Stromal Cell Line Research Articles

Induction of Human T Cell Development In Vitro with OP9-DL4-7FS Cells Expressing Human Cytokines.

For nearly a generation now, OP9-DL1 and OP9-DL4 cells have provided an efficient and reliable cell system to generate T cells from mouse and human hematopoietic stem cells (HSCs) and pluripotent stem cells. OP9-DL1 and OP9-DL4 were originally derived from the OP9 mouse bone marrow stromal cell line, which was transduced to ectopically express Delta-like 1 or 4 proteins, respectively. OP9-DL cells mimic the thymic microenvironment in that when cocultured with mouse or human (h) HSCs, they interact with and activate Notch receptors present on HSCs, required for T cell differentiation. The HSC/OP9-DL cocultures require additional cytokines that are necessary for survival and proliferation of hematopoietic cells. For hHSCs, these factors are interleukin-7 (IL-7), stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (FLT3L) that are normally exogenously added to the cocultures. In this chapter, we describe methods for establishing a novel and improved version of OP9-DL4 cells, called OP9-DL4-7FS cells that circumvent the addition of these costly cytokines, by transducing OP9-DL4 cell line to express human IL-7, FLT3L, and SCF (7FS). Herein, we describe the protocol for the generation of OP9-DL4-7FS cells and the conditions for OP9-DL4-7FS/hHSC coculture to support T cell lineage initiation and expansion while comparing it to the now "classic" OP9-DL4 coculture. The use of OP9-DL4-7FS cell system will provide an improved and cost-effective method to the commonly used OP9-DL/HSC coculture for studying both mouse and human T cell development.

Biological, Functional and Genetic Characterization of Bone Marrow Mesenchymal Stromal Cells from Adult Ph+ Acute Lymphoblastic Leukemia

Objectives: To clarify the biological characteristics of bone marrow mesenchymal stromal cells (MSCs) in Ph+ acute lymphoblastic leukemia (Ph+ ALL) patients in different disease states, and compare protective effects of MSCs in different disease states on leukemia cells and explore the possible mechanisms. Methods: Bone marrow specimens from Ph+ ALL patients in various disease status (newly diagnosis, complete remission, relapse/ refractory) and healthy donors were collected. Bone marrow mononuclear cells (BMNCs) were isolated and cultured for primary MSCs. (1) CCK-8 method was used to compare proliferation ability of MSCs in vitro. (2) IC50 concentrations of dasatinib (Dasa), imatinib (Ima) and dexamethasone (Dex) in SUP-B15 were determined. SUP-B15 were co-cultured with HS-5 (a human stromal cell line) and MSCs respectively, then the apoptosis ratio of SUP-B15 cells after drug treatment was detected. (3) Transcriptome sequence (RNA-seq) of MSCs was performed, and differentially-expressed genes (DEGs) and signal pathway enrichment analysis among MSCs and BMNCs groups were performed. (5) qRT-PCR was used to detect the expression levels of candidate genes in MSCs. Results: (1) The proliferation ability of DR-MSCs in vitro was stronger than ALL-MSCs (P<0.01, Figure 1A). Proliferation ability of MSCs from refractory and relapsed group (RR-MSCs) were stronger than that from complete remission group (CR-MSCs) and newly diagnosed group (ND-MSCs)(P<0.05, P<0.05, Figure 1A). There was no significant difference between newly diagnosed-complete remission (ND-CR) paired MSCs (P>0.05). (2) The ratios of apoptotic SUP-B15 cells were significant decreased when co-cultured with HS-5 with and without the presence of Dasa, Ima, and Dex (P＜0.05, P＜0.0001, P＜0.001, P＜0.01, Figure 1B). (3) Ratios of apoptotic SUP-B15 cells were significantly reduced when co-cultured with different MSCs under the treatment of blank control, Dasa, Ima, and Dex. The abilities to prevent SUP-B15 cell apoptosis of DR-MSCs and ND-MSCs was stronger than that of CR-MSCs (P<0.001, P<0.05, P<0.01, P<0.0001, P<0.05, P<0.05, P<0.05, P<0.001) (Figure 1B). (4) The RNA-seq results showed that the gene expression thermogram among DR-MSCs, ND-MCSs and CR-MCSs were quite different (Figure 1C-D). Expression of BMP4 in ND-MSCs was significantly up-regulated (FC=3.311, P. adj=0.024) comparing with DR-MSCs. RR-MSCs expressed higher level of BMP4 than CR-MSCs (P＜0.05). The expression level of BMP6 in CR-MSCs was lower than that in ND-MSCs (P<0.05). DEGs were mainly enriched in BMP/SMADs signaling pathway by GO enrichment analysis (P. adj＜0.0001). (5) Comparing with DR-MSCs, DEGs in ND-MSCs were enriched in cytokine receptors and immune gene-related pathways by Reactome enrichment analysis (P. adj=0.023). (6) TIDE scores of CR-MSCs and RR-MSCs were higher than those of ND-MSCs (P=0.045 and P =0.06, respectively). Conclusion: Compared with DR-MSCs, Ph+ ALL-MSCs had a weaker proliferative capacity in vitro. MSCs derived from different disease status have protective effects on the apoptosis of SUP-B15 induced by drugs, the effects of DR-MSCs and ND-MSCs were stronger than that of CR-MSCs. The protective effect may be achieved through the activity of BMP/SMADs signaling pathway. Figure Legend: A. In Vitro proliferation of MSCs;B.The effect of MSCs cells on apoptosis of SUP-B15;C.Differential gene expression thermogram of DR-MSCs and ND-MCSs;D.Differential gene expression thermogram of ND-CR compared samples. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

