Stromal Line Research Articles

Extracellular matrices of stromal cell subtypes regulate phenotype and contribute to the stromal microenvironment in vivo

BackgroundBone marrow stromal cells (BMSCs) are highly heterogeneous, which may reflect their diverse biological functions, including tissue maintenance, haematopoietic support and immune control. The current understanding of the mechanisms that drive the onset and resolution of heterogeneity, and how BMSCs influence other cells in their environment is limited. Here, we determined how the secretome and importantly the extracellular matrix of BMSCs can influence cellular phenotype.MethodsWe used two immortalised clonal BMSC lines isolated from the same heterogeneous culture as model stromal subtypes with distinct phenotypic traits; a multipotent stem-cell-like stromal line (Y201) and a nullipotent non-stem cell stromal line (Y202), isolated from the same donor BMSC pool. Label-free quantitative phase imaging was used to track cell morphology and migration of the BMSC lines over 96 h in colony-forming assays. We quantified the secreted factors of each cell line by mass spectrometry and confirmed presence of proteins in human bone marrow by immunofluorescence.ResultsTransfer of secreted signals from a stem cell to a non-stem cell resulted in a change in morphology and enhanced migration to more closely match stem cell-like features. Mass spectrometry analysis revealed a significant enrichment of extracellular matrix (ECM) proteins in the Y201 stem cell secretome compared to Y202 stromal cells. We confirmed that Y201 produced a more robust ECM in culture compared to Y202. Growth of Y202 on ECM produced by Y201 or Y202 restored migration and fibroblastic morphology, suggesting that it is the deficiency of ECM production that contributes to its phenotype. The proteins periostin and aggrecan, were detected at 71- and 104-fold higher levels in the Y201 versus Y202 secretome and were subsequently identified by immunofluorescence at rare sites on the endosteal surfaces of mouse and human bone, underlying CD271-positive stromal cells. These proteins may represent key non-cellular components of the microenvironment for bona-fide stem cells important for cell maintenance and phenotype in vivo.ConclusionsWe identified plasticity in BMSC morphology and migratory characteristics that can be modified through secreted proteins, particularly from multipotent stem cells. Overall, we demonstrate the importance of specific ECM proteins in co-ordination of cellular phenotype and highlight how non-cellular components of the BMSC microenvironment may provide insights into cell population heterogeneity and the role of BMSCs in health and disease.

MarrowCellDLD: a microfluidic method for label-free retrieval of fragile bone marrow-derived cells

Functional bone marrow studies have focused primarily on hematopoietic progenitors, leaving limited knowledge about other fragile populations, such as bone marrow adipocytes (BMAds) and megakaryocytes. The isolation of these cells is challenging due to rupture susceptibility and large size. We introduce here a label-free cytometry microsystem, MarrowCellDLD, based on deterministic lateral displacement. MarrowCellDLD enables the isolation of large, fragile BM-derived cells based on intrinsic size properties while preserving their viability and functionality. Bone marrow adipocytes, obtained from mouse and human stromal line differentiation, as well as megakaryocytes, from primary human CD34+ hematopoietic stem and progenitor cells, were used for validation. Precise micrometer-range separation cutoffs were adapted for each cell type. Cells were sorted directly in culture media, without pre-labeling steps, and with real-time imaging for quality control. At least 106 cells were retrieved intact per sorting round. Our method outperformed two FACS instruments in purity and yield, particularly for large cell size fractions. MarrowCellDLD represents a non-destructive sorting tool for large, fragile BM-derived cells, facilitating the separation of pure populations of BMAds and megakaryocytes to further investigate their physiological and pathological roles.

The Role of Fetal Membranes during Gestation, at Term, and Preterm Labor.

