples, resulting in the useful material for the precision medicine. Citation Format: Kazuhiro Saso, Norikatsu Miyoshi, Masaru Sasaki, Shiki Fujino, Hidekazu Takahashi, Naotsugu Haraguchi, Taishi Hata, Chu Matsuda, Tsunekazu Mizushima, Masaki Mori, Yuichiro Doki. Efficiency improvement niche factors in drug susceptibility test using two dimension organoids derived from colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 108.

Abstract 1925: Establishment and characterization of 3D cancer organoids as clinically relevant ex vivo drug screening tools for cancer translational research and drug discovery

Abstract Patient derived cancer organoids (PDO), a form of comprehensive 3D spheroid cultures that harbor cancer multicellular components and mimic cancer lesion structures and tumor heterogeneity, have been proven to be innovative preclinical model system for use in both academic research and industrial cancer drug development. It has been demonstrated by a large amount of independent literature reports that 3D PDOs can better predict drug response than 2D cell line settings due to their clinical relevant tumor micro-environment. We have the world’s largest patient derived xenograft (PDX) tumor model collection (~2500 models), which were annotated and validated genomically and phenotypically. However, there appears to be limitations when using PDX models to evaluate compounds in large scale fashion with narrow time windows and limited budget allocation. To address these limitations and to fully leverage preclinical values of these PDX model assets, we have established and characterized a series of PDX-derived cancer organoid (PDXO) models so that they could be used as scalable and high throughput compatible drug screening platforms. As first phase of development, we started with low passage PDX primary tumors from 5 major cancers types, including colorectal, lung, breast cancer, gastric and pancreatic carcinoma. The PDX tumor tissues were mechanically and enzymatically dissociated and the digested tumor fragments were cultured and passaged in Matrigel containing culture medium supplemented with stem cell niche factors. Technical protocols for 3D cultures including culture propagation procedures and medium optimization have been established. Characterization and QC of these PDXO models were done by gross morphology, histopathology, and genomic profiling. Histopathological analysis showed cellular/structural similarities (ductal, mucous or carcinoid) between PDXO models and original PDX tumor tissues, suggesting that tissue specific structural features were maintained in the 3D organoids. Moreover, ~10 PDXO models were tested for ex vivo cytotoxicity with SOC drugs. The preliminary data indicated that majority of (73%, 24/33) the PDXO ex vivo drug sensitivity IC50 datasets correlated with the in vivo PDX drug sensitivity TGI data. The negative and positive predictive values for PDXO are 94% and 53%, respectively. Genomic characterization including RNAseq and WES, is in progress. In summary, we successfully established PDXO models that were stably passaged from PDX primary tumors and resembled original PDX tumors both morphologically and histopathologically. In addition, the PDXOs apparently had encouraging predictive values for SOC drug response. Further in-depth model validation/characterization is warranted. Citation Format: Xiaoxi Xu, Satya Pathi, Limei Shang, Yan Liu, Peng Han, Likun Zhang, Binchen Mao, Davy Ouyang, Henry Li, Wenqing Yang. Establishment and characterization of 3D cancer organoids as clinically relevant ex vivo drug screening tools for cancer translational research and drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1925.