First Documentation of Cell-to-Cell Peroxisome Transfer in Hematopoietic Stem and Progenitor Cells: A Method to "Relieve Stress"?

Cell to cell communication is key to survival. Recently it has been demonstrated that intercellular transfer of mitochondria occurs and can aid in marrow recovery after irradiation. Peroxisomes are a single membrane bound organelle responsible for various functions including lipid metabolism and protecting the cell from oxidative damage by reactive oxygen species (ROS). We have created the first mouse model in which peroxisomes can be tracked in living tissues using GFP tagged with a peroxisome targeting signal sequence (PTS), under the control of the Rosa promoter. Such GFP-PTS animals showed widespread peroxisomal GFP expression in all organs including hematopoietic stem and progenitor cells (HSPC) such as LSK-SLAM. Marrow stroma cell lines were established from GFP-PTS animals. In vitro peroxisome transfer from stroma to HSPC showed a transfer rate of up to 0.5 %, which could be achieved only when cell-to-cell contact took place; the use of transwells inhibited transfer. When HSPCs were simulated to proliferate, peroxisome transfer dropped by 50.1% (p<0.0001). The ability of HSPC to take up peroxisomes in vivo was tested in a model of hematopoietic cell transplant (HCT). Using GFP-PTS mice as recipients with wild-type (WT) marrow donors, we found that after busulfan or radiation (9 Gy) conditioning, peroxisome transfer could occur into WT adoptively HSPC within 48 hours after HCT at a rate of 1%. At 3 months post HCT, bone marrow mononuclear cells (BMMC) and Lin-SCA1+CKIT+ (LSK) were 100% donor and maintained GFP+peroxisomes at a frequency of 0.9% which were transferred from surrounding niche/stroma cells. To test HSPC to HSPC peroxisome transfer, sublethal conditioning (4.5 Gy) of WT was used to generate mixed chimerism models with marrow from GFP-PTS mice used as donor. Total peripheral or marrow engraftment was 45-55% donor as expected at 3 months post-HCT. WT BMMC could take up peroxisomes at a 0.9% frequency, while LSK had a significantly higher peroxisome uptake of 3.9% (p=0.02) indicating greater efficiency. Results were similar if GFP-PTS+ animals were recipients in a sublethal transplant setting. Remarkably, we could also demonstrate that peroxisome transfer can occur between GFP-PTS donor hematopoietic cells and WT cells in the liver, spleen, thymus, and brain after HCT. The genetic deletion of peroxisomes (via pex1 knockout) in animals resulted in ROS levels 10.3-fold over that of wild-type in PBMC, BMMC, and LSK (p<0.0001) as assessed by CellROX assay. Such animals had a severe defect in growth, had normal hematopoiesis, and increased radiation sensitivity indicating the importance of peroxisomes in reducing oxidative stress. To test the hypothesis that peroxisome transfer is utilized in situations of increased oxidative stress, pex1 KO stroma lines were exposed to increasing doses of radiation followed by incubation with GFP-PTS+ BMMC. Radiation treatment enhanced peroxisome transfer 5.1-fold over no-irradiated control (p<0.01), and cells receiving donor peroxisomes had greater viability (84% vs 68% viable, p<0.01) and reduced levels oxidative stress 24.8% compared to cell not receiving peroxisomes (p=0.03). The entire system was reproduced in a zebrafish model expressing GFP-PTS. Sublethally irradiated WT zebrafish engrafted donor GFP-PTS+ marrow at 50% and showed a significantly high rate of peroxisome uptake of into WT HSPS of 30% at 3 months post-HCT, suggesting some species may perform peroxisome transfer at much greater rates than others. In conclusion, we have demonstrated, for the first time, that peroxisome transfer can occur between a wide variety of cell types in vitro and in vivo, and HSPC-to-HSPC peroxisome transfer seems to be the most robust. The ability of peroxisome transfer to rescue cells from radiation-induced ROS and death was demonstrated in vitro while in vivo experiments are proceeding. There may be other far-reaching effects in terms of altering cellular metabolism occurring as a result of peroxisome transfer remaining to be discovered.