During pregnancy, the fetal membranes (i.e., amniochorionic membranes) surround the intrauterine cavity and provide mechanical, immune, and endocrine support to protect the fetus. Though they are a vital component of the intrauterine cavity, the fetal membranes are largely overlooked as an extension of the placenta, leading to a poor understanding of their role during gestation, parturition, or preterm birth. The fetal membranes are comprised of fetal cellular and stromal layers and line up with maternal decidua forming the feto-maternal interface during pregnancy. This interface plays a large role during pregnancy and the induction of term or preterm parturition (e.g., labor). Here we summarize the function of the fetal membranes focusing on their role during gestation at term, and during preterm births.

Tumor-infiltrating lymphocyte signature in epithelial and stromal compartments of an esophageal squamous cell carcinoma acidic microenvironment mediated by MCT4

Tumor-infiltrating lymphocytes (TILs), including but not limited to neutrophils, M2 macrophages, cytotoxic CD8 T cells and dendritic cells, will play a role in the acidic tumor microenvironment mediated by monocarboxylate transporter 4 (MCT4) in esophageal squamous cell carcinoma (ESCC). However, the roles they play and their significance in ESCC remain less clear. To understand the clinicopathological and prognostic significance of neutrophils, M2 macrophages, CD8 T cells and dendritic cells in the tumor acidic microenvironment mediated by MCT4, we investigated the distribution of these TILs in the epithelial and stromal compartments of ESCC by means of multiplexed immunohistochemistry on a tissue microarray containing 87 paired dots of ESCC and its adjacent normal tissue (ANT) and an additional 6 cases of unpaired ESCC dots. The density of cells stained with MCT4 in the epithelium was significantly associated with overall survival. Dendritic cells stained with S100 in epithelial compartmentalization were found to markedly correlate with clinical stage and tumor invasion depth. No other significant association could be identified in terms of prognostic and clinicopathological significance. The potential correlation between the number of cells stained with MCT4 versus the number of TILs was also explored, showing that only in epithelial cells were there significant and positive correlations identified between the number of cells stained with MCT4 versus the number of neutrophils stained with CD15, M2 macrophages stained with CD163 and CD8 T cells stained by CD8a. However, no significant correlation was found along the stromal line. Together, the data we described here, although somewhat discouraging, showed that in epithelial cells from which ESCC originated, acidicity mediated by MCT4 may be responsible for lactate release and may have an effect on the infiltration of TILs we assessed.

Extramedullary hematopoiesis: mesenchymal stromal cells from spleen provide an in vitro niche for myelopoiesis

Murine spleen has been shown to harbour stromal cells that support hematopoiesis with production of myeloid antigen–presenting cells. Similar stromal lines have now been isolated from long-term cultures (LTC) of human spleen. When human progenitor populations from spleen, bone marrow and cord blood were employed as a source of progenitors for co-culture above splenic stromal lines, myelopoiesis was supported. Human splenocytes gave production of predominantly myeloid dendritic-like cells, with minor subsets resembling conventional dendritic cells (cDC) cells, and myeloid or monocyte-derived DC. Human bone marrow progenitors gave rise to myelopoiesis from hematopoietic progenitors, while human cord blood supported limited myelopoiesis from existing myeloid precursors. Transcriptome analysis compared two stromal lines differing in myelopoietic support capacity. Gene profiling revealed both stromal lines to reflect perivascular reticular cells with osteogenic characteristics. However, the 5C6 stroma which failed to support hematopoiesis uniquely expressed several inhibitors of the WNT pathway. Combined data now show that splenic stroma of both human and murine origin provides a mesenchymal stromal cell microenvironment which is WNT pathway–dependent, and which supports in vitro myelopoiesis with production of specific subsets of myeloid and dendritic-like cells.

Role of SVEP1 in Stroma-Dependent Hematopoiesis In vitro.