PF465 UPREGULATION OF CD276 IN ENDOTHELIAL CELLS OF MURINE AND PDX ACUTE MYELOID LEUKAEMIA MODELS

Background:Haematopoietic stem cells (HSCs) reside atop of the blood hierarchy and localise in the bone marrow (BM). Acute myeloid leukaemia (AML) is caused by accumulation of mutations in the stem cell compartment, which blocks the differentiation of the myeloid lineage. AML also modulates the BM microenvironment but the mechanisms of action are largely unknown. We and others, demonstrated recently that BM marrow vasculature is damaged in AML engrafted mice (Passaro et al. 2017; Duarte et al. 2018). The arterioles are increased, while the sinusoidal endothelial cells are vastly decreased. In order to understand the mechanism underlining this change, we previously performed RNA‐sequencing of CD31+ endothelial cells (ECs) extracted from healthy and leukemic mice.Aims:This project aims to identify key processes that play a role in the crosstalk between AML cells and BM microenvironment and test whether modulating these niche factors can improve the current therapeutic regimen. Thus, herein we focused on the potential role of CD276 on ECs of AML engrafted mice.Methods:RNAseq of BM ECs from non‐transplanted or AML Patients’ Derived Xenograft (PDX) mice was performed. Flow cytometry and immunostaining of BM ECs were performed. ShRNA against CD276 is being used to define the effect of its knockdown in ECs on leukaemia engraftment in our 3D humanised scaffold model. Blocking antibodies against CD276 will also be tested in vivo.Results:Analysis of the RNAseq data showed that CD276 (B7‐H3) gene is among the top 100 differentially expressed genes and is particularly upregulated in ECs of AML engrafted mice. It has been demonstrated that CD276 is overexpressed specifically in ECs of solid tumours and its blockade leads to tumour shrinkage (Seaman et al. 2017). The protein network map of CD276 indicated that it can participate in immune responses as well as adhesion signalling pathways. Indeed, there are several such genes deregulated in the leukemic ECs suggesting that CD276 might have a functional role in transforming the BM niche. CD276 overexpression was validated in ECs PDX and murine AML models. Moreover, we also observed this upregulation in human ECs by using our humanised scaffolds seeded with AML cell lines.Summary/Conclusion:So far, our RNAseq analysis of healthy and leukemic PDX mice shows that CD276 is being upregulated in ECs. We confirmed this upregulation at the protein level both using mouse and human ECs. Our next step is to determine the consequence of CD276 downregulation on leukaemia engraftment.

Cancer cell niche factors secreted from cancer-associated fibroblast by loss of H3K27me3

ObjectiveCancer-associated fibroblasts (CAFs), a major component of cancer stroma, can confer aggressive properties to cancer cells by secreting multiple factors. Their phenotypes are stably maintained, but the mechanisms are not...

APRIL signaling in human antibody secreting cells promotes cellular response via p38 and differential immediate early gene regulation

Abstract Long-lived antibody secreting cells (ASCs) persist as quiescent plasma cells (PCs) in niche microenvironments. However, we know little about how ASCs integrate these signals to drive changes in function. APRIL is an important in vivo niche factor, for which the primary receptor is BCMA, but to our knowledge APRIL-induced signaling and consequent gene regulation in primary human ASCs are poorly defined. In particular, little is known of signals that APRIL generates at the transition from proliferative plasmablast to quiescent plasma cell, an important transition in ASC differentiation. Using a model system of in vitro PC differentiation, which utilizes APRIL signaling as a key survival promoting factor, we demonstrate that significant levels of multimerized mega-APRIL are needed to deliver signals into ASCs. This in part reflects a process of cell intrinsic shedding of BCMA, the APRIL receptor, as previously reported by others. Blockade of proteolytic shedding by gamma secretase inhibitors enhances surface BCMA expression and mega-APRIL induced PC survival, generating phenotypically mature PCs with prolonged longevity from in vitro and ex vivo plasmablasts. At the plasmablast to plasma cell transition APRIL induces a selective pattern of MAP kinase activation in human ASCs, preferentially activating p38. This contrasts to another niche signal, SDF1, which preferentially targets ERK. Differential gene regulatory responses are evident for immediate early genes, with APRIL producing a preferential activation of FOSB relative to FOS or EGR1. The results provide the basis for systematically exploring the signaling and gene regulatory networks involved in the response of human ASCs to niche factors supporting PC survival.

Engineering a haematopoietic stem cell niche by revitalizing mesenchymal stromal cells.

Haematopoietic stem cells (HSCs) are maintained by bone marrow (BM) niches in vivo1,2, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors by Nestin-GFP+ mesenchymal-derived stromal cells (MSCs) is downregulated upon culture, suggesting that transcriptional rewiring may contribute to this reduced HSC maintenance potential. Using an RNA sequencing screen, we identified 5 transcription factors (Klf7, Ostf1, Xbp1, Irf3, Irf7) that restored HSC niche function in cultured BM-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed higher repopulation capacity and protected lethally irradiated recipient mice. Competitive reconstitution assays revealed ~7-fold expansion of functional HSCs by rMSCs. rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. Analysis of the reprogramming mechanisms uncovered a role for myocyte enhancer factor 2c (Mef2c) in the revitalization of MSCs. These results provide insight in the transcriptional regulation of the niche with implications for stem cell-based therapies.

Mitochondrial Dynamics: Biogenesis, Fission, Fusion, and Mitophagy in the Regulation of Stem Cell Behaviors.