A composite, off-the-shelf osteoinductive material for large, vascularized bone flap prefabrication

We previously described an immortalized, genetically-engineered human Mesenchymal stromal cell line to generate BMP2-enriched Chondrogenic Matrices (MB-CM), which after devitalization and storage could efficiently induce ectopic bone tissue by endochondral ossification. In order to increase the efficiency of MB-CM utilization towards engineering scaled-up bone structures, here we hypothesized that MB-CM can retain osteoinductive properties when combined with an osteoconductive material. We first tested different volumetric ratios of MB-CM:SmartBone® (as clinically used, osteoconductive reference material) and assessed the bone formation capacity of the resulting composites following ectopic mouse implantation. After 8 weeks, as little as 25% of MB-CM was sufficient to induce bone formation and fusion across SmartBone® granules, generating large interconnected bony structures. The same composite percentage was then further assessed in a scaled-up model, namely within an axially-vascularized, confined, ectopically prefabricated flap (0.8 cm3) in rats. The material efficiently induced the formation of new bone (31% of the cross-sectional area after 8 weeks), including bone marrow and vascular elements, throughout the flap volume. Our findings outline a strategy for efficient use of MB-CM as part of a composite material, thereby reducing the amount required to fill large spaces and enabling utilization in critically sized grafts, to address challenging clinical scenarios in bone reconstruction. Statement of significanceMost bone repair strategies rely either on osteconductive properties of ceramics and devitalized bone, or osteoinductive properties of growth factors and extracellular matrices (ECM). Here we designed a composite material made of a clinically accepted osteoconductive material and an off-the-shelf tissue engineered human cartilage ECM with strong osteoinductive properties. We showed that low amount of osteoinductive ECM potentiated host cells recruitment to form large vascularized bone structures in two different animal models, one being a challenging prefabricated bone-flap model targeting challenging clinical bone repair. Overall, this study highlights the use of a promising human off-the-shelf material for accelerated healing towards clinical applications.

Doxorubicin induces prolonged DNA damage signal in cells overexpressing DEK isoform-2.

DEK has a short isoform (DEK isoform-2; DEK2) that lacks amino acid residues between 49-82. The full-length DEK (DEK isoform-1; DEK1) is ubiquitously expressed and plays a role in different cellular processes but whether DEK2 is involved in these processes remains elusive. We stably overexpressed DEK2 in human bone marrow stromal cell line HS-27A, in which endogenous DEKs were intact or suppressed via short hairpin RNA (sh-RNA). We have found that contrary to ectopic DEK1, DEK2 locates in the nucleus and nucleolus, causes persistent γH2AX signal upon doxorubicin treatment, and couldn't functionally compensate for the loss of DEK1. In addition, DEK2 overexpressing cells were more sensitive to doxorubicin than DEK1-cells. Expressions of DEK1 and DEK2 in cell lines and primary tumors exhibit tissue specificity. DEK1 is upregulated in cancers of the colon, liver, and lung compared to normal tissues while both DEK1 and DEK2 are downregulated in subsets of kidney, prostate, and thyroid carcinomas. Interestingly, only DEK2 was downregulated in a subset of breast tumors suggesting that DEK2 can be modulated differently than DEK1 in specific cancers. In summary, our findings show distinct expression patterns and subcellular location and suggest non-overlapping functions between the two DEK isoforms.