Study of the microenvironment that supports hematopoietic stem cell (HSC) development in vivo is very difficult involving small numbers of interacting cells which are usually not well defined. While much is known about HSC niches located within the bone marrow in terms of contributing cell types and signalling molecules, very little is known about equivalent niches within spleen. Extramedullary hematopoiesis in spleen contributes myeloid cells important in the mobilisation of an immune response. As a result, it is important to develop in vitro models to identify the cells which constitute HSC niches in spleen and to identify the regulatory molecules supporting myeloid cell development. Studies described here document a model system to study the maintenance and differentiation of HSC by splenic stromal cells in vitro. The splenic stromal lines 5G3 and 3B5 differ in hematopoietic support capacity. SVEP1 and IGF2 are molecules of interest specifically expressed by 5G3 stroma. Gene knockdown technology using shRNA plasmids has been used to reduce gene expression in 5G3 and to determine specific effects on myeloid cell development following co-culture with overlaid hematopoietic progenitors in vitro. Knockdown of Svep1 gave specific inhibition of a dendritic cell (DC) population described previously in spleen (L-DC). Knockdown of Igf2 resulted in loss of production of a minor subset of conventional (c) DC. SVEP1 is now considered a marker of mesenchymal stromal cells with osteogenic differentiative capacity reflective of perivascular stromal cells. The power of this in vitro model is evidenced by the fact that it has been used to define SVEP1 as a specific adhesion molecule that regulates the hematopoietic process dependent on stromal niche interaction. The identification of stromal cells and molecules that contribute to the hematopoietic process in spleen, brings us closer to the realm of therapeutically regulating hematopoiesis in vivo, and to inhibiting niches which support cancer stem cells.

Understanding the natural selection of human embryos: Blastocyst quality modulates the inflammatory response during the peri-implantation period.

Decidualized cells display an active role during embryo implantation sensing blastocyst quality, allowing the implantation of normal developed blastocysts and preventing the invasion of impaired developed ones. Here, we characterized the immune microenvironment generated by decidualized cells in response to soluble factors secreted by blastocysts that shape the receptive milieu. We used an in vitro model of decidualization based on the Human Endometrial Stromal Cells line (HESC) differentiated with medroxiprogesterone and dibutyryl-cAMP, then treated with human blastocysts-conditioned media (BCM) classified according to their quality. Decidualized cells treated with BCM from impaired developed blastocysts increased IL-1β production. Next, we evaluated the ability of decidualized cells to modulate other mediators associated with menstruation as chemokines. Decidualized cells responded to stimulation with BCM from impaired developed blastocysts increasing CXCL12 expression and CXCL8 secretion. The modulation of these markers was associated with the recruitment and activation of neutrophils, while regulatory T cells recruitment was restrained. These changes were not observed in the presence of BCM from normal developed blastocysts. Soluble factors released by impaired developed blastocysts induce an exacerbated inflammatory response associated with neutrophils recruitment and activation, providing new clues to understand the molecular basis of the embryo-endometrial dialogue.

The Relevance of SOCS1 Methylation and Epigenetic Therapy in Diverse Cell Populations of Hepatocellular Carcinoma.

The suppressor of cytokine signaling 1 (SOCS1) is a tumor suppressor gene found to be hypermethylated in cancers. It is involved in the oncogenic transformation of cirrhotic liver tissues. Here, we investigated the clinical relevance of SOCS1 methylation and modulation upon epigenetic therapy in diverse cellular populations of hepatocellular carcinoma (HCC). HCC clinical specimens were evaluated for SOCS1 methylation and mRNA expression. The effect of 5-Azacytidine (5-AZA), a demethylation agent, was assessed in different subtypes of HCC cells. We demonstrated that the presence of SOCS1 methylation was significantly higher in HCC compared to peri-HCC and non-tumoral tissues (52% vs. 13% vs. 14%, respectively, p < 0.001). In vitro treatment with a non-toxic concentration of 5-AZA significantly reduced DNMT1 protein expression for stromal subtype lines (83%, 73%, and 79%, for HLE, HLF, and JHH6, respectively, p < 0.01) compared to cancer stem cell (CSC) lines (17% and 10%, for HepG2 and Huh7, respectively), with the strongest reduction in non-tumoral IHH cells (93%, p < 0.001). 5-AZA modulated the SOCS1 expression in different extents among the cells. It was restored in CSC HCC HepG2 and Huh7 more efficiently than sorafenib. This study indicated the relevance of SOCS1 methylation in HCC and how cellular heterogeneity influences the response to epigenetic therapy.