Stem cells have the unique capacity to differentiate into many cell types during embryonic development and postnatal growth. Through coordinated cellular behaviors (self-renewal, proliferation, and differentiation), stem cells are also pivotal to the homeostasis, repair, and regeneration of many adult tissues/organs and thus of great importance in regenerative medicine. Emerging evidence indicates that mitochondria are actively involved in the regulation of stem cell behaviors. Mitochondria undergo specific dynamics (biogenesis, fission, fusion, and mitophagy) during stem cell self-renewal, proliferation, and differentiation. The alteration of mitochondrial dynamics, fine-tuned by stem cell niche factors and stress signaling, has considerable impacts on stem cell behaviors. Here, we summarize the recent research progress on (1) how mitochondrial dynamics controls stem cell behaviors, (2) intrinsic and extrinsic factors that regulate mitochondrial dynamics, and (3) pharmacological regulators of mitochondrial dynamics and their therapeutic potential. This review emphasizes the metabolic control of stemness and differentiation and may shed light on potential new applications in stem cell-based therapy.

Phenotypic heterogeneity of 2D organoid reflects clinical tumor characteristics

Unlike cancer cell lines, tumors and primary cultured cells exhibit phenotypic heterogeneity. Although methods for establishing organoids within three-dimensional (3D) gels are well-known, the growth is slower than that of two-dimensional (2D) cultured cells and many niche factors need to be added. In this study, we established primary cultured organoid in 2D culture (2D organoid; 2DO) in a reproducible manner and with clinical phenotypic heterogeneity. The 2DO contained cancer stem cells (CSCs) expressing CD44 and CD133. The addition of basic fibroblast growth factor and transforming growth factor-β as niche factors was necessary to establish the 2DO and maintain the CSC population. The established 2DO showed sufficient proliferation, and the culture could be transferred to the 3D culture. Morphological analysis of the xenograft induced by 2DO reflected parental tumor differentiation, and gene expression in the 2DO was similar to that in the parental tumor. In vitro drug sensitivity analysis using the 2DO reflected individual clinical courses. The 2DO is an in vitro personal cancer model, and the results of the drug sensitivity assessment may be useful for introducing clinical chemotherapy agents in personalized medicine.

Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment.

BackgroundGlioblastoma (GBM), a highly malignant brain tumor, invariably recurs after therapy. Quiescent GBM cells represent a potential source of tumor recurrence, but little is known about their molecular underpinnings.MethodsPatient-derived GBM cells were engineered by CRISPR/Cas9-assisted knock-in of an inducible histone2B-GFP (iH2B-GFP) reporter to track cell division history. We utilized an in vitro 3D GBM organoid approach to isolate live quiescent GBM (qGBM) cells and their proliferative counterparts (pGBM) to compare stem cell properties and therapy resistance. Gene expression programs of qGBM and pGBM cells were analyzed by RNA-Seq and NanoString platforms.FindingsH2B-GFP-retaining qGBM cells exhibited comparable self-renewal capacity but higher therapy resistance relative to pGBM. Quiescent GBM cells expressed distinct gene programs that affect cell cycle control, metabolic adaptation, and extracellular matrix (ECM) interactions. Transcriptome analysis also revealed a mesenchymal shift in qGBM cells of both proneural and mesenchymal GBM subtypes. Bioinformatic analyses and functional assays in GBM organoids established hypoxia and TGFβ signaling as potential niche factors that promote quiescence in GBM. Finally, network co-expression analysis of TCGA glioma patient data identified gene modules that are enriched for qGBM signatures and also associated with survival rate.InterpretationOur in vitro study in 3D GBM organoids supports the presence of a quiescent cell population that displays self-renewal capacity, high therapy resistance, and mesenchymal gene signatures. It also sheds light on how GBM cells may acquire and maintain quiescence through ECM organization and interaction with niche factors such as TGFβ and hypoxia. Our findings provide a starting point for developing strategies to tackle the quiescent population of GBM.FundNational Institutes of Health (NIH) and Deutsche Forschungsgemeinschaft (DFG).