Mining of transcriptome identifies CD109 and LRP12 as possible biomarkers and deregulation mechanism of T cell receptor pathway in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a heterogeneous disease with highest mortality compared to other types of leukemia. There is a need to find the gene abnormalities and mechanisms behind them due to their heterogenic nature. The present study is aimed to understand genes, pathways and biomarker proteins influenced by transcriptomic deregulation due to AML. Differentially expressed gene (DEG), protein-protein interaction network, gene ontology, KEGG pathway, variant analysis and secretome analyses were performed using different AML RNAseq datasets. A total of 655 DEGs including 291 up-regulated and 364 down-regulated genes, which were satisfied with a fold change of 1.5 were identified. Top hub genes for AML were identified as TP53, PTPRC and AKT1. This integrative bioinformatics approach revealed the deregulation of T Cell Receptor (TCR) pathway and altered immune response related genes. The survival analysis revealed the associated deregulation of multiple TCR pathway related genes. Variant analysis identified the benign and likely benign nature of many important target genes and markers screened, which were found to have an important role in the progression of AML. DEGs and secretome analysis found out a set of seven molecules represents potential biomarkers for AML. In vitro analytical validation showed overexpression pattern of CD109 and LRP12 in AML cell line and HL-60 cells than the normal human bone marrow-derived stromal cell line HS-5. Here we report first time for CD109 and LRP12 as a possible biomarkers for the diagnostic significance. Amino acid substitutions detected by variant analysis and deregulation of immune checkpoint molecules revealed their role in reducing immune response and inability to fight cancer cells. In conclusion, this study highlights the possibility of new biomarkers for AML and the mechanism of decrease in immune response due to the downregulation of co-stimulatory immune molecules, which needs further clinical validation investigations.

Bmal1 Regulates Prostate Growth via Cell-Cycle Modulation.

The circadian clock system exists in most organs and regulates diverse physiological processes, including growth. Here, we used a prostate-specific Bmal1-knockout mouse model (pBmal1 KO: PbsnCre+; Bmal1fx/fx) and immortalized human prostate cells (RWPE-1 and WPMY-1) to elucidate the role of the peripheral prostate clock on prostate growth. Bmal1 KO resulted in significantly decreased ventral and dorsolateral lobes with less Ki-67-positive epithelial cells than the controls. Next, the cap analysis of gene expression revealed that genes associated with cell cycles were differentially expressed in the pBmal1 KO prostate. Cdkn1a (coding p21) was diurnally expressed in the control mouse prostate, a rhythm which was disturbed in pBmal1 KO. Meanwhile, the knockdown of BMAL1 in epithelial RWPE-1 and stromal WPMY-1 cell lines decreased proliferation. Furthermore, RWPE-1 BMAL1 knockdown increased G0/G1-phase cell numbers but reduced S-phase numbers. These findings indicate that core clock gene Bmal1 is involved in prostate growth via the modulation of the cell cycle and provide a rationale for further research to link the pathogenesis of benign prostatic hyperplasia or cancer with the circadian clock.

Life in an optical fiber: Monitoring of cell cultures with microcavity in-line Mach-Zehnder interferometer

Monitoring cell adhesion and growth are crucial for various applications involving drug screening, cytotoxicity, and cytocompatibility studies. However, acquiring accurate information about the growing state and responsiveness to a treatment of a cell system in a real-time and label-free manner is still a challenge. This work presents the first research on direct, real-time, and label-free adherent cell culture monitoring using a microcavity in-line Mach–Zehnder interferometer (μIMZI) fabricated in an optical fiber. The sensing solution based on μIMZI offers a great advantage over many other monitoring concepts tracking the changes taking place on the microcavity's bottom surface and within its volume, thus offering a greater penetration depth. In this study, we verified performance of the approach using a non-cancer bone marrow stromal cell line HS-5. The results demonstrate that the changes of the acquired signal are closely related to the different states of cells' adhesion, proliferation, morphology, and variation of mass. Thus, this label-free, real-time μIMZI-based monitoring technique gives a great promise to the analysis or monitoring of relevant new treatments in future scientific, as well as clinical applications.

Echiumin E, an Aryl Dihydronaphthalene Lignan from the Australian Invasive Plant Paterson's Curse (Echium plantagineum).