CALCIFICATION OF ARTERIES IN PATIENTS WITH ATHEROSCLEROSIS: CAUSES AND MECHANISMS OF DEVELOPMENT

The paper shows analogies between the mechanisms of calcification of arteries, various tissues and bone mineralization. In calcification the same mechanisms are involved, as at ossification of an organic matrix of a bone, including participation of stem cells. In the arteries and capillaries of various organs found polypotent precursors – mesenchymal stem cells, presumably responsible for the pathological mineralization of the arterial wall. Circulating in the blood and present in the intima of the human atheromatous aorta colony-forming stem cells of the stromal line of differentiation. Adventitia cells or pericytes are considered as a pluripotent mesenchymal reserve for replenishment of some cellular forms of connective tissue. Bone marrow stem cells of the stromal line of differentiation with the presence of pluripotent stromal cells in the blood and granulomas (plaques) and their transformation into bone tissue cells are involved in atherogenesis. Smooth muscle cells (SMCs) and myofibroblasts of the arterial wall adventitia have an osteoblast-like phenotype. Collagen and elastic fibers are involved in the calcification of blood vessels and soft tissues. Calcification of granulomas (plaques) accompanies their inflammatory morphogenesis, accompanied by the development of scar tissue. It often accompanies and completes the inflammation in them. This process is observed with the gradual replacement of the parenchyma of some organs with connective tissue. The mineral phase in bones and soft tissues, represented by calcium (Ca) and phosphate (P), contacts with nucleators - specific areas of collagen fibers. Non-collagen proteins are also involved in arterial calcification. With the progression of atherosclerosis and calcification of granulomas/plaques by cells present in the arterial wall, osteonectin is expressed. Osteopontin content is associated with Ca deposits in them; in particular, it correlates with the Ca level in the coronary arteries. In the processes of ossification and calcification, phosphatases play a certain role. In areas of calcification of granulomas/plaques, osteoprotegerin is found, which inhibits the activity of alkaline phosphatase in the aorta and prevents calcification of the media. Arterial wall calcification and inflammation are inhibited by fetuin-A and matrix γ-carboxyglutaric protein (MGP). The rate of bone formation and tissue calcification depends on the concentration of Ca and P in plasma and extracellular fluid. With their high concentration in the extracellular fluid, the mineral phase appears where it normally does not exist. Lipids are among the initiators of granuloma / plaque calcification in the arteries. The role of inflammation and necrosis in the calcification of the arteries is confirmed by experiments on animals with repeated intravenous injections of adrenaline, which causes focal necrosis of the middle membrane, which then undergoes calcification. In arterial walls, heart muscle, bone and cartilage tissue, osteonectin, osteocalcin, osteopontin, bone sialoprotein are often formed. In general, the mechanisms of arterial and soft tissue calcification are poorly understood. There are no effective remedies for calcification.

Micellar Hyaluronidase and Spiperone as a Potential Treatment for Pulmonary Fibrosis.