A Comparative Immunofluorescence Study Demonstrating that Peri‐arteriolar Glioma Stem Cell Niches in Human Glioblastoma are a Mimic of Peri‐arteriolar Hematopoietic Stem Cell Niches in Human Bone Marrow

Glioblastoma is the most aggressive type of brain tumor with poor patient survival. A fraction of glioblastoma cells within glioblastoma tumors consists of slowly dividing and therefore therapy‐resistant glioma stem cells (GSCs) residing in protective peri‐arteriolar niches and are held responsible for tumor recurrence. The hypothesis of this study is that GSC niches are a mimic of hematopoietic stem cell (HSC) niches in human bone marrow as in both types of niches, similar niche factors and molecular interactions are involved in retention and release of stem cells. Immunofluorescence was performed on paraffin sections of 10 glioblastoma patient samples and 2 human bone marrow samples to localize GSC niches and HSC niches and to determine whether the two types of niches are similar or not. Smooth muscle actin (SMA) was used to detect smooth muscle cells in the arteriolar wall. The markers CD133 and CD150 were used as HSC markers and CD244 as hematopoietic progenitor cell marker. CD133 and SOX2 were used as markers to detect GSCs and CD105 and CD73 were used as markers to detect mesenchymal stem cells (MSCs). In addition, expression of chemoattractants stromal‐derived factor‐1α (SDF‐1α) and osteopontin (OPN), and their receptors, C‐X‐C receptor type 4 (CXCR4) and CD44, respectively, was localized. Expression of hypoxia‐inducible factor(HIF)‐1α, HIF‐2α and vascular endothelial growth factor (VEGF) was localized to determine hypoxic conditions. We found CD133‐CD150‐CXCR4‐CD44‐positive HSCs localized adjacent to bone around SMA‐positive arterioles in SDF‐1α‐ and OPN‐rich niches. CD244‐positive hematopoietic progenitor cells were localized at a distance from bone near sinusoids. CD105‐ and CD73‐positive MSCs were localized in peri‐arteriolar niches as well. Expression of HIF‐1α, HIF‐2α and VEGF confirmed hypoxia. Similarly, we found CD133‐SOX2‐CXCR4‐CD44‐positive GSCs around SMA‐positive arterioles in SDF‐1α‐ and OPN‐rich niches. CD105‐positive MSCS were localized around the arterioles. Hypoxic conditions were confirmed by HIF‐1α, HIF‐2α and VEGF expression in GSC niches. In both niche types, SDF‐1α and OPN retain HSCs and GSCs in hypoxic peri‐arteriolar niches by interacting with their receptors CXCR4 and CD44 on the HSCs and GSCs, respectively. MSCs are major SDF‐1α producers, attracting and maintaining HSCs and GSCs in niches. We conclude that GSC niches in glioblastoma are a mimic of HSC niches in bone marrow. We aim to disrupt interactions between GSCs and their protective niches to induce GSC differentiation and proliferation to render GSCs more sensitive to anti‐glioblastoma therapies.Support or Funding InformationKWF; UVA 2014‐6839This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rap1 signal modulators control the maintenance of hematopoietic progenitors in bone marrow and adult long-term hematopoiesis.

Adult long‐term hematopoiesis depends on sustaining hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM) niches, where their balance of quiescence, self‐renewal, and hematopoietic differentiation is tightly regulated. Although various BM stroma cells that produce niche factors have been identified, regulation of the intrinsic responsiveness of HSPC to the niche factors remains elusive. We previously reported that mice deficient for Sipa1, a Rap1 GTPase‐activating protein, develop diverse hematopoietic disorders of late onset. Here we showed that transplantation of BM cells expressing membrane‐targeted C3G (C3G‐F), a Rap1 GTP/GDP exchanger, resulted in the progressive decline of the numbers of HSPC repopulated in BM with time and impaired long‐term hematopoiesis of all cell lineages. C3G‐F/HSPC were sustained for months in spleen retaining hematopoietic potential, but these cells inefficiently contributed to overall hematopoietic reconstitution. C3G‐F/HSPC showed enhanced proliferation and differentiation with accelerated progenitor cell exhaustion in response to stem cell factor (SCF). Using a Ba/F3 cell line, we confirmed that the increased basal Rap1GTP levels with C3G‐F expression caused a markedly prolonged activation of c‐Kit receptor and downstream signaling through SCF ligation. A minor population of C3G‐F/HSPC also showed enhanced proliferation in the presence of thrombopoietin (TPO) compared to Vect/HSPC. Current results suggest an important role of basal Rap1 activation status of HSPC in their maintenance in BM for sustaining long‐term adult hematopoiesis.