A new aryl dihydronaphthalene lignan, echiumin E (1), and four known compounds, echiumin A, globoidnan A, (-)-rabdosiin, and rosmarinic acid (2-5), were isolated from the Australian invasive plant Echium plantagineum (Paterson's curse) for the first time. Echiumin E (1) was characterized by 1D/2D NMR spectroscopy and MS spectrometry, with its absolute configuration assigned through comparison of experimental and TDDFT-calculated ECD data. Echiumin E (1) along with compounds 3-5 were screened in vitro against three cancer cell lines (SH-SY5Y, HeLa, and PC-3) and a prostate stromal (normal) cell line (WPMY-1) using a resazurin reduction assay. Echiumin E (1) was found to be active toward HeLa cells (IC50 0.21 μM).

TEM8 Tri-specific Killer Engager binds both tumor and tumor stroma to specifically engage natural killer cell anti-tumor activity

BackgroundThe tumor microenvironment contains stromal cells, including endothelial cells and fibroblasts, that aid tumor growth and impair immune cell function. Many solid tumors remain difficult to cure because of tumor-promoting...

Expression of Concern: "Neferine inhibits growth and migration of gastrointestinal stromal tumor cell line GIST-T1 by up-regulation of miR-449a" [Biomed. Pharmacother. 109 (2019) 1951-1959

Expression of Concern: "Neferine inhibits growth and migration of gastrointestinal stromal tumor cell line GIST-T1 by up-regulation of miR-449a" [Biomed. Pharmacother. 109 (2019) 1951-1959

Bisphenols A, F, S and AF trigger apoptosis and/or endoplasmic reticulum stress in human endometrial stromal cells

Disruption of non-differentiated endometrial stromal cells could have noxious consequences in female reproduction, impairing endometrial remodelling and implantation. Following the classification of bisphenol A (BPA) as an endocrine disrupting chemical, it started to be gradually withdrawn from the market, being substituted by structural analogues, whose effects in human health are not fully understood. This work used a telomerase-immortalized human endometrial stromal cell line (St-T1b) to study the effects of BPA and its three most commercialized structural analogues (ranked: bisphenols S, F and AF) on endometrial stromal cells to understand their effects on female reproductive function. Bisphenols showed dissimilar effects. All four compounds generated endoplasmic reticulum (ER) stress. In addition, bisphenols A, F and AF induced apoptosis through different mechanisms, with bisphenol AF causing cell cycle arrest at G2/M phase. Bisphenol AF decreased mitochondrial transmembrane potential and bisphenols A, F and AF produced oxidative stress.

Processing methods for human amniotic membrane as scaffold for tissue engineering with mesenchymal stromal human cells.

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function. The aims of this work were to compare chemically and physically processed human Amniotic Membranes (hAM) and analyze the cytocompatibility and proliferation rate (PR) of two primary human mesenchymal stromal cell lines, from different sources and donor conditions seeded over these scaffolds. The evaluated hAM processes were: cold shock to obtain a frozen amniotic membrane (FEAM) with remaining dead epithelial cells, denudation of hAM with trypsin for 20/10min (DEAM20/10) or treatment with sodium dodecyl sulfate to decellularized hAM (DAM). All samples were sterilized with gamma radiation. The selection of the treated hAM to then generate composites was performed by scanning and transmission electron microscopy and characterization by X-ray diffraction, selecting DEAM10 and FEAM as scaffolds for cell seeding. Two sources of primary human stromal cells were used, both developed by our researchers, human Dental Pulp Stem Cells (hDPSC) from living donors and human Mesenchymal Stromal Cells (hMSC) from bone marrow isolated from brain dead donors. This last line of cells conveys a novel source of human cells that, to our knowledge, have not been tested as part of this type of construct. We developed four in vitro constructs without cytotoxicity signs and with different PR depending on the scaffolds and cells. hDPSC and hMSC grew over both FEAM and DEAM10, but DEAM10 allowed higher PR.

Functionalized 3D-Printed ST2/Gelatin Methacryloyl/Polcaprolactone Scaffolds for Enhancing Bone Regeneration with Vascularization.