Concentration of hyaluronic acid (HA) in the lungs increases in idiopathic pulmonary fibrosis (IPF). HA is involved in the organization of fibrin, fibronectin, and collagen. HA has been proposed to be a biomarker of fibrosis and a potential target for antifibrotic therapy. Hyaluronidase (HD) breaks down HA into fragments, but is a subject of rapid hydrolysis. A conjugate of poloxamer hyaluronidase (pHD) was prepared using protein immobilization with ionizing radiation. In a model of bleomycin-induced pulmonary fibrosis, pHD decreased the level of tissue IL-1β and TGF-β, prevented the infiltration of the lung parenchyma by CD16+ cells, and reduced perivascular and peribronchial inflammation. Simultaneously, a decrease in the concentrations of HA, hydroxyproline, collagen 1, total soluble collagen, and the area of connective tissue in the lungs was observed. The effects of pHD were significantly stronger compared to native HD which can be attributed to the higher stability of pHD. Additional spiperone administration increased the anti-inflammatory and antifibrotic effects of pHD and accelerated the regeneration of the damaged lung. The potentiating effects of spiperone can be explained by the disruption of the dopamine-induced mobilization and migration of fibroblast progenitor cells into the lungs and differentiation of lung mesenchymal stem cells (MSC) into cells of stromal lines. Thus, a combination of pHD and spiperone may represent a promising approach for the treatment of IPF and lung regeneration.

Abstract PO-025: Radiation-induced and FACS-sorted senescent tdTOMp16+ cells upregulate profibrotic genes in C57BL/6 stromal target cells

Abstract Acute Radiation Syndrome and the multiorgan failure from delayed effects of acute radiation exposure present challenging consequences of radiation terrorism or a radiological accident. Recent research suggests that radiation-induced cellular senescence plays an important role in radiation-induced pulmonary fibrosis (RIPF), and that clearance of senescent cells (SCs) could be an effective therapeutic strategy. However, the identification and targeted removal of only senescent cells using drugs have been difficult. Here we have established a bone-marrow stromal cell line from a tdTOMp16+ mouse. We show that 5 Gy irradiation induces ~9% cellular senescence in tdTOMp16+ stromal line after 10 days and can be isolated as a pure population of red tdTOMp16+ cells by FACS sorting. To confirm that these irradiated and then sorted red cells are senescent, we cultured them as single cells in 96-well plates and followed for four weeks. None of the 960 irradiated red cells we plated divided after 2 weeks, 137 of these cells remained intact and assumed large and flat cellular morphology. In contrast, 50 of 800 cells that were irradiated but didn’t turn red, divided, as did 104 of 560 nonirradiated control cells (0 Gy) divided. There was significant upregulation of senescent cell markers including SA-ß-gal, p16, and p21 in the irradiated and sorted red cells when compared to irradiated nonred cells. Sorted irradiated red, non-irradiated, or irradiated non-red cells were placed on the top well of transwells. There was a significant induction of fibrotic genes Ctgf, Tgf- ß, collagen 1a, and collagen 3 in C57/B6 stromal target cells in the bottom layer by irradiated red cells, but not the other cell populations irradiated non-red. Thus, radiation-induced biomarkers of fibrosis are induced by senescent cells. Citation Format: Amitava Mukherjee, Michael Epperly, Donna Shields, Wen Hou, Renee Fisher, Diala Hamade, Joel S. Greenberger. Radiation-induced and FACS-sorted senescent tdTOMp16+ cells upregulate profibrotic genes in C57BL/6 stromal target cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-025.

Molecular mechanism study of Shouhui Tongbian Capsules on promoting energy metabolism of gastrointestinal stromal cells

Mechanism study was performed to explore how Shouhui Tongbian Capsules promotes energy metabolism of gastrointestinal stromal cells. In this study, gastrointestinal stromal cells line GIST-882 was used as the model to explore energy metabolism regulation effects of Shouhui Tongbian Capsules extract(10, 20, 50 and 100 μg·mL~(-1)) by measuring the cell proliferation, ATP level, mitochondrial membrane potential, and mitochondrial isocitrate dehydrogenase activity. Meanwhile, Western blot was used to detect the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Our results showed that Shouhui Tongbian Capsules promoted cell proliferation and increased ATP level of gastrointestinal stromal cells. In addition, Shouhui Tongbian Capsules obviously improved mitochondrial structural integrity, and increased mitochondrial membrane potential in GIST-882 cells. Mechanism study revealed that Shouhui Tongbian Capsules increased mitochondrial isocitrate dehydrogenase activity and up-regulated the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Collectively, our study indicated that Shouhui Tongbian Capsules promoted the energy metabolism for gastrointestinal stromal cells proliferation by activating mitochondrial isocitrate dehydrogenase to induce ATP production, as well as activating SCF/c-Kit and CDK2/cyclin A signaling pathways.