Abstract WP88: Relationship Between Small Vessel Ischemic White Matter Disease and Collateral Recruitment in Patients With Acute Embolic Ischemic Stroke

Introduction: White matter hyperintensities (WMH) is the most prominent neuroimaging feature of small vessel disease (SVD). SVD is thought to be a result of arteriolosclerosis, chronic hypoperfusion of white matter, selective oligodendrocyte death and degeneration of myelinated fibers. The risk of ischemic stroke is significantly increased with the presence of WMH. The presence of intracranial collaterals is a strong predictor of salvageable neurological function in acute stroke; however, the factors affecting the ability to recruit collaterals are poorly understood. We wanted to study if the presence and severity of small vessel white matter disease had an adverse effect on cerebral collateral flow in acute embolic large vessel stroke. Methods: We identified a consecutive series of adult subjects with acute embolic large vessel strokes confirmed on CT angiography (CTA) from a 10-year institutional database. Patients without a brain MRI within six months of CTA were excluded. Collateral scores of the affected hemisphere were calculated on CTA using the modified Tan scale (0-4) by two independent trained reviewers and confirmed by a neuroradiologist. The WMH was quantified using the Fazekas scale (0-3) incorporating both deep and periventricular components. Areas of restricted diffusion on brain MRI in the peri-stroke period were eliminated from the assessment of Fazekas score. Spearman’s correlation tests were performed for the association between collateral scores and WMH. Results: A total of 234 patients (mean age 70±14 years, 47% [110/234] women) satisfied the inclusion criteria. The prevalence of periventricular and deep WMH was 83% and 87% respectively. Aging had a significant impact on periventricular WMH (r=0.48, p <0.001) and deep WMH (r=0.47, p <0.001). However, collateral scores exhibited no statistically significant relationship with the presence or severity of periventricular scores (r=0.08, p=0.17) or deep WMH scores (r=0.01, p=0.77). Conclusions: Our results indicate that the presence of SVD does not affect cerebral collateral flow. The vascular niche and risk factors of SVD appear to be distinct from that of intracranial vessels responsible for collateral reperfusion of ischemic territory in acute large vessel embolic stroke.

Systematic variation in North American tree species abundance distributions along macroecological climatic gradients

AbstractAimThe species abundance distribution (SAD) is a fundamental pattern in macroecology. Understanding how SADs vary spatially, and identifying the variables that drive any change, is important from a theoretical perspective because it enables greater understanding of the factors that underpin the relative abundance of species. However, precise knowledge on how the form of SADs varies across large (continental) scales is limited. Here, we use the shape parameter of the gambin distribution to assess how meta‐community‐scale SAD shape varies spatially as a function of various climatic variables and dataset characteristics.LocationEastern North America (ENA).Time periodPresent day.Major taxa studiedTrees.MethodsUsing an extensive continental‐scale dataset of 863,930 individual trees in plots across ENA sampled using a standardized method, we use a spatial regression framework to examine the effect of temperature and precipitation on the form of the SAD. We also assess whether the prevalence of multimodality in the SAD varies spatially across ENA as a function of temperature and precipitation, in addition to other sample characteristics.ResultsWe found that temperature, precipitation and species richness can explain two‐thirds of the variation in tree SAD form across ENA. Temperature had the largest effect on SAD shape, and it was found that increasing temperature resulted in more logseries‐like SAD shapes (i.e. SADs with a relatively higher proportion of rarer species). We also found spatial variation in SAD multimodality as a function of temperature and species richness.Main conclusionsOur results indicate that temperature is a key environmental driver governing the form of ENA tree meta‐community‐scale SADs. This finding has implications for our understanding of local‐scale variation in tree abundance and suggests that niche factors and environmental filtering are important in the structuring of ENA tree communities at larger scales.

Cancer Stem Cells: Concepts, Challenges, and Opportunities for Cancer Therapy.

Cancer stem cells (CSCs) are a subpopulation of cancer cells with self-renewal capacity, thatfuel tumor growth and contribute to the heterogeneous nature of tumors. First identified in hematological malignancies, CSC populations have to date been proposed in solid tumors in various organs. In vitro and in vivo assays, mouse genetic models, and more recently single-cell sequencing technologies and other '-omics' methodologies have not only facilitated the identification of novel CSC populations but also revealed and clarified novel properties of CSCs. Increasingly, both cell-autonomous and CSC niche factors are recognized as important contributors of CSC properties. The deepened understanding of CSC properties and characteristics would enable and facilitate the rational design of CSC-specific therapeutics that would, ideally, have high selectivity for cancer cells, eliminate tumor bulk, and prevent tumor recurrence. Addressing these issues would form some of the key challenges of the CSC research field in the coming years.