Growth factors were often used to improve the bioactivity of biomaterials in order to fabricate biofunctionalized bone grafts for bone defect repair. However, supraphysiological concentrations of growth factors for improving bioactivity could lead to serious side effects, such as ectopic bone formation, radiculitis, swelling of soft tissue in the neck, etc. Therefore, safely and effectively applying growth factors in bone repair biomaterials comes to be an urgent problem that needs to be addressed. In this study, an appropriate concentration (50 ng/mL) of Wnt3a was used to pretreat the 3D-bioprinting gelatin methacryloyl(GelMA)/polycaprolactone(PCL) scaffold loaded with bone marrow stromal cell line ST2 for 24 h. This pretreatment promoted the cell proliferation, osteogenic differentiation, and mineralization of ST2 in the scaffold in vitro, and enhanced angiogenesis and osteogenesis after being implanted in critical-sized mouse calvarial defects. On the contrary, the inhibition of Wnt/β-catenin signaling in ST2 cells reduced the bone repair effect of this scaffold. These results suggested that ST2/GelMA/PCL scaffolds pretreated with an appropriate concentration of Wnt3a in culture medium could effectively enhance the osteogenic and angiogenic activity of bone repair biomaterials both in vitro and in vivo. Moreover, it would avoid the side effects caused by the supraphysiological concentrations of growth factors. This functionalized scaffold with osteogenic and angiogenic activity might be used as an outstanding bone substitute for bone regeneration and repair.

3D Living Dressing Improves Healing and Modulates Immune Response in a Thermal Injury Model.

Thermal injury trauma can induce a state of immunosuppression, causing wounds to become chronic in nature. Stem cell-based therapies represent a promising new approach to treat such wounds due to their capacity to self-renew and their multi-lineage potential. Mesenchymal stem cells (MSCs) are known to secrete endogenous factors that stimulate wound healing by promoting angiogenesis, extracellular matrix remodeling, skin regeneration, and by dampening down inflammation. MSC delivery in a biomaterial construct can augment their wound-healing capacity by concentrating cells at the burn site and upregulating trophic factor secretion. The work presented is the first to evaluate repair in an in vitro raft thermal injury model using a regenerative, dual cell delivery three-dimensional (3D) core/shell (c/s) "living dressing" construct. This previously characterized 3D c/s bioprinted construct, which delivers both MSCs and endothelial cells, was used to treat an in vitro 3D raft skin thermal injury wound model. The mesenchymal stromal cell line (T0523) was encapsulated within a gelatin-based shell bioink, and human umbilical vein endothelial cells within a chitosan-based core bioink to biofabricate a living dressing for enhanced thermal injury repair and regeneration. We hypothesized that the cell-laden c/s tissue engineered construct (TEC) would strengthen the wound's proangiogenic, anti-inflammatory, and skin regeneration potential. An in vitro thermal injury in a 3D raft skin model showed a slight delay in wound closure in the presence of the c/s TEC but was augmented by corresponding increases in the release of wound-healing factors, epidermal growth factor, matrix metalloproteinases-9, transforming growth factor-α, platelet-derived growth factor; a decrease in pro-inflammatory factor interleukin-6, and evidence of neovascularization.

The protective effects of Azilsartan against hypoxia in endometrial stromal cells: an implication in endometriosis

IntroductionEndometriosis is a reproductive disorder closely associated with hypoxia stress. Increasing evidences have implied the important roles of angiotensin II (ANG II) receptors in the pathophysiology of endometriosis. Thus, we speculated that Azilsartan (AZL), an ANG II receptor blocker, might have an effective function in controlling endometriosis.Material and methodsThe endometriotic animal model was established in female SD rats (6-8 weeks old, 200-220 g). Rats were divided into sham group and endometriosis (EMS) group. Rats in EMS group were anaesthetized by halothane and a mid-ventral incision was performed to expose the bowels. Human endometrial stromal cell line T-HESC was used for the in vitro assays. The T-HESC cells were cultured in DMEM-F12 mediumwith 10% fetal bovine serum (FBS, Hyclone), and 4 mM L-glutamine, 0.25% HEPES plus necessary antibiotics (Sigma-Aldrich, USA), at 37°C in a humidified atmosphere of 5% CO2.ResultsThe results show that upregulation of ANG II type 1 (AT1) receptor was observed in the endometriotic rat models. Treatment with AZL prevented the development of endometriotic lesions and suppressed the expressions of HIF-1α and cyclooxygenase 2 (COX-2) in endometriotic rats. In vitro assays proved that hypoxia-induced proliferation, migration, and invasion of T-HESC cells were attenuated by AZL. AZL inhibited the expression levels of hypoxia-inducible factor-1α (HIF-1α), COX-2, and prostaglandin E2 (PGE2) production in hypoxia-induced T-HESC cells. Overexpression of HIF-1α blocked the effects of AZL on T-HESC cells in response to hypoxia.ConclusionsAZL showed therapeutic function on endometriosis through inhibiting hypoxia-induced cell proliferation, migration, and invasion of T-HESC cells via HIF-1α/COX-2/PGE2 signaling.