Digital Image Analysis Applied to Tumor Cell Proliferation, Aggressiveness, and Migration-Related Protein Synthesis in Neuroblastoma 3D Models.

Patient-derived cancer 3D models are a promising tool that will revolutionize personalized cancer therapy but that require previous knowledge of optimal cell growth conditions and the most advantageous parameters to evaluate biomimetic relevance and monitor therapy efficacy. This study aims to establish general guidelines on 3D model characterization phenomena, focusing on neuroblastoma. We generated gelatin-based scaffolds with different stiffness and performed SK-N-BE(2) and SH-SY5Y aggressive neuroblastoma cell cultures, also performing co-cultures with mouse stromal Schwann cell line (SW10). Model characterization by digital image analysis at different time points revealed that cell proliferation, vitronectin production, and migration-related gene expression depend on growing conditions and are specific to the tumor cell line. Morphometric data show that 3D in vitro models can help generate optimal patient-derived cancer models, by creating, identifying, and choosing patterns of clinically relevant artificial microenvironments to predict patient tumor cell behavior and therapeutic responses.

Generation of T cells from Human and Nonhuman Primate Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes. In addition, we describe a protocol for robust T cell generation from hPSCs conditionally expressing ETS1. The presented protocols provide a platform for T cell production for disease modeling and evaluating their use for immunotherapy in large animal models.

Elevated insulin levels compromise endometrial decidualization in mice with decrease in uterine apoptosis in early-stage pregnancy.

Women with hyperinsulinism and insulin resistance have reduced fertility, but the underlying mechanism is still poorly understood. Aberrant endometrial decidualization in early pregnancy was linked to pregnancy complications. In this study, we aimed to test whether elevated insulin levels compromise decidualization in early-stage pregnancy. C57BL/6Jmice in high insulin-exposed group were given a subcutaneous injection of recombinant insulin at a concentration of 0.05IU daily. During decidualization in early pregnancy, serum levels of insulin, E2, P4, LH, FSH and blood glucose were significantly altered in mice treated with high insulin levels. The number of embryo implantation sites and endometrial decidual markers BMP2, ER, PR was significantly decreased by high insulin levels in vivo. Artificial decidual induction in primary mouse endometrial stromal cells and immortal human endometrial stromal cells line were all compromised after treated with 100nmol/L insulin levels. All these results on flow cytometry, transmission electron microscopy and western blotting of Bax, Bcl2, cleaved Caspase3, cleaved PARP proteins level showed that decidual cells apoptosis was significantly decreased. Mitochondrial transmembrane potential also significantly increased by the influence of high insulin levels. PI3K and p-Akt were much higher after insulin exposure and the compromised decidualization by high insulin treatment was rescued by PI3K/Akt inhibitor LY294002 both in vitro and in vivo. In conclusion, we demonstrated that elevated insulin levels could compromise mice decidualization in early-stage pregnancy and PI3K/p-Akt-regulated apoptosis was essential for this role. It provides a clue for future investigation on compromised reproduction in women with hyperinsulinemia.

Transplanted spleen stromal cells with osteogenic potential support ectopic myelopoiesis.