Prostaglandin I2 in the Endosteal Bone Marrow Niche As a Novel Regulator of Hematopoietic Stem Cells

Haematopoietic stem cells (HSCs) are regulated by their immediate microenvironment or niche. The most potent functional HSCs are enriched at the endosteum near the bone, which comprises ~10% of total bone marrow (BM). To identify novel niche factors that regulate HSCs, we performed a gene expression microarray seeking genes that were >2-fold overexpressed in the endosteal BM relative to the central BM. In this screen, we uncovered known essential HSC niche factors overexpressed in the endosteal BM such as Scf, Cxcl12, and Angpt1, which validated our approach.Among the genes overexpressed in the endosteal BM, prostaglandin I2 (PGI2) synthase (Ptgis) was one of the highest enriched genes in the endosteum (>10-fold by qRT-PCR, p<0.001). Within the endosteal BM, Ptgis was most abundantly expressed by osteoblasts, followed by mesenchymal stem/progenitor cells and endothelial cells. Ptgis is the sole enzyme responsible for biosynthesis of PGI2, originally discovered as a potent anti-thrombotic and vasodilating agent. PGI2 has no reported roles in HSC regulation in the BM and was chosen as candidate niche factor for further investigation. Due to the extreme lability of PGI2, we employed a clinically approved PGI2 analogue iloprost for all subsequent experiments.In initial experiments where we cultured fluorescence activated cell sorted (FACS) BM lineage- Kit+ Sca-1- (LKS+) haematopoietic stem and progenitor cells (HSPCs) for 7 days in serum free conditions, we found iloprost treatment potently reduced proliferation and differentiation compared to vehicle controls, assessed by flow cytometry phenotyping (p<0.0001). To determine whether iloprost treatment alters HSC function, we pulse treated whole BM cells with iloprost for 1 hour before competitive transplant and found iloprost pulse-treatment resulted in 14-fold increased multilineage reconstitution potential (p<0.05). This suggests that PGI2 enhances HSC function by dampening differentiation.We next sought to determine whether iloprost affects HSC function in vivo. In steady state, low dose 0.1mg/kg iloprost administration to mice for 15 days did not alter BM HSPC phenotypes by flow cytometry nor HSC reconstitution potential in competitive transplants compared to vehicle controls suggesting PGI2 does not alter HSC function in homeostasis. To further test the effect of PGI2 following stress, mice were sub-lethally (6.5 Gy) irradiated or administered pro-inflammatory granulocyte colony stimulating factor (G-CSF) for 3 days. We found low dose iloprost administration partially rescued BM HSC reconstitution potential 21 days following irradiation (p<0.05) or 3 days following G-CSF administration (p<0.01). These data suggest that PGI2 protects HSC from stress.To understand the extrinsic mechanisms through which PGI2 regulates HSC in the BM following stress, we performed a HSC homing assay using naïve donors transplanted into 2-day G-CSF administered recipients. Analysis of HSPC homing to the BM at 4 hours post-transplantation revealed ~4-fold greater proportion of LKS+ HSPC homing to the BM of iloprost co-administered recipients compared to vehicle controls (p<0.01). We assessed mRNA expression of essential HSC niche factors in the BM of 2-day G-CSF-administered mice and found that G-CSF administration decreased Scf and Cxcl12 expression, which were both partially rescued with iloprost co-administration (p<0.05). Together the data suggest that preservation of the BM niche function is one of the mechanisms through which PGI2 protects HSC during stress.In summary, we have identified PGI2 as a novel HSC niche factor abundant in the endosteal BM. PGI2 analogues like Iloprost are well-tolerated and used clinically to treat vascular diseases such as pulmonary arterial hypertension and Raynaud's phenomenon. Our research suggests that PGI2 analogues can be rapidly repurposed in the clinic to improve HSC transplant outcomes and protect against BM failure following acute stressors such as accidental irradiation or inflammation. DisclosuresLevesque:GlycoMimetics: Equity Ownership, Patents & Royalties.