SIRT1 suppresses in vitro decidualization of human endometrial stromal cells through the downregulation of forkhead box O1 expression

SIRT1 regulates survival, DNA repair, and metabolism in human cells and has pleiotropic effects on age-related diseases through either deacetylating target proteins or inhibiting gene transcription. Forkhead box O1 (FOXO1) is one of the most important transcription factors during decidualization. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) are well-known FOXO1-dependent genes in decidualizing cells. To determine whether SIRT1 plays a role in decidualization, we investigated morphological changes in cells following artificially stimulated decidualization and expression levels of PRL, IGFBP1, and FOXO1 in the immortalized non-neoplastic human endometrial stromal cell line T HESCs. SIRT1 expression decreased in the decidualization condition and SIRT1 inhibited morphological changes caused by decidualization of T HESCs. SIRT1 suppressed PRL, IGFBP1, and FOXO1 expression; inhibited FOXO1, PRL, and IGFBP1 promoter activity; and decreased histone protein acetylation of the FOXO1 promoter. We found that FOXO1 expression increased in the secretory phase compared with the proliferative phase, whereas SIRT1 expression decreased in the secretory phase in the human endometrium. We also revealed that SIRT1 may inhibit embryo implantation according to the blastocyst-like spheroid implantation assay. Collectively, these results indicate that SIRT1 suppresses decidualization of human endometrial stromal cells by inhibiting FOXO1 expression.

Inhibition of Canonical Wnt Signaling Promotes Ex Vivo Maintenance and Proliferation of Hematopoietic Stem Cells in Zebrafish.

The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow; however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution. Here, using a stromal cell line from the zebrafish kidney, zebrafish kidney stromal (ZKS), we uncover that an inhibitor of canonical Wnt signaling, IWR-1-endo, is a potent regulator of HSCs. Coculture assays revealed that ZKS cells were in part supportive of maintenance, but not expansion, of gata2a:GFP+runx1:mCherry+ (gata2a+runx1+) HSCs. Transcriptome analysis revealed that, compared with candidate niche cells in the kidney, ZKS cells weakly expressed HSC maintenance factor genes, thpo and cxcl12, but highly expressed canonical Wnt ligand genes, wnt1, 7bb, and 9a. Thpo supplementation in ZKS culture slightly increased, but inhibition of canonical Wnt signaling by IWR-1-endo treatment largely increased the number of gata2a+runx1+ cells (>2-fold). Moreover, we found that gata2a+runx1+ cells can be maintained by supplementing both IWR-1-endo and Thpo without stromal cells. Collectively, our data provide evidence that IWR-1-endo can be used as a novel supporting factor for HSCs.

P694: INVESTIGATING THE ROLE OF CELL ADHESION MOLECULES IN TUNNELING NANOTUBES FORMATION IN CHRONIC MYELOID LEUKEMIA MICROENVIRONMENT