Spleen stromal lines which support in vitro hematopoiesis are investigated for their lineage origin and hematopoietic support function in vivo. Marker expression and gene profiling identify a lineage relationship with mesenchymal stem cells and perivascular reticular cells described recently in bone marrow. Stromal lines commonly express Cxcl12, Pdgfra and Pdgfr typical of bone marrow derived perivascular reticular cells but reflect a unique cell type in terms of other gene and marker expression. Their classification as osteoprogenitors is confirmed through ability to undergo osteogenic, but not adipogenic or chondrogenic differentiation. Some stromal lines were shown to form ectopic niches for HSCs following engraftment under the kidney capsule of NOD/SCID mice. The presence of myeloid cells and a higher representation of a specific dendritic-like cell type over other myeloid cells within grafts was consistent with previous in vitro evidence of hematopoietic support capacity. These studies reinforce the role of perivascular/perisinusoidal reticular cells in hematopoiesis and implicate such cells as niches for hematopoiesis in spleen.

Histone demethylase KDM4A regulates adipogenic and osteogenic differentiation via epigenetic regulation of C/EBPα and canonical Wnt signaling.

Epigenetic modifications play a central role in cell differentiation and development. In the current study, we have recognized lysine demethylase 4A (KDM4A) as a novel epigenetic regulator of osteoblast and adipocyte differentiation. Kdm4a expression was upregulated during osteogenesis and adipogenesis of primary marrow stromal cells and established stromal ST2 line. Overexpression of wild-type Kdm4a promoted adipogenic differentiation and blocked osteogenic differentiation of the progenitor cells. This effect was largely alleviated when the catalytically dead mutation was made. Conversely, depletion or inactivation of Kdm4a in undifferentiated progenitor cells inhibited the formation of adipocytes and promoted the differentiation of osteoblasts. Mechanism explorations showed that overexpression of Kdm4a upregulated the expression of secreted frizzled-related protein 4 (Sfrp4) and CCAAT/enhancer-binding protein α (C/ebpα). Chromatin immunoprecipitation assay demonstrated that KDM4A directly bound the promoters of Sfrp4 and C/ebpα, removed the histone methylation mark H3K9me3, and reduced DNA methylation levels of CpG in promoter regions of C/ebpα and Sfrp4. Furthermore, overexpression of Kdm4a inactivated canonical Wnt signaling. Moreover, activation of canonical Wnt signaling through silencing of Sfrp4 in ST2 attenuated the inhibition of osteogenic differentiation and the enhancement of adipogenic differentiation by KDM4A. These data have identified KDM4A as a novel regulator of osteoblast and adipocyte differentiation and suggest KDM4A inhibition as a potential therapeutic target for treating metabolic disorders such as osteoporosis.

PB1878 β‐CATENIN IS AN EFFECTOR OF THE CROSS‐TALK BETWEEN CLL B‐CELLS AND THE MICROENVIRONMENT

Background:CLL is a lymphoproliferative disorder characterized by the accumulation of CD5+ B cells in the blood and lymphoid organs (bone marrow, lymph nodes and spleen). This accumulation is mainly due to an increased survival and proliferation of tumor B cells within the lymphoid organs through interaction with the microenvironment as isolated B‐CLL cells rapidly undergo apoptosis in vitro. In the quest for the molecular basis of the communication between B‐CLL cells and the microenvironment, we focused our attention on β‐catenin that participates to the stabilization of cell‐cell contacts and signal transduction. Cytosolic β‐catenin level is maintained low following its phosphorylation that triggers the degradation by the proteasome. Several molecules, such as Wnt ligands inhibit β‐catenin phosphorylation and induce its stabilization.Aims:The aim of our study is to evalute the role of β‐catenin in the cross talk between CLL B cells and the microenvironnement.Methods:Co‐cultures of primary B‐CLL cells and human bone marrow stromal cells line (HS‐5).Results:Using a model of co‐culture of primary B‐CLL cells and human marrow stromal cells we observed that the co‐culture rapidly stabilizes β‐catenin in B‐CLL cells. Since in B‐CLL cells the pathway linked to antigen stimulation is essential for survival, we explored whether β‐catenin stabilization involve BCR pathway effectors. Interestingly, we observed that the inhibition of the kinase BTK hampers β‐catenin stabilization in presence of stromal cells. Concomitant with the participation of BTK, a phosphatase activity is involved in β‐catenin stabilization. We are currently addressing whether and how the stabilization of β‐catenin regulates cell survival and the interactions between B‐CLL and stromal cells.Summary/Conclusion:Our results will convey new elements to describe the cross‐talk between tumor cells and the microenvironment to define new targets that interfere with this dialogue as alternative therapeutic strategies.

Paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS): clinical findings in twelve patients including recurrence after keratoplasty.

To describe the ocular findings of 12 subjects with paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS). Ocular examination included corneal spectral domain optical coherence tomography. In three individuals with an initial diagnosis of a lattice or Thiel-Behnke corneal dystrophy, the TGFBI gene was screened by conventional Sanger sequencing. We confirmed a diagnosis of MGUS by systemic examination and serum protein electrophoresis in 12 individuals (9 males and 3 females), with a mean age at presentation of 52.2years (range 24-63years) and mean follow-up 6.4years (range 0-17years). The best-corrected visual acuity (BCVA) at presentation ranged from 1.25 to 0.32. In all individuals, the corneal opacities were bilateral. The appearances were diverse and included superficial reticular opacities and nummular lesions, diffuse posterior stromal opacity, stromal lattice lines, superficial and stromal crystalline deposits, superficial haze and a superficial ring of hypertrophic tissue. In one individual, with opacities first recorded at 24years of age, we documented the progression of corneal disease over the subsequent 17years. In another individual, despite systemic treatment for MGUS, recurrence of deposits was noted following bilateral penetrating keratoplasties. The three individuals initially diagnosed with inherited corneal dystrophy were negative for TGFBI mutations by direct sequencing. A diagnosis of MGUS should be considered in patients with bilateral corneal opacities. The appearance can mimic corneal dystrophies or cystinosis. In our experience, systemic treatment of MGUS did not prevent recurrence of paraproteinemic keratopathy following keratoplasty.

Histone demethylase KDM7A reciprocally regulates adipogenic and osteogenic differentiation via regulation of C/EBPα and canonical Wnt signalling.

Recent emerging evidences revealed that epigenetic methylation of histone and DNA regulates the lineage commitment of mesenchymal progenitor cells. This study was undertaken to delineate the actions of histone lysine demethylase 7A (KDM7A) on osteogenic and adipogenic differentiation. Kdm7a expression was up‐regulated in primary marrow stromal cells and established stromal ST2 line after adipogenic and osteogenic treatment. Silencing of endogenous Kdm7a in the cells blocked adipogenic differentiation whereas promoted osteogenic differentiation. Conversely, overexpression of wild‐type Kdm7a in the progenitor cells enhanced adipogenic differentiation whereas inhibited osteogenic differentiation. However, the effect of KDM7A on cell differentiation was largely attenuated when the point mutation was made that abolishes enzymatic activity of KDM7A. Mechanism investigations revealed that silencing of Kdm7a down‐regulated the expression of the CCAAT/enhancer binding protein α (C/EBPα) and secreted frizzled‐related protein 1 (Sfrp1). Chromatin immunoprecipitation (ChIP) assay revealed that KDM7A directly binds to the promoters of C/EBPα and Sfrp1 and removes the histone methylation marks H3K9me2 and H3K27me2. Furthermore, silencing of Kdm7a activated canonical Wnt signalling. Thereafter, activation of canonical Wnt signalling through silencing of Sfrp1 in ST2 attenuated the stimulation of adipogenic differentiation and inhibition of osteogenic differentiation by KDM7A. Our study suggests that KDM7A balances adipogenic and osteogenic differentiation from progenitor cells through epigenetic control of C/EBPα and canonical Wnt signalling and implicates that control of KDM7A action has an epigenetic perspective of curtailing metabolic disorders like osteoporosis.