MGP Interacts with BMP-4 and BMP-2 and Supports Normal and Malignant Hematopoietic Stem/Progenitor Cells

The general transcriptional coregulatory complex Mediator, subcomplex of the RNA polymerase II holoenzyme, is the endpoint convergence of a variety of intracellular signals, and initiates transcription through recruitment of general initiation factors and formation of a functional preinitiation complex. Among the Mediator subunits, MED1 is crucial for hematopoiesis: it is known to be employed in transcription of genes involved in hematopoietic niche function, as well as in differentiation of hematopoietic precursor cells (e.g., RARα- and VDR-mediated myelomonopoiesis, GATA1-mediated erythromegakaryopoiesis, and iNKT cell development). Among the attenuated genes in Med1-/- mesenchymal stromal cells was matrix Gla protein (MGP), modulator of the BMP-SMAD signals. As MGP is abundantly expressed in bone tissues and reportedly interacts with niche factors BMP-4 and BMP-2, we hypothesized that MGP might modulate niche function in hematopoiesis through the interaction with these BMPs. We tested this possibility by an in vitro niche model composed of a coculture of bone marrow (BM) mesenchymal stromal cells and BM hematopoietic cells. When mouse BM hematopoietic cells were cocultured with OP-9 or MS-5 mouse BM stromal cells in the presence of the blocking antibody against MGP, the growth and DNA synthesis of BM hematopoietic cells were attenuated and the number of long-term culture-initiating cells (LTC-ICs), which corresponded to the supported hematopoietic stem/progenitor cells (HSPCs), decreased. We next asked if MGP also functioned as a niche factor for malignant HSPCs, To this end, we utilized MB-1 cells, blast crisis CML-derived myeloblastic leukemia cells. MB-1 cells are dependent on cocultured BM stromal cells and possess characteristics of leukemia stem cells (LSCs). When MB-1 cells were cocultured with OP-9 or MS-5 BM stromal cells in the presence of the anti-MGP antibody, the number of MB-1 cells, as well as the number of cobblestone formation, i.e., LSC nature, of MB-1 cells, was profoundly attenuated. Therefore, MGP appears to have a trophic effect on hematopoietic cells and to support normal and malignant HSPCs in our niche model. MGP was, both at mRNA and protein levels, expressed abundantly in BM stromal cells but scarcely in normal hematopoietic cells. However, MB-1 leukemia cells (presumably ectopically) expressed a meaningfully high level of MGP. During the coculture of normal BM hematopoietic cells with OP-9 or MS-5 BM stromal cells, MGP was induced prominently and transiently after one day. MGP, expressed in MS-5 or OP-9 BM stromal cells, was likewise transiently induced after 1 day of culture with BM hematopoietic cells that were physically dissociated from these BM stromal cells by the transwell apparatus, indicating that secreted humoral factor(s) induced the MGP expression in the BM stromal cells. As for a malignant situation, the expressions of MGP in both MB-1 leukemia cells and OP-9 or MS-5 BM stromal cells were fluctuated in an oscillated days period. Since the support of HSPCs required their association with BM stromal cells, the induced MGP expression was apparently insufficient for HSPCs support, and the action of induced MGP may be indirect. Therefore, we next asked if BMP-4 and BMP-2, which reportedly associate with MGP, were also induced by the coculture and were employed in HSPCs support jointly with MGP. Indeed, during the coculture, BMP-4 was rapidly and transiently induced within a few days, simultaneously with MGP, followed by a subsequent and sustained induction of BMP-2 that lasted for over a week. However, neither BMP-4 nor BMP-2 was produced by BM stromal cells when cultured with dissociated BM hematopoietic cells using the transwell apparatus. Therefore, the induction of BMP2/BMP4 was dependent on physical association of hematopoietic cells and stromal cells. GST-pulldown assays and mammalian two-hybrid assays confirmed that MGP specifically interacted with both BMP-4 and BMP-2. To elucidate the mechanism of action of the blocking antibody against MGP, we tested if the antibody inhibited the interaction between MGP and BMP-4. Indeed, serial GST pulldown assays disclosed that the addition of the antibody specifically abolished the interaction between GST-MGP and BMP-4. These results indicate that MGP might act as a niche factor for normal and malignant HSPCs, at least in part, through interacting with, and modulating the action of, BMP-4 and BMP-2. DisclosuresNo relevant conflicts of interest to declare.