Background: The cross-talk and interactions between leukemia cells and their microenvironment play an important role in the development of drug resistance. Tunneling nanotubes (TNTs) represent a novel type of direct cell-cell communication way allowing for direct transfer of different types of cargo between distant cells. Our previous studies (Kolba et al., 2019) have shown that these structures are formed between bone marrow stromal HS-5 cells and chronic myeloid leukemia (CML) K562 cells. Significantly, TNT-mediated transfer of membrane vesicles from stromal to leukemic cells resulted in the stroma-mediated protection of CML cells from imatinib-induced apoptosis. Despite the efforts made to understand the molecular mechanisms regulating TNTs formation, they still remain unclear - especially in the leukemia microenvironment. We have shown that TNTs are formed between stromal and leukemic cells, following direct cell-cell interaction and cell dislodgement. Thus, we have focused our attention on cell adhesion molecules, as potential key players in tunneling nanotubes formation and TNT-mediated vesicles transfer between leukemic and stromal cells. Aims: The aim of presented studies was to elucidate the role of CD44 and ITGβ1 - one of the most important cell adhesion molecules, in TNTs formation between leukemic and stromal cells and TNT-mediated exchange of vesicles. Methods: To examine this, we established the HS-5 stromal cell line with silenced CD44 protein expression. Then, we co-cultured control (wt) or CD44-silenced HS-5 cells with leukemic K562 cells. For ITGβ1 studies, we used blocking anti-ITGβ1 antibody. Using confocal microscopy, three types of TNTs in co-cultures were counted: homotypic TNTs formed between stromal cells, homotypic TNTs formed between CML cells and TNTs formed between stromal and CML cells (heterotypic). Moreover, the TNT-mediated transfer of cellular vesicles from stromal to leukemic cells was assessed in these co-cultures using flow cytometry. Since CD44 expression increases in CML cells after co-culture with stroma, we examined its potential role in the drug resistance in CML acceptor cells. To do this, rhodamine-123 efflux assay was used. Results: We found that both, tunneling nanotubes number and uptake of cellular vesicles by leukemia, decreased after co-culture with CD44-silenced stromal cells and after blocking with anti-ITGβ1 antibody. Flow cytometry analyses of rhodamine-123 efflux showed significantly increased efflux pump activity in leukemic acceptor cells. Summary/Conclusion: Our data suggest that CD44 and ITGβ1 can play an important role in formation of tunneling nanotubes between leukemic and stromal cells and TNT-mediated cellular vesicles exchange. Moreover, CD44 presented in acceptor cells is involved in the development of drug resistance in leukemic cells, by promoting activity of efflux transporters.

Abstract 6374: BMP6 overcomes multiple myeloma-induced bone disease by inducing the expression of small leucine-rich proteoglycans

Abstract Multiple myeloma (MM), the second most common hematologic malignancy in adults, is characterized by clonal proliferation of malignant plasma cells and tumor-induced complications, including MM-induced bone disease (MBD). Up to 90% of MM patients develop MBD during the course of their disease, which is a major cause of morbidity and mortality. MM therapy focuses primarily on agents that seek to eradicate the tumor cells and others which inhibit bone resorption. In contrast, no drugs are currently available to promote bone formation in MM patients and reverse MBD. Towards addressing this unmet therapeutic need, we applied a modified version of our “humanized” bone marrow (BM)-like scaffold (huBMsc)-based xenograft model, where MM cells and bone marrow mesenchymal stromal cells (BMMSCs) are co-implanted. In this system, we investigated the impact of MM cells on BMMSCs to identify markers that can be targeted with the intent to reverse MBD. After confirming that MM cells indeed inhibit bone formation in vivo, we performed transcriptional analysis of ex vivo expanded “unexposed” control BMMSCs vs. “MM-exposed” BMMSCs and found 332 differentially expressed genes. Next, we compared these data with transcriptional profiles of BMMSCs after in vitro differentiation towards osteoblasts, as well as publicly available data of transcriptional profiles of BMMSCs obtained from MM, ALL, and AML patients. Combined, this analysis identified 8, MM-specific, differentially expressed genes, including the small leucine-rich proteoglycans (SLRPs), ASPN, OGN and OMD, that are 1) suppressed in the presence of MM (xenograft model and patient-derived BMMSCs); 2) induced during in vitro osteogenic differentiation; and 3) not dysregulated in BMMSCs in other hematological malignancies. As reported previously, we observed that bone morphogenetic protein (BMP)6 can upregulate the expression of these SLRPs in BMMSCs, while inducing osteogenic differentiation. Moreover, incubation of MM cells with BMP6 in either mono- or co-cultures with stromal cell lines, resulted in reduced MM cell viability. In addition, pretreatment of BMMSCs with BMP6 completely abolished their support of MM proliferation. Interestingly, BMP6, in contrast to BMP2 and BMP4, did not induce, but inhibited receptor activator of nuclear kappa-B ligand (RANKL)-dependent osteoclast generation. Collectively, our analyses identify BMP6 as a master regulator that promotes osteogenic differentiation, inhibits RANKL-dependent osteoclastogenesis and can directly induce MM cell death. Therefore, by reshaping the BM microenvironment to become less tumor-supportive, BMP6 represents an attractive candidate for MM treatment. Citation Format: Jiaxian Wang, Thomas Baardemans, Ricardo de Matos Simoes, Olga Dashevsky, Huipin Yuan, Joost D. de Bruijn, Anton C. Martens, Constantine S. Mitsiades, Richard W. Groen. BMP6 overcomes multiple myeloma-induced bone disease by inducing the expression of small leucine-rich proteoglycans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6